Abstracts of the 3rd International Electronic Conference on Microbiology ^†

Abstract

The current proceedings summarize the presentations delivered during the third International Electronic Conference on Microbiology (ECM 2025), which was held online from 1 to 3 April 2025, via the SciForum platform. This virtual event brought together researchers from around the world to share recent advances in microbiological sciences. The ECM 2025 highlighted recent developments across a broad spectrum of microbiological research, including antimicrobial resistance, gut microbiota, infectious diseases, and environmental microbiomes. Participants shared their work through online presentations and abstracts, with selected submissions invited for full publication. The event fostered global collaboration, promoted open-access science, and showcased innovative tools for studying and managing microbial systems in health, agriculture, and industry. The multidisciplinary program was organized into several thematic sessions: S1. Gut Microbiota and Health Disease. S2. Foodborne Pathogens and Food Safety. S3. Antimicrobial Agents and Resistance. S4. Emerging Infectious Diseases. S5. Microbiome and Soil Science. S6. Microbial Characterization and Bioprocess. S7. Microbe–Plant Interactions. This conference report presents summaries of the contributions made by participating authors over the three-day event.

1. Session: Gut Microbiota and Health Disease

1.1. Dissemination of Enterococcus spp. Across One Health Settings: Insights from Fingerprinting Analysis

Joana Monteiro Marques ^1,2, Nicolau Fialho ^1,2, Maria do Mar Duarte ^1,2, Maria Teresa Barreto Crespo ^3,4, Teresa Semedo-Lemsaddek ^1,2

¹ 

Centre for Interdisciplinary Research in Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Av. da Universidade Técnica de Lisboa, 1300-477 Lisbon, Portugal

² 

Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), 1300-477 Lisbon, Portugal

³ 

iBET—Institute of Experimental Biology and Technology, 2781-901 Oeiras, Portugal

⁴ 

ITQB NOVA—Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal

Enterococcus spp. are ubiquitous Gram-positive bacteria present in the gastrointestinal tract of humans and animals, as well as in various environmental niches. Over recent decades, Enterococcus spp. have emerged as significant opportunistic pathogens in healthcare-associated infections, with vancomycin-resistant E. faecium identified as a critical health threat. In this context, the One Health concept underscores the interconnection of human, animal, and environmental health, highlighting the importance of intervention fields such as antimicrobial resistance and food safety.

This study aimed to isolate and analyze the genetic diversity of enterococci from a diverse range of samples across One Health domains: sick (n = 18) and healthy (n = 17) humans, sick (n = 13) and healthy (n = 14) animals, canteen food (n = 11), public transportation surfaces (n = 8), and surface water (n = 8). Samples were cultured on enterococci-selective media, with and without vancomycin supplementation. Approximately 20% of characteristic colonies were selected and confirmed at the genus level, followed by DNA extraction and RAPD-PCR using primers OPC15 and GTG5 in independent reactions. Amplification analysis and dendrogram construction were performed using the BioNumerics software to obtain relationship patterns within and across One Health sectors.

A total of 148 isolates were obtained from selective media, comprising 81 environmental, 27 animal-, and 35 human-derived isolates, including 24 from vancomycin-supplemented media. Overall, no distinct clustering by sample type or One Health sector was observed, suggesting the widespread dissemination of genetically diverse enterococci across these settings. However, isolates from surface water and clinical samples showed high genetic similarity (≥80–95%), with some clinical clusters also containing isolates from healthy humans.

These findings confirm the widespread dissemination of enterococci across One Health sectors, including presumptive vancomycin-resistant enterococci, and underscore the need for further biosurveillance studies.

1.2. Comparative Analysis of Gut Microbiota Metabolism in Healthy and Pathological Conditions

Ekaterina Sorokina, Ekaterina Chernevskaya, Maria Getsina

• Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, Moscow, Russia

Metabolite analysis is a promising method and is increasingly being used to identify dysfunctions of various organs in patients. One of the sources of metabolites in the body is the gut microbiota. An increase in sepsis-associated aromatic microbial metabolites is a prognostically unfavorable factor and increases the risk of death in patients with sepsis.

The aim of this work was to evaluate the composition and function of the gut microbiota in patients with sepsis compared to healthy donors using chromatograph mass spectrometry.

The study included 10 patients with sepsis and 9 healthy donors, comparable in gender and age. The incubation of feces samples was carried out in thioglycollate medium (TG (at 37 °C)) with the addition of sepsis-associated microbial metabolites (25 μM phenyllactic acid (PLA) or 25 μM 4-hydroxyphenyllactic acid (4-HPLA)). Metabolite concentrations were determined using a GC-2010 Plus gas chromatograph and a GCMS-QP2020 mass spectrometer (Shimadzu, Kyoto, Japan). The proportion of sepsis-associated aromatic metabolites in patients with sepsis was significantly higher than normal with a significance of 40%, while in donors, it did not exceed 5%. Comparison of the metabolomic profiles of normobiota and pathobiota in an experiment showed that when loaded with sepsis-associated metabolites of PLA and 4-HPLA, the microbiota of a healthy subject biotransforms them into the end products of microbial metabolism, whereas the pathobiota of a septic patient is unable to perform this function. Thus, in sepsis, the normobiome is transformed into a pathobiome, which reflects the dysfunction of the gut microbiota.

1.3. Decreased Levels of Aromatic Microbial Metabolites as a Reflection of Microbiota Dysfunction in Cancer Patients

Maria Getsina, Ekaterina Chernevskaya

• Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, 25-2 Petrovka Str., 107031 Moscow, Russia

Introduction

There is growing evidence that cancer patients have altered metabolisms due to disruption of the microbiome. Serum concentrations of aromatic microbial metabolites may reflect microbiota dysfunction.

Aim

The aim of this study was to evaluate the serum concentrations of aromatic microbial metabolites in patients undergoing treatment for malignant oncological diseases.

Methods

Two cohorts of patients were studied: patients with pancreatic cancer before surgery (n = 64) and children with various malignant oncological diseases (leukemia, lymphoma, nephroblastoma, ependymoma, etc.) (n = 40). The study used two comparison groups: healthy donors (n = 18) and practically healthy children referred for preventive examination (n = 18). Aromatic microbial metabolites such as phenyllactic acid, hydroxyphenyllactic acid and hydroxyphenylacetic acid were identified by GC-MS.

Results

The concentration of metabolites in the serum of patients with cancer in both studies is statistically significantly lower than that of healthy subjects. The sum of concentrations of the aromatic microbial metabolites was 1.9 (1.5; 2.2) µmol/L in healthy donors and 1.4 (0.9; 2.0) µmol/L in pancreatic cancer patients before surgery (p-value 0.001). The sum of concentrations of the aromatic microbial metabolites in children was 2.2 (1.5; 2.6) µmol/L in the control group vs. 1.5 (1.1; 1.8) µmol/L in the cancer group (p-value = 0.001).

Conclusions

Our study reveals the profound metabolic dysfunction of microbiota in cancer, as serum aromatic microbial metabolites were significantly different in patients compared to healthy subjects. It is promising to study the relationship between metabolic profiles and gut microbiota composition for a deep understanding of cancer pathogenesis.

1.4. Evaluation of Molecular Hydrogen in the Treatment of Intestinal Inflammation in a Murine Model

Oumaima Anachad, Wafaa Taha, Imad Fenjar, Hicham Wahnou, Faiza Bennis, Fatima Chegdani

• Laboratory of Immunology and Biodiversity, Faculty of Sciences Aïn Chock, Hassan II University of Casablanca, Casablanca, Morocco

Intestinal inflammation is central to the pathogenesis of various gastrointestinal disorders, making the exploration of effective treatment strategies essential. This study investigates the effects of molecular hydrogen in alleviating intestinal inflammation induced in a murine model. We aim to assess its therapeutic potential through histopathological analysis, gene expression evaluation, and bacterial load quantification.

Intestinal inflammation was induced in 15 female mice using kappa-carrageenan over 3 months. Following induction, the mice were assigned to four treatment groups: diclofenac (20 mg/kg), molecular hydrogen, negative control, and positive control. Treatment lasted 15 days. Colon length was measured as an indicator of inflammation, and histopathological analysis was conducted on the colon, liver, and intestines. RNA was extracted from the colon for quantitative reverse transcription PCR to assess the expression of inflammation-related genes (TNFα, IL1β, IL6, IL10, and SOD1), while DNA from fecal samples was analyzed for E. coli and Bifidobacterium presence. Bacterial load quantification is being optimized using qPCR, with a focus on refining the melting temperature protocol for specificity.

The histopathological analysis revealed significant tissue damage in the inflammation-induced group, with partial recovery observed in the diclofenac- and molecular hydrogen-treated groups. The qRT-PCR results showed a more pronounced inflammatory response in the untreated group, while IL10 was lower in the treated groups, suggesting the modulation of the inflammatory response. IL6 was moderately reduced by diclofenac, while molecular hydrogen had only mild effects. Interestingly, IL1β was elevated in both treatment groups, indicating possible pro-inflammatory effects. Additionally, SOD1 levels were lower in treated groups, indicating reduced antioxidant activity.

The preliminary findings suggest that both diclofenac and molecular hydrogen have therapeutic potential in reducing intestinal inflammation. Diclofenac primarily acts through anti-inflammatory pathways, while molecular hydrogen may modulate oxidative stress and gut microbiota. Further studies are needed to clarify the mechanisms of action for these treatments in gastrointestinal diseases.

1.5. Gut Microbiota-Supporting Beverage: Kombucha’s Activity Against Gram-Positive Bacteria

Maria Leszczyńska ¹, Paulina Lenart ¹, Maciej Grzegorczyk ¹, Agnieszka Grzegorczyk ², Martyna Kasela ², Sylwia Andrzejczuk ²

¹ 

Student’s Scientific Association “mikroGRAM” at the Department of Pharmaceutical Microbiology, Medical University of Lublin, 20-093 Lublin, Poland

² 

Department of Pharmaceutical Microbiology, Medical University of Lublin, 20-093 Lublin, Poland

Fermented foods have been rapidly gaining popularity recently due to their use in medicine and dietetics. Kombucha is a fermented beverage containing numerous probiotic bacteria and their metabolites, which have beneficial effects on the digestive system. The purpose of this study was to determine the antimicrobial activity of kombucha containing probiotic strains against bacteria that cause gastrointestinal diseases.

The activity of kombucha was tested before fermentation and during fermentation on days 1, 2, 3, 7, 8, and 14 against reference strains of Gram-positive bacteria from the American Type Culture Collection (ATCC) such as methicillin-sensitive Staphylococcus aureus (MSSA) ATCC 29213, two methicillin-resistant Staphylococcus aureus (MRSA) strains (ATCC 43300 and ATCC BAA-1707), Bacillus cereus ATCC 10876, and Enterococcus faecalis ATCC 29212. The well-diffusion method was used for this assessment, and the antimicrobial activity of this beverage was evaluated by the zone of growth inhibition (mm) around the kombucha well.

The highest activity was observed after 14 days of kombucha fermentation against almost all tested bacteria with a zone of growth inhibition of 18-21 mm. The highest activity was observed against S. aureus ATCC 29213 and S. aureus ATCC 43300.

These data show that kombucha is a beverage that can support the treatment of food poisoning caused by pathogenic microorganisms such as methicillin-sensitive S. aureus (MRSA), methicillin-resistant S. aureus (MRSA), B. cereus, and E. faecalis.

1.6. Microbial Signatures in High- and Low-Blood-Glucose Participants of the Generation 100 Study

Natalia G Bednarska ¹, Asta Kristine Håberg ²

¹ 

London School of Hygiene and Tropical Medicine, Department of Infectious Tropical Diseases, London, UK

² 

NTNU, Department Neuromedicine and Movement Science, Norwegian University of Science & Technology (NTNU), 7034 Trondheim, Norway

Introduction: Type 2 diabetes mellitus (T2DM) is an age-related metabolic disease that is often considered inflammatory, as various cytokine profiles are associated with its progression. T2DM typically follows a prediabetic stage, during which insulin resistance develops. This stage can often be corrected early in life, potentially preventing or delaying the onset of T2D. Intestinal dysbiosis has been linked to metabolic disorders, including insulin resistance and T2D. A healthy, diverse gut microbiome, composed primarily of four phyla—Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria—is essential for maintaining the gut epithelial barrier. Phylogenetically related bacterial groups, such as Proteobacteria and Enterobacteriaceae, have been linked to poor glycaemic control and negative metabolic outcomes, including obesity, insulin resistance, and an impaired lipid profile. Methods: This study involved 73 older adults (ages 76–83) from the randomized controlled trial entitled the Generation 100 Study. We performed high-throughput sequencing of the bacterial 16S rRNA gene to obtain metagenomic microbial profiles for all participants. These profiles were then correlated with clinical measures. Results: We observed distinct patterns of microbial beta diversity between the high- and normal-glucose groups. Overall, the microbial diversity was significantly reduced in the high-glucose group. At the highest taxonomic level (Phylum), we found that Synergistes, Elusimicobia, Euryarchaeota, Verrucomicrobia, and Proteobacteria were all significantly decreased in participants with high blood glucose. Additionally, P. copri was significantly elevated in the high-glucose (10-fold increase) and high-CRP (777-fold increase) groups, suggesting that it may serve as an early inflammatory and diabetic marker. These findings are consistent with previous research identifying P. copri as a pro-inflammatory pathogen. We also found that the Fusobacterium genus was significantly increased in the normal glucose group, with a 151-fold increase compared to the high-glucose group (p 0.005). Conclusions: Our results indicate significant changes in the microbiome that may provide valuable insights for early intervention in pre-diabetic states.

1.7. The Role of Personalized Nutrition to Modulate Gut Microbiota for Disease Prevention

A. Perez-Vazquez ¹, P. Barciela ¹, M. Carpena ¹, F. Chamorro ¹, A.O.S Jorge ^1,2, P. Donn ¹, Rafael Nogueira-Marques ¹, M.A. Prieto ¹

¹ 

Universidade de Vigo, Nutrition and Bromatology Group, Department of Analytical Chemistry and Food Science, Instituto de Agroecoloxía e Alimentación (IAA)—CITEXVI, 36310 Vigo, Spain

² 

REQUIMTE/LAQV, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, R. Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal

The human body hosts a diverse community of microorganisms, with the gastrointestinal tract being the most densely colonized, hosting a thousand microbial species collectively referred to as the gut microbiota. Recent studies have demonstrated that the gut microbiota maintains multidirectional and communicational connections with various organs through metabolic, endocrine, neural, humoral, and immunological pathways. Alterations in this gut–organ axis can lead to a wide range of health issues beyond gastrointestinal disorders which affect other organ systems. Emerging evidence highlights the intricate relationship between diet and microbiota in the onset and progression of diseases. Personalized nutrition has gained attention as a strategy to identify specific microbiome traits that predict responses to dietary components. This approach holds promise for designing targeted dietary interventions that promote favorable health outcomes by modulating the gut microbiota. This systematic review explores current insights into the mechanisms underlying gut–organ interactions and examines the potential of personalized nutrition for disease prevention taking advantage of these mechanisms. Furthermore, it addresses existing limitations in understanding the mechanistic pathways and the implementation of personalized nutrition as a preventive strategy. Finally, by summarizing the latest findings, this review aims to provide a clearer understanding of the gut microbiota’s role in systemic health and its modulation through dietary strategies, paving the way for innovative approaches in preventive medicine.

2. Session: Foodborne Pathogens and Food Safety

2.1. Antimicrobial Activity of Peptides Produced by Saccharomyces cerevisiae, Wickerhamomyces anomalus and Tetrapisispora phaffii Against Foodborne Pathogens

Patrícia Branco ^1,2, Diogo Mendes ¹, Elisabete Muchagato Maurício ^1,3,4

¹ 

BIORG—Bioengineering and Sustainability Research Group, Faculdade de Engenharia, Universidade Lusófona, Av. Campo Grande 376, 1749-024 Lisbon, Portugal

² 

Linking Landscape, Environment, Agriculture and Food (LEAF), Associated Laboratory TERRA, Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017 Lisboa, Portugal

³ 

CBIOS—Research Center for Biosciences & Health Technologies, Universidade Lusófona, Campo Grande 376, 1749-024 Lisbon, Portugal

⁴ 

Elisa Câmara, Lda, Dermocosmética, Centro Empresarial de Talaíde, n°7 e 8, 2785-723 Lisbon, Portugal

In the food industry, synthetic preservatives are commonly used to inhibit microbial spoilage, prevent chemical and nutritional changes, and extend shelf life. However, growing consumer demand for healthier, minimally processed foods without synthetic additives has led to a search for natural preservation alternatives, such as antimicrobial peptides from yeasts. Pathogenic microorganisms such as Escherichia coli, Listeria monocytogenes, and Salmonella spp. represent major contamination risks to food products. To address these risks, various bioactive compounds with antimicrobial properties, including antimicrobial peptides, bacteriocins, and mycocins, have been identified as potential natural preservatives. This study aimed to assess the antimicrobial efficacy of peptide fractions from Saccharomyces cerevisiae, Wickerhamomyces anomalus, and Tetrapisispora phaffii against E. coli (ATCC 25922), L. monocytogenes (ISA 4008), and Salmonella spp. (ISA 4008). MIC tests revealed varying levels of activity, with peptides from S. cerevisiae demonstrating the strongest effect, achieving an MIC of 250 µg/mL against all three pathogens. Peptides from W. anomalus showed MICs of 2300 µg/mL for both E. coli and L. monocytogenes, and 1150 µg/mL against Salmonella spp. In contrast, peptides from T. phaffii required 1250 µg/mL to inhibit E. coli and Salmonella spp., and 2500 µg/mL to inhibit L. monocytogenes, indicating differing levels of antimicrobial efficacy. Additionally, a challenge test was conducted to determine the shelf life of the juice. The objective was to assess the antimicrobial efficacy of yeast-derived peptides for preserving watermelon juice at 25 °C. The results showed that S. cerevisiae peptides achieved a >5-log CFU/mL reduction in E. coli within seven days, reducing the pathogen to undetectable levels and suggesting irreversible cellular damage. Peptides from W. anomalus also exhibited antimicrobial effects, though less pronounced, with a reduction of 1.1 log CFU/mL. These findings suggest that yeast-derived peptides, particularly from S. cerevisiae, could serve as effective natural biopreservatives to enhance the microbial safety of fruit juices.

2.2. Essential Oils for the Inhibition of Listeria monocytogenes in Food Applications

Ana Olívia Serra Jorge ¹, M. Carpena ², S. Seyyedi-Mansour ², P. Barciela ², A. Perez-Vazquez ², Rafael Nogueira-Marques ³, M. Beatriz P. P. Oliveira ¹, M.A. Prieto ²

¹ 

REQUIMTE/LAQV, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, R. Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal

² 

Universidade de Vigo, Nutrition and Bromatology Group, Department of Analytical Chemistry and Food Science, Instituto de Agroecoloxía e Alimentación (IAA)—CITEXVI, 36310 Vigo, Spain

³ 

REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida 431, 4249-015 Porto, Portugal

Listeria monocytogenes remains a critical foodborne pathogen responsible for listeriosis, a severe illness with high mortality rates, particularly among immunocompromised individuals, pregnant women, and the elderly. The persistence of this pathogen in food production environments presents a serious challenge to public health and food safety. While traditional chemical preservatives have been widely used to control microbial contamination, growing concerns regarding their potential health risks and environmental impact have intensified the search for natural alternatives.

Among these alternatives, essential oils (EOs) have emerged as promising antimicrobial agents due to their potent bioactive properties. Derived from aromatic plants such as Zingiber officinale (ginger), Origanum vulgare (oregano), and Thymus vulgaris (thyme), EOs contain key antimicrobial compounds—thymol, carvacrol, and eugenol—that exert bactericidal effects by disrupting bacterial cell membranes and interfering with essential metabolic functions. This mode of action has been extensively studied, with numerous reports confirming their inhibitory effects against L. monocytogenes in diverse food matrices, including dairy products, meat, and fresh produce.

Despite their potential, challenges remain regarding the stability, volatility, and sensory impact of EOs in food applications. Recent advances in encapsulation technologies, such as nanoemulsions and liposomal delivery systems, have been explored to enhance their controlled release and improve efficacy in food systems. This systematic review consolidates current research on the antimicrobial properties of EOs, their mechanisms of action, and their practical applications in food preservation. Additionally, it outlines technological innovations that aim to optimize EO-based preservation strategies, emphasizing the need for further studies to ensure their commercial viability and regulatory compliance.

2.3. Evaluation of Lactose-Sulfite-Phenol Red-Rifampicin (LSPR) Agar for the Co-Enumeration of Salmonella enterica and Shiga-toxigenic Escherichia coli in Sausage Batter

Boluwatife Esther Ajayi, Thomas Matthew Taylor

• Animal Science, Texas A & M University, College Station, USA

Accurate enumeration of Salmonella enterica and Shiga-toxigenic Escherichia coli (STEC) is critical for validating pathogen control in dry fermented sausage production. Lactose-Sulfite-Phenol Red-Rifampicin (LSPR) agar is a selective medium designed to recover and enumerate these pathogens under environmental stress. S. enterica produces black colonies on LSPR, while STEC produces cream-colored colonies, enabling visual differentiation. This study evaluates LSPR’s performance for recovering S. enterica and STEC after lactic acid (pH 3.0) and salt (35.9 g NaCl/100 mL) stress and its suitability for co-enumeration in sausage batter.

Cocktails of seven rifampicin-resistant serotypes of S. enterica and STEC were stressed for 0, 30, 60, and 120 s, then plated on BHI, LSPR, and modified LSPR (MLSPR). Initial bacterial loads averaged 7.5 log CFU/mL. After 120 s of acid stress, STEC declined by 4.2 log CFU/mL, while S. enterica declined by 3.1 log CFU/mL (p 0.05). Under salt stress, reductions were 2.8 and 2.1 log CFU/mL, respectively (p > 0.05). Two-way ANOVA revealed that exposure time significantly affected bacterial survival (p = 0.0027), with no significant differences in recovery between media types (p = 0.0776). MLSPR did not enhance recovery over LSPR (p = 0.238).

The interaction between media and exposure time was not statistically significant for either acid (p = 0.2380) or salt stress (p = 0.9031), indicating consistent bacterial reductions across media.

The findings indicate that media type had no statistically significant effect on bacterial counts, as LSPR, BHI, and MLSPR supported similar levels of pathogen recovery showing the suitability of the media for co-enumeration of Salmonella and STEC in sausage batter. However, exposure time to stress solution showed a significant impact on bacterial survival, with longer exposure times resulting in greater reductions in STEC and S. enterica counts.

2.4. Microbiological Assessment and Acceptability of Minced Meat from Hypermarkets: Pathogenic and Spoilage Microorganisms

Joana Paiva ^1,2,3, Cristina Saraiva ^2,3,4

¹ 

University of Trás-os-Montes e Alto Douro

² 

Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), 5001-801 Vila Real, Portugal

³ 

Centro de Ciência Animal e Veterinária (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), 5001-801 Vila Real, Portugal

⁴ 

Veterinary and Animal Research Centre, Associate Laboratory for Animal and Veterinary Science (AL4AnimalS), University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal

Minced meat contains spoilage microorganisms, reducing shelf-life and causing economic and environmental impacts. It can also harbor pathogens that pose risks to public health, leading to foodborne illnesses and product recalls. The aim of this study was to evaluate the deteriorative and pathogenic microbiota of minced meat obtained in hypermarkets. Thirty samples of minced meat from bovine (n = 15), swine (n = 6), and poultry (n = 9), produced from hypermarkets in Vila Real, were analyzed for the presence of three important foodborne pathogenic bacteria, i.e., L. monocytogenes, S. aureus, and E. coli. In parallel, total aerobic microorganisms at 30 °C (mesophiles), Enterobacteriaceae, Lactic Acid Bacteria (LAB), and Pseudomonas spp., were also enumerated as hygiene/safety indicator organisms. Microbial concentrations (log10 cfu/g) of 6.01 ± 0.19 (total mesophiles), 3.79 ± 1.27 (Enterobacteriaceae), 4.14 ± 0.96 (LAB), 1.35 ± 1.31 (Pseudomonas spp.), 0.07 ± 0.29 (L. monocytogenes); 0.21 ± 0.66 (S. aureus), and 0.52 ± 0.81 (E. coli) were found. Significant differences in microbial counts were found only for total mesophiles and E. coli, with poultry showing higher levels than beef and pork (p 0.05). Although poultry meat had the highest microbial counts overall, which is associated with its lower acceptability, followed by beef and pork minced meat; this difference was not statistically significant (p > 0.05). Enterobacteriaceae was the microorganism with the lowest acceptability, contributing to a reduction in the general product acceptability (83.3%) compared to the higher rates for other microorganisms. These findings emphasize the role of minced meat in the transmission of pathogenic and deteriorative microorganisms, highlighting the importance of proper handling and thorough cooking to prevent foodborne diseases.

2.5. Natural Extracts as Antimicrobial Agents: A Comparative Study of Castanea sativa, Hibiscus sabdariffa L., and Punica granatum L.

Joana C.L Martins ^1,2,3,4,5, Juliana Garcia ^1,2,5, Rafaela Guimarães ^1,5, Maria José Alves ^1,5,6,7, Maria José Saavedra ^2,3,4

¹ 

AquaValor—Centro de Valorização e Transferência de Tecnologia da Água, Portugal

² 

CITAB—Centre for the Research and Technology of Agro-Environment and Biological Sciences and Institute for Innovation, Capacity Building and Sustainability of Agri-Food Production (Inov4Agro), University of Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal

³ 

CECAV—Veterinary and Animal Research Centre and Associate Laboratory for Animal and Veterinary Science (AL4AnimalS), University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal

⁴ 

AB2Unit—Antimicrobials, Biocides & Biofilms Unit and Veterinary Sciences Department University of Trás-os-Montes and Alto Douro (UTAD), 5001-801 Vila Real, Portugal

⁵ 

LiveWell—Research Centre for Active Living & Wellbeing, Instituto Politécnico de Bragança, 5300-253 Bragança, Portugal

⁶ 

CIMO—Centro de Investigação de Montanha, Instituto Politécnico de Bragança, Bragança, Portugal

⁷ 

Laboratório Associado para a Sustentabilidade e Tecnologia em Regiões de Montanha (SusTEC), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal

Food safety and food security cannot be considered without considering the One Health concept (1). This approach underscores the interdependence of health challenges compromising people, animals, plants, and the environment in which they coexist. Antimicrobial resistance is one of these threats, and the food chain is considered an important reservoir of antibiotic resistance genes (2). The food industry produces wastes and by-products from a variety of sources which have the potential to mitigate environmental impacts and be effective against pathogenic foodborne bacteria (3,4). In addition to by-products, natural sources have demonstrated significant antimicrobial and antioxidant properties, emphasizing the potential of natural extracts derived from the food chain to enhance food safety (5). This study aimed to assess the antimicrobial activity of natural products from northern Portugal, including Hibiscus sabdariffa L., Punica granatum L., and Castanea sativa hedgehog, to understand their potential as bioactive compounds. To prepare the crude extract, we employed a solid–liquid extraction process followed by freeze-drying to obtain the dried extracts. Initially, a screening of the extracts was conducted using the MIC method to evaluate their antimicrobial activity against key pathogens, including Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and more. Subsequently, the best-performing extract was analyzed for its physicochemical properties, including antioxidant capacity and phenolic compound content. Hibiscus sabdariffa L. exhibited MIC values of 7.81 mg/mL against both Gram-positive and Gram-negative bacteria, demonstrating bactericidal activity. Punica granatum L. showed MIC values ranging from 1.95 mg/mL to 3.91 mg/mL, while the hedgehog extract of Castanea sativa, displayed the most effective activity, with MIC values between 0.250 mg/mL and 1.00 mg/mL. Castanea sativa hedgehog extract was further analyzed for its antioxidant activity and phenolic compound contents. Overall, this study highlights the promising role of these natural extracts as antimicrobial agents against pathogenic bacteria and the necessity for further investigations.

2.6. Strategies for the Decontamination of Acanthamoeba spp. and Naegleria fowleri in Different Water Systems in Bombali and Tonkolili Districts (Sierra Leone): Lessons from the UK Recovery Handbook for Biological Incidents

Antonio Peña-Fernández ^1,2, María de los Ángeles Peña ³, Raoul Emeric Guetiya Wadoum ⁴, Umar Anjum ²

¹ 

Department of Surgery, Medical and Social Sciences, Faculty of Medicine and Health Sciences, University of Alcalá, Ctra. Madrid-Barcelona, Km. 33.600, 28871 Alcalá de Henares, Madrid, Spain

² 

Leicester School of Allied Health Sciences, De Montfort University, Leicester, LE1 9BH, UK

³ 

Departamento de Ciencias Biomédicas, Universidad de Alcalá, Crta. Madrid-Barcelona Km, 33.6, 28871 Alcalá de Henares, Madrid, Spain

⁴ 

Department of Public Health, Microbiology and immunology, Ernest Bai Koroma University of Science and Technology, Makeni, Sierra Leone

The US Environmental Protection Agency’s Contaminant Candidate List includes Acanthamoeba spp. and Naegleria fowleri due to their ability to withstand harsh environmental conditions and their function as reservoirs for various important human pathogens. These human-pathogenic free-living amoebas (FLAs) pose significant challenges for their elimination from water systems because of their resistance to commonly used disinfectants, including those employed in treating drinking water. Our research group has previously isolated and detected cysts/amoebas of Acanthamoeba spp. in water from various open water systems across Bombali and Tonkolili Districts in Sierra Leone (SL), emphasizing a potential public health risk to the Sierra Leonean population. We have used the emerging guidelines to decontaminate different environments, including open and drinking water systems, available in the UK Recovery Handbook for Biological Incidents (UKRHBI) to identify applicable recovery options (ROs) to mitigate the presence of FLAs in water systems in SL. The UKRHBI provides customized ROs for decontaminating/managing environments affected by any biological hazard, which are appropriately selected according to the physiological characteristics of the biological hazard (in this case, FLAs) and the characteristics of the site, following an accessible methodology. The following ROs might be applicable to the characteristics of the water systems and areas monitored: restricting water use, encouraging bottled water for drinking, removing contamination sources, modifying water treatment systems (e.g., integrating ozone-based treatments), and sludge treatment (use of ultrasonic disintegration treatment and topsoil stabilization treatment). Emerging technologies, such as photolysis of sodium chloride and sodium hypochlorite, have demonstrated efficacy in inactivating Acanthamoeba castellanii and have the potential to be adapted to target other chlorine-resistant pathogens. The adoption of these interventions by the relevant authorities in SL could lead to the effective protection of vulnerable populations, including children, the elderly, immunocompromised individuals, and Ebola survivors.

3. Session: Antimicrobial Agents and Resistance

3.1. Vertical Transmission of Microbiota and Metabolites: Impact of Maternal Obesity on Neonates

Aparna Krishnakumar ¹, Juan Manuel Vélez Ixta ¹, Carmen Josefina Juárez Castelán ¹, Tizziani Benítez Guerrero ¹, Alberto Piña Escobedo ¹, Enrique Rico Arzate ², Jose Javier Castro Arellano ², Silvia Romero Maldonado ³, Yair Cruz Narváez ², Maria Luisa Pizano-Zárate ³, Jaime García-Mena ¹

¹ 

Departamento de Genética y Biología Molecular, Av Instituto Politécnico Nacional 2508, San Pedro Zacatenco, Gustavo A. Madero, 07360 Ciudad de México, CDMX, Mexico

² 

ESIQIE-IPN, Av Instituto Politécnico Nacional s/n, Lindavista, Gustavo A. Madero, 07700 Ciudad de México, CDMX, Mexico

³ 

Instituto Nacional de Perinatología, C. Montes Urales 800, Lomas—Virreyes, Lomas de Chapultepec IV Secc, Miguel Hidalgo, 11000 Ciudad de México, CDMX, Mexico

Introduction: This study investigates the effects of maternal obesity on microbiota composition and metabolite profiles in neonates and breast milk. Maternal obesity can alter the bacterial landscape and bioactive compounds in breast milk, increasing the risk of obesity in infants. The microbiota and metabolites in breast milk and neonatal fecal samples from obese and normal-weight Mexican mothers were compared. Methodology: Fecal and milk samples were collected from binomials. DNA extraction was performed, followed by the PCR amplification of the V3-16S ribosomal RNA gene for next-generation sequencing (NGS) analysis. Taxonomic microbiota profiling was conducted, and metabolic pathway analysis was carried out using PICRUST to identify pathways associated with different bacterial taxa. Metabolites were extracted and analyzed via FT-ICR mass spectrometry. Comprehensive bioinformatics analyses were conducted to assign taxonomy and assess bacterial diversity. Results: The findings suggest that the vertical transmission of bacteria and metabolites from mother to infant exists. Patescibacteria and Firmicutes were identified as dominant phyla in human milk. Fecal samples from neonates born to obese mothers exhibited a lower abundance of Bifidobacterium and Lactobacillus than those born to normal-weight mothers. In addition, fecal samples from neonates and milk from obese mothers were characterized by an increased abundance of Lactobacillus and Staphylococcus. Conclusions: This study highlights breastfeeding’s crucial role in transferring maternal microbiota and metabolites to infants, showing strong microbial and metabolic connections between breast milk and meconium. Maternal BMI does not affect overall bacterial composition, but distinct metabolites linked to maternal obesity were found, suggesting potential neonatal health impacts. The research suggests a pathway for intergenerational obesity risk transmission via breastfeeding, emphasizing the need for further investigation into the long-term health effects on infants of obese mothers. This work was financed by CONACyT 163235 INFR-2011-01 and CONACyT FORDECYT-PRONACES/6669/2020_Programa Presupuestario F003-Ciencia de frontera 2019.

3.2. Staphylococcus aureus Biofilm Gene Analysis: Risks for Patients with Chronic Lymphocytic Leukemia (CLL)

Maciej Grzegorczyk ¹, Agnieszka Grzegorczyk ²

¹ 

Students Scientific Association “mikroGRAM” at Department of Pharmaceutical Microbiology, Medical University of Lublin, 20-093 Lublin, Poland

² 

Department of Pharmaceutical Microbiology, Medical University of Lublin, 20-093 Lublin, Poland

Biofilms have a complex multicellular structure composed of bacteria and extracellular polymeric substances, which collectively display adhesive properties and allow bacterial cells to stick to each other and to given surfaces. Biofilms may be formed on the abiotic surfaces of medical instruments such as catheters or implants, as well as on biotic surfaces, e.g., living tissues. Biofilm structures are formed in order to increase bacteria’s chances of survival in unfavorable environmental conditions, prevent immune cells from eliminating bacterial cells, and to provide antibiotic resistance. Staphylococcus aureus is one of the most common sources of upper respiratory tract infections and is a procaryote with the ability to form biofilm structures. Biofilm is considered to be, among others, a virulence factor that contributes to difficulties and therapeutic failures in treating antibiotic-resistant staphylococcal infections. This can pose a huge risk for individuals with an impaired function of the immune system, such as patients with CLL.

A total of 51 S. aureus strains were isolated from 34 patients with CLL treated in the Saint Jan of Dukla Oncology Centre of the Lublin Region (Poland). DNA from bacteria was extracted using the spin-column method and was later used to perform PCR reactions in order to detect the icaA, icaD, icaB, and icaC genes responsible for the formation of biofilm.

An analysis of the electrophoretic separation of amplification products in the studied S. aureus strains showed the presence of icaA genes in 49 (96.08%) isolates, while icaD, icaB, and icaC genes were detected in 45 (88.24%), 47 (92.16%), and 41 (80.4%) S. aureus isolates, respectively.

Our study found a very significant prevalence of all genes responsible for biofilm formation among isolated S. aureus strains, which, in turn, may contribute to chronic staphylococcal infections, difficulties in antibiotic therapies, and may also delay anticancer treatment, undermining its efficiency and even threatening the lives of patients.

3.3. Effects of Environmental Antimicrobial Residues on the Conjugative Transfer of Antimicrobial Resistance Genes in the Gut Microbiota

Amer Abdelgany ^1,2, Jiewen Guan ¹

¹ 

Canadian Food Inspection Agency, Ottawa, Canada

² 

Biology Department, Carleton University, Ottawa, Canada

Antimicrobial resistance (AMR) poses a significant public health challenge, as AMR bacteria and their genes persist in environments such as farms and water systems. AMR bacteria can infect humans through contaminated food, water, or direct contact. We hypothesize that both intermittent exposure to clinical antimicrobials and environmental residues might exacerbate this colonization, leading to gut dysbiosis and facilitating the spread of resistance genes. This could enhance the survival and persistence of AMR bacteria and promote AMR gene transfer among bacterial communities in the gut.

To investigate this, we utilized a mouse model to examine the conjugative transfer of a mobile AMR plasmid from a Salmonella Heidelberg donor to Enterobacteriaceae in the gut under treatments of intermittent clinical doses of ampicillin and sub-clinical levels of tetracycline in drinking water. Fecal samples were collected from mice at regular intervals to isolate AMR bacteria on selective agar media. DNA was extracted from these samples for future 16S rRNA sequencing to analyze microbial community shifts and qPCR targeting ARGs to examine plasmid transfer dynamics.

Preliminary results show that the donor Salmonella Heidelberg persisted for four months in all treatment groups except the control, which had no antimicrobial exposure. The donor reached a high density immediately after each ampicillin treatment and gradually became undetectable. The AMR plasmid was transferred to commensal E. coli and to an introduced recipient Salmonella Heidelberg. While detailed sequencing and bioinformatics analyses are pending, initial findings highlight the significant impacts of antimicrobial exposure on the persistence of AMR bacteria and the transfer of AMR plasmids within the gut microbiota.

3.4. From Surface to Infection: Biofilm Formation of Coagulase Negative Staphylococci

Catarina Ribeiro Freitas ^1,2, Vanessa Silva ^1,2,3,4, Eliana Costa ⁵, Ana Fontes ⁵, Ana Bento-Pinto ⁵, Gilberto Igrejas ^2,3,4, Patrícia Poeta ^1,6,7

¹ 

Microbiology and Antibiotic Resistance Team (MicroART), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

² 

Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal

³ 

Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

⁴ 

LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, University NOVA of Lisbon, Caparica, Portugal

⁵ 

Hospital Centre of Trás-os-Montes and Alto Douro, Clinical Pathology Department, Vila Real, Portugal

⁶ 

CECAV—Veterinary and Animal Research Centre, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal

⁷ 

Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

Biofilm formation by Coagulase Negative Staphylococci (CoNS) represents a critical challenge in device-associated infections, often leading to treatment failures. While Staphylococcus epidermidis has been extensively studied, the biofilm-forming potential of other CoNS species remains underexplored. This study analyzed 152 isolates belonging to 11 CoNS species obtained from individuals with infections, with the aim of assessing interspecies variability in biofilm formation and its potential clinical implications.

The 152 isolates were evaluated for their capacity to form biofilms using the microtiter biofilm assay. Statistical analyses were conducted to ascertain associations between biofilm formation in the various CoNS species.

The majority of CoNS isolates (64,47%) demonstrated a high capacity for biofilm production, while only 7.24% were classified as non-producers. Despite the variability observed between the isolates, no statistically significant differences were identified in the ability to form biofilms between the different species.

The results obtained in this study offer significant insights into the biofilm forming capacity of CoNS, which may have substantial ramifications for the formulation of novel therapeutic strategies aimed at mitigating biofilm formation and, by extension, the reduction in the prevalence of infections within hospital environments.

This work was supported by project UIDB/00772/2020 funded by the Portuguese Foundation for Science and Technology (FCT). This work was also supported by LAQV-REQUIMTE, which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020).

3.5. Isolation of Fungal Endophytes from Monsonia angustifolia and Screening for Their Antimicrobial and Extracellular Enzymatic Activities

Maruping Hoffman Frost, Khumiso Dithebe, Elbert Jansen Van Rensburg, Raymond Makola

• Department of Biochemistry Microbiology and Biotechnology, University of Limpopo, Private Bag X1106, Sovenga 0727, South Africa

Endophytes reside in healthy plant tissue without causing visible harm. Fungal en-dophytes produce structurally and chemically diverse secondary metabolites, including those similar to the host plant, with antimicrobial and anti-inflammatory activities. Fungal endophytes also produce various extracellular enzymes with applications in various in-dustries including the pharmaceutical industry. Despite this knowledge, the diversity and bioactivities of fungal endophytes of South African medicinal plants are relatively under-explored. Thus, the aim of this study was to isolate fungal endophytes from the medicinal plant Monsonia angustifolia and evaluate their antimicrobial and extracellular enzymatic activities. A total of 29 morphologically distinct fungal endophytes with 21 isolates related to filamentous fungi and 8 to yeasts were isolated from the surface-sterilized leaves, stems and spikes. The majority of the isolates (59%) were obtained from the stems. The sequenc-ing of the internal transcribed spacer region revealed that all the filamentous fungi and four yeast isolates belonged to the Phylum Ascomycota, while the other four yeast isolates reside with Basidiomycota. The broth micro-dilution assay revealed that the ethyl acetate crude extracts of the endophytes possessed varying inhibitory activity, with minimum in-hibitory concentrations ranging from 0.02 to 2.50 mg/mL against six clinically significant pathogens. Agar-plate screening for amylase, cellulase, laccase, lipase, pectinase, protease and xylanase enzymes revealed that the isolates had the ability to produce at least one of the tested enzymes, with four filamentous fungi exhibiting the ability to produce all the screened enzymes. This study highlights the significance of fungal endophytes as sources of antimicrobial secondary metabolites and industrially significant enzymes.

3.6. Achieving Natural Control of the Phytopathogenic fungus Cylindrocladium sp. Using Two Different Thyme Essential Oils Applied Under In Vitro Conditions

María Cecilia Prieto ¹, Nicolas Ortiz ², María José Duarte ², Pedro Sansberro ², Ernestina Galdeano ²

¹ 

Departamento de Agroalimentos, Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Córdoba, Argentina

² 

Instituto de Botánica del Nordeste, Consejo Nacional de Investigaciones Científicas y Técnicas, Corrientes 3400, Argentina

Ilex paraguariensis (Ip) is a crop with economic and cultural importance in South America, where its leaves are used to prepare an infusion called “mate”. This crop is affected by different phytopathogenic microorganisms. Cylindrocladium (Cy) are the causal agent of the black spot of Ip. The objective of this work was to evaluate the use of two thyme (Thymus vulgaris) essential oils (EOs) as control agents of Cy. The thyme EOs (T1 and T2) were characterized by means of GC-MS. Cy was isolated from infected leaves of Ip. Cy, and a 14-day culture was used to obtain the work solution (100,000 spores/mL). The EOs were diluted in dimethyl sulfóxide at a rate of 1:4, and nine two-fold serial dilutions were performed. Treatments consisted of the addition of 15 µL of work solution to 165 µL broth with Ip and 20 µL of each EO dilution, which were then incubated for 72 h. Four rep-lications were carried out. Negative (Ip broth) and positive controls (without EOs) were performed. The minimum inhibitory concentration (MIC) was determined as the minimal concentration in which no micelial growth was observed. Tubes without micelial growth were sowed in Ip medium and incubated for 48 h. The minimum fungicide concentration (MFC) was defined as the minimal EO concentration at which no Cy colonies were grown. T1 was mainly composed of thymol (40.32%) and p-cymene (36.43%), while T2 comprised p-cymene (59.65%), thymol (11.78%), and eucalyptol (5.35%). Both EOs exhibited fungi-static and fungicidal activity, with MIC values of 2.35 µL/mL for T1 and 31.25 µL/mL for T2, and MFCs of 2.93 µL/mL and 50 µL/mL, respectively. Despite both EOs showing good antifungic activity, T1 showed a greater bioactivity, which was correlated with the higher relative amount of thymol compared with T2. These results demonstrate the effectiveness of thyme EOs as natural control agents against Cy.

3.7. Antibacterial and Antifungal Properties of Aqueous and Alcoholic Extracts of Arnica montana L.

Sylwia Andrzejczuk ¹, Magdalena Sozoniuk ², Martyna Kasela ¹

¹ 

Department of Pharmaceutical Microbiology, Medical University of Lublin, Poland

² 

Institute of Plant Genetics, Breeding and Biotechnology, University of Life Sciences in Lublin, Poland

Arnica montana L. is renowned for its biological properties, including its antimicrobial effects, which are attributed to the presence of sesquiterpene lactones or flavonoids. The aim of this study was to assess the antimicrobial activity of ethanolic (EtE) and aqueous (AqE) extracts of Arnica montana L. against a reference strains of microbes, employing a double broth dilution method (concentration of 7.81–30000 µg/mL); identifiyng MICs and MBC/MFCs. Amoxicillin, nystatin, and 70% ethanol were used as positive controls. The MBC(MFC)/MIC ratios were used to assess the activity of EtE/AqE (4 bactericid-al/fungicidal; ≥4—static). As a result, a differential antimicrobial activity was demon-strated in MIC = 1000–2000 µg/mL and MIC = 8000–16000 µg/mL for Gram(+) bacteria for EtE and AqE, respectively. EtE was more effective against Salmonella Typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus mirabilis (MIC = 1000–2000 µg/mL), compared to AqE (MIC > 16000 µg/mL). EtE was bactericidal against staphylococ-ci and Enterobacteriaceae rods, while AqE was bactericidal against Bacillus cereus. Unlike EtE, which was effective to most Candida spp. (MIC = 500–800 µg/mL), AqE had no anti-fungal properties.

This study suggests that the extracts and essences of A. montana have several benefi-cial properties such as antibacterial and antifungal effects. It highlights that essence ex-tracts (EtEs) are more potent than aqueous extracts (AqEs) and can serve as natural anti-microbial agents. The raw material is mainly used in topical preparations due to its high toxicity, but can also be taken orally in low concentrations as a homeopathic remedy. The results of this study may pave the way for further research into activity against other mi-crobial strains, and the development of new formulations for different applications. How-ever, the challenge is to optimize solvent concentrations that are most suitable for biologi-cal experiments, particularly for alcohol extracts, which are good for solubilisation but can be cytotoxic if not carefully controlled.

3.8. Antibiofilm Activity of Rosaceae Honeys Against Acinetobacter baumannii and Their Prebiotic Effect on Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus

Francesca Coppola ^1,2, Manar Abdalrazeq ³, Florinda Fratianni ¹, Maria Neve Ombra ¹, Gokhan Zengin ⁴, Filomena Nazzaro ¹

¹ 

Institute of Food Science, CNR, Avellino, Italy

² 

Department of Agricultural Sciences, University of Naples “Federico II”, 80055 Portici, Italy

³ 

An-Najah National University, Nablus, Palestine

⁴ 

Department of Biology, Faculty of Science, Selcuk University, Konya, Turkey

Introduction

Natural products are an important source of therapeutic agents for their antimicrobial and prebiotic properties in an era of increasing antibiotic resistance. The antimicrobial and prebiotic properties of honeys from hawthorn, cherries, raspberries, almonds and ap-ples were investigated.

Methods

The antimicrobial activity of the honey, diluted 1:1 w: vol with deionized water and filtered, was assessed (20 mg/mL) by evaluating, through the crystal violet test, its ability to inhibit the biofilm of the high-risk pathogen Acinetobacter baumannii, and, through the MTT test, its capacity to affect the metabolism of the A. baumannii sessile cells. The prebiotic po-tentiality of the honey was assessed on Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus, to evaluate the capacity to affect their growth and to inhibit the biofilm of that pathogen.

Results

The honeys inhibited the biofilm of A. baumannii with percentages ranging from 43.49% (cherry) to 59.43% (apple). The honeys did not affect the metabolism of the sessile cells of the pathogen (except cherry honey, 27.16%), so other inhibition mechanisms should be investigated. Rosaceae honey positively affected the growth of the two lactobacil-li, with an increase ranging from 8-fold to even 13-fold (when L. rhamnosus grew in the presence of cherry honey), compared to the control, grown in MRS medium. The biofilm inhibitory activity of the supernatants of lactobacilli showed that all types of honey were more effective than the control, with inhibition percentages of up to 34.24% (supernatant of L. plantarum grown with apple honey), and was especially evident on the metabolism of sessile cells, as demonstrated by the high inhibition percentages observed (up to 61.31%), higher than the supernatant of the two strains conventionally grown.

Conclusions

Future work will focus on the effect of Rosaceae honey on other pathogenic bacteria and probiotics, with the aim of expanding knowledge of its biological properties and assessing the consequent possibilities of its use.

3.9. Bark Extract of Melia azedarach Inhibits Motility and Viability of Ralstonia solanacearum Growing in Humid Substrate

María Cecilia Prieto ¹, Silvia Schaller ², Verónica Obregón ³, Nelson Rubén Grosso ⁴, Ernestina Galdeano ²

¹ 

Departamento de Agroalimentos, Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Córdoba, Argentina.

² 

Instituto de Botánica del Nordeste; Consejo Nacional de Investigaciones Científicas y Técnicas, Corrientes 3400, Argentina

³ 

EEA Bella Vista; Instituto Nacional de Tecnología Agropecuaria, Bella Vista 3432, Argentina

⁴ 

Departamento de Fundamentación Biológica, Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Córdoba 5000, Argentina

Bacterial wilt of tomato is caused by the soilborn bacterium Ralstonia solanacearum (Rs). The disease is difficult to manage and produces serious yield losses. Natural products, such as plant extracts, represent new environmentally sustainable control strategies. The aim of this study was to evaluate the antibacterial efficiency of Melia azedarach bark extracts against the phytopathogenic bacteria Rs. Three bark extracts were performed using water and ethanol as solvents. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of bark extract against Rs were defined using the broth microdillution method. Motility inhibition and changes in colony morphology were assessed, and bacterial survival was evaluated on humid substrate supplemented with different bark extract amounts. Bark extract affected the viability, colony morphology (where changes in the shape, cell density, and edge morphology of the colony were observed), and motility (with swarming motility reduced by 47.61% with ½ bark extract MIC, and 66.67% with bark extract MIC) on Rs. The application of the extract on the humid substrate controlled Rs in fewer amounts than MIC (0.176 g/l). Bacterial motility has been described as a key feature for pathogenicity in Rs. In this context, the inhibitory effect of M. azedarach bark extract on bacterial motility, and its bactericidal activity when applied on the substrate (where Rs survives between growing seasons), showed that this natural product is a promising tool to be used as an efficient sustainable strategy for the control of bacterial wilt of tomato.

3.10. Celery (Apium graveolens) Seed Extracts: A Natural and Effective Strategy Against Helicobacter pylori Infection

Esther Rodríguez González ¹, Mónica Rodríguez García Risco ¹, Jose Manuel Silvan Jimenez ², Adolfo Martinez Rodriguez ²

¹ 

Department of Production and Characterization of Novel Foods. Institute of Food Science Research (CIAL, CSIC-UAM), Cantoblanco Campus, Autonomous University of Madrid, C/Nicolás Cabrera 9, 28049 Madrid, Spain

² 

Microbiology and Food Biocatalysis Group (MICROBIO), Department of Biotechnology and Food Microbiology, Institute of Food Science Research (CIAL, CSIC-UAM), Cantoblanco Campus, Autonomous University of Madrid, C/Nicolás Cabrera 9, 28049 Madrid, Spain

Helicobacter pylori (H. pylori) is a significant human pathogen that infects approximately 50–70% of the global population. It colonizes the gastric epithelium, causing histological alterations that can lead to diseases ranging from chronic gastritis to gastric cancer. Recent research has focused on exploring bioactive compounds from natural sources as potential therapeutic strategies against H. pylori due to their promising biological properties. This study investigated bioactive extracts from celery seeds (Apium graveolens) to determine their potential antibacterial, antioxidant, and anti-inflammatory effects against H. pylori.

The extracts were obtained using various solvents through the solid–liquid extraction technique assisted by ultrasound, and both their chemical composition and biological activities, including antioxidant, anti-inflammatory, and antibacterial effects, against a specific strain of H. pylori, were evaluated.

The results showed that the chemical composition of the extracts varied depending on the solvent polarity employed, which also influenced their biological activity. Ethanolic extracts exhibited the highest phenolic content (42.8 ± 0.9 mg GAE/g extract) and were found to be rich in luteolin and apigenin derivatives, which are known for their potent antioxidant properties. In contrast, hexane extracts contained higher levels of essential oil compounds. Regarding biological activities, the ethanolic extract demonstrated the highest antioxidant activity (IC₅₀ 2 mg/mL), while aqueous and ethanolic extracts showed the strongest anti-inflammatory activity (90% NO production inhibition). Both ethanolic and hexane extracts exhibited the greatest antibacterial activity against H. pylori (MIC 0.005 mg/mL).

Based on these results, the ethanolic extract of celery seeds appears to be the most promising for use against H. pylori due to its chemical composition and potent antioxidant, anti-inflammatory, and antibacterial activities against this pathogen.

3.11. Deciphering Microbiome Dynamics and Antibiotic Resistome Profiles in Anaerobic Bioreactors Treating Swine Wastewater Containing Different Sulfonamides

Qinmao Zhou ¹, Hebin Liang ², Jin Huang ³, Uli Klümper ⁴, Peiju Fang ⁵, Zehui Yu ⁵, Bing Li ¹

¹ 

Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, China

² 

College of Resources and Environment, Anhui Agricultural University, Hefei, China

³ 

Central South University, Changsha, China

⁴ 

Institute for Hydrobiology, Technische Universität Dresden, Germany

⁵ 

Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, China

Residual antibiotics in swine wastewater (SW) promote antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), threatening the environment and human health. Anaerobic digestion (AD) is a promising green technology for treating SW, but the impact of antibiotics on the microbial community and resistome during AD is unclear.

To address this gap, anaerobic batch experiments treating SW containing different sulfonamides (SMs) were conducted, with samples collected for DNA extraction and analysis of the microbial community and antibiotic resistome. ARG-carrying mobile genetic elements (MGEs) were detected, and ARG hosts were identified using metagenomic binning.

The results showed a significant negative correlation between the Abundance-based Coverage Estimator (ACE) index and the batches, with notable variability in the Shannon index. Enterococcus emerged as the dominant genus under SMs stress. A total of 24 types and 440 subtypes of ARGs were identified, with SMs stress significantly increasing the abundance of sul1, sul2, and sul3. The highest ARG abundance was observed in the sulfamonomethoxine (SMM) group during the initial stage and in the sulfamerazine (SMR) group after long-term anaerobic culture. Plasmid identification revealed that ARGs on chromosomes are much higher than those on plasmids. However, ARGs are more likely to co-occur on plasmids. Hybrid assembly identified two complete integrons and two composite transposons. Binning reconstructed 34 and 33 medium- to high-quality ARGs host genomes from all samples, determining their taxonomic classification and potential pathogenicity.

In conclusion, SMs stress intermittently disrupts the microbial community structure during AD. ARG composition and distribution are influenced by antibiotic pressure and initial ARGs in seed sludge. Prolonged antibiotic exposure increases the risk of ARG mobilization via MGEs. Specific ARB without ARGs but with inherent resistance also require attention. This study offers new insights into the occurrence and impact of antibiotics during the AD treatment of SW.

3.12. Evaluating the Efficacy of Dielectric Barrier Discharge Plasma Against Planktonic and Biofilm Cultures of Staphylococcus aureus

Irena Maliszewska, Anna Zdubek

• Department of Organic and Medicinal Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland

Background. It is well-documented that numerous pathogenic microorganisms present in hospital settings exhibit diminished susceptibility to various biocidal agents, including but not limited to hydrogen peroxide, alcohols, chlorhexidine, iodophors, triclosan, and benzalkonium compounds. Previous reports have also highlighted the cross-resistance between biocide exposure and bacterial resistance to antibiotics. Therefore, it is necessary to search for effective methods of pathogen eradication. One of such effective methods is nonthermal plasma. It seems that due to the complex and diverse mechanisms of pathogen destruction, the development of bacterial resistance/tolerance to this eradication method is unlikely.

The aim of this study was to evaluate the effect of sublethal exposures of S. aureus to nonthermal plasma on some cell surface properties, biofilm formation efficiency, and the possibility of inducing tolerance of pathogenic bacteria to this biocidal technique.

Methods. Plasma inactivation of S. aureus inoculated on the surface of glass plates was performed using a DBD reactor operating at atmospheric pressure with air as working gas. The effects of multiple exposures to sublethal doses of plasma on the ability of cells to auto-aggregate, surface hydrophobicity, the ability to form biofilm on different surfaces, and changes in decimal reduction time (D) were assessed.

Results. Exposure of planktonic bacteria to nonthermal plasma for 2 min and 11 s results in 90% bacterial mortality (D). Destruction of the biofilm formed on the glass surface turned out to be more difficult. It was shown that the auto-aggregation properties and the hydrophobicity of cells significantly decreased after plasma treatments compared to control cells and the degree of these changes increased with the number of exposures to plasma. These changes in the surface structures of bacteria suggested that the ability of these pathogens to form biofilms may be limited. Our results showed that DBD plasma remained an effective biocidal method even after multiple exposures

3.13. Exploring Beer By-Products as Novel Antibacterial Ingredients for Healthcare Products

Carolina Benera ¹, Patrícia Branco ^2,3, Adília Charmier ², Catarina Pinto Reis ^4,5, Elisabete Muchagato Maurício ^2,6,7

¹ 

Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal

² 

BIORG—Bioengineering and Sustainability Research Group, Faculdade de Engenharia, Universidade Lusófona, Av. Campo Grande 376, 1749-024 Lisbon, Portugal

³ 

Linking Landscape, Environment, Agriculture and Food (LEAF), Associated Laboratory TERRA, Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017 Lisboa, Portugal

⁴ 

Instituto de Biofísica e Engenharia Biomédica (IBEB), Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal

⁵ 

Nanomedicine and Biomedical Imaging Group, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal

⁶ 

CBIOS—Research Center for Biosciences & Health Technologies, Universidade Lusófona, Campo Grande 376, 1749-024 Lisbon, Portugal

⁷ 

Elisa Câmara, Lda, Dermocosmética, Centro Empresarial de Talaíde, n°7 e 8, 2785-723 Lisbon, Portugal

Beer is a widely consumed carbonated beverage made from natural ingredients, including malted cereal, hops, yeast and water. It is rich in nutrients and contains carbohydrates, minerals, vitamins, amino acids and polyphenols. The brewing process, however, generates a significant amount of solid waste, including hot trub, a slurry of entrained wort, hop particles and mainly unstable high-molecular-weight colloidal proteins that coagulate during the boiling of the wort. Given the environmental impact of agro-industrial waste, finding sustainable methods to reuse this waste by transforming it into bio-products is crucial.

The aim of this study was to assess the potential of converting these by-products into biologically active extracts suitable for use as functional ingredients in cosmetic and pharmacological formulations. The extracts consisted of one alcoholic and one hydroalcoholic extract prepared by Soxhlet extraction and one hydroalcoholic extract prepared by maceration. The extracts’ antibacterial activity against the Gram-positive bacteria S. aureus, S. aureus (MRSA), S. epidermidis, B. cereus, E. faecalis, S. mitis, S. pyogenes, and S. mutans, as well as against the Gram-negative bacteria E. coli and P. aeruginosa and against the yeast C. albicans, was determined by the agar diffusion method and the minimum inhibitory concentration (MIC) by the microdilution method. The minimum inhibitory concentration and inhibition zones values obtained showed significant inhibitory effects against the Gram-positive bacteria tested, with the Soxhlet extracts presenting the best inhibitory results for both hydroalcoholic and alcoholic extracts. In both methods, the extracts showed the best antibacterial activity against the microorganisms S. epidermidis and B. cereus, with an MIC of 0.63 and 0.31 mg/mL, respectively. The tested extracts seem to be promising, low-cost antibacterial agents that can be incorporated in cosmetic and pharmacological formulations. Additional studies will be conducted to assess their antioxidant activity and safety by using in vitro and in vivo models and investigating the chemical composition of the extracts.

3.14. Extended-Spectrum β-Lactamase-Encoding Genes in the Pig Production Chain in Brazil

Gracielle Rodrigues Pereira ¹, Marcelo Luiz Lima BrandÃo ², Ana Beatriz Portes ^3,4, Bernardete Ferraz Spisso ⁵

¹ 

Oswaldo Cruz Foundation

² 

Instituto de Tecnologia em Imunobiológicos/Fundação Oswaldo Cruz (BioManguinhos/Fiocruz), Rio de Janeiro, Brazil

³ 

Instituto de Microbiologia Paulo de Góes/Universidade Federal do Rio de Janeiro (IMPG/UFRJ), Rio de Janeiro, Brazil

⁴ 

Laboratory of Advanced Analysis in Biochemistry and Molecular Biology (LAABBM), Department of Biochemistry, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil

⁵ 

Instituto Nacional de Controle de Qualidade em Saúde/Fundação Oswaldo Cruz (INCQS/Fiocruz), Rio de Janeiro, Brazil

Animal husbandry is a key promoter of antibiotic resistance (AMR) and contributes to the spread of AMR genes throughout the environment and food supply. The pig industry is one of the leading sectors in animal-based food production worldwide, with Brazil ranking fourth in terms of pork production and export. The emergence of extended-spectrum β-lactamase (ESBL)-producing bacteria in this sector represents a significant public health concern. These enzymes confer resistance to a broad range of β-lactams, threatening the effectiveness of antibiotic therapy for infections in both humans and animals. A systematic review was conducted to address the current knowledge of AMR within the Brazilian pig production chain. The presence of ESBL-encoding genes in bacteria isolated from pig feces, intestine, carcass, and urine, as well as pork-based food, was investigated. TEM-type genes were detected in 11 of the 12 studies, including blaTEM-1A (n = 33 strains) and blaTEM-1B (n = 44 strains). Both genes were found in Salmonella isolated from all sample categories. Additionally, blaTEM-1 was detected in E. coli (n = 4 strains from feces) and Salmonella spp. (n = 2 from carcass; n = 5 from feces). CTX-type genes were detected in Proteus mirabilis (blaCTX-M-65; n = 6 from food) and Salmonella (blaCTX-M-8; n = 1 from feces; n = 1 from pig carcass). blaCMY-2 was detected in Salmonella from feces (n = 1) and carcass (n = 1), whereas blaCMY-M2 was detected in E. coli (n = 5) from pig feces. blaPSE-1 and blaSHV-1 genes were investigated using PCR in E. coli strains isolated from pig feces; however, these were not detected. In conclusion, the pig industry may contribute to the spread of ESBL-encoding genes, posing a public health risk. Data reveal a significant presence of blaTEM genes across various samples, alongside other ESBL-encoding gene types in the Brazilian pig production chain. These findings highlight the need for integrated strategies to control AMR, particularly to reduce risks that may arise from animal -based food production systems.

3.15. From Desert to Lab: Evaluating Camel Urine’s Antibacterial Efficacy Against Carbapenem-Resistant Gram-Negative Bacilli

Amina Ressmi ¹, Habiba Raqraq ¹, Rafiq Aniba ¹, Asmaa Dihmane ¹, Nabila Soraa ², Abouddihaj Barguigua ¹

¹ 

Team of Biotechnology and Sustainable Development of Natural Resources, Biology-Geology Department, Polydisciplinary Faculty, Sultan Moulay Slimane University, Beni Mellal 23023, Morocco

² 

Faculty of Medicine and Pharmacy of Marrakech Cadi Ayyad University, Microbiology Department, Mohamed VI University Hospital Center, Marrakech 40000, Morocco

Antimicrobial resistance currently contributes to over seven million deaths annually worldwide, and that number is projected to rise to ten million by 2050. The increasing prevalence of multidrug-resistant (MDR) bacteria, compounded by the widespread misuse of antibiotics, underscores the urgent need for alternative treatment strategies. Natural products, including camel urine, are emerging as promising candidates to combat this growing public health crisis. By exploring these alternative agents, we may be able to effectively address multidrug resistance and improve global health outcomes.

Using the well diffusion method, sterilized camel urine from male and female camels was tested against eight multidrug-resistant bacterial strains isolated from clinical samples. The strains were first identified through phenotypic characterization on selective media, followed by confirmation using MALDI-TOF mass spectrometry and the BD Phoenix system.

Our findings revealed that female camel urine exhibited a stronger antibacterial effect than male camel urine. Specifically, the largest inhibition zones were observed on Mueller–Hinton agar, with diameters of 32 mm for NDM-1-producing Enterobacter cloacae, 30 mm for NDM-1-producing Klebsiella oxytoca, 29 mm for VIM-producing Acinetobacter baumannii and Citrobacter freundii, 25 mm for OXA-48-producing Pseudomonas aeruginosa and Escherichia coli, and 22 mm for NDM-1-producing Klebsiella pneumoniae. The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were ranging from 0.08 to 12.5 mg/L and from 0.8 to 25 mg/L, respectively.

This investigation demonstrated that sterilized female camel urine exhibits significant in vitro antibacterial activity against various carbapenem-resistant bacteria isolated from clinical samples which may serve as a promising source of new compounds to combat bacterial infections.

3.16. Gut Dysbiosis Promotes Dissemination of Antimicrobial Resistance Genes

Jiewen Guan ¹, Mingsong Kang ¹, Calvin Lau ², Catherine Carrillo ², Edward Topp ³

¹ 

Ottawa Laboratory (Fallowfield), Canadian Food Inspection Agency, Ottawa, ON, Canada

² 

Ottawa Laboratory (Carling), Canadian Food Inspection Agency, Ottawa, ON, Canada

³ 

Agroecology research unit, INRAE, University of Burgundy, Dijon, France

Introduction: The global spread of antimicrobial resistance (AMR) genes (ARGs) poses a major challenge to bacterial infection treatment. Antibiotic use, while targeting pathogens, also disrupts commensal gut bacteria, with effects varying by antibiotic type. The impact of gut dysbiosis on ARG mobility from foodborne bacteria, especially with or without food matrix microbes, remains unclear.

Methods: Using a murine model, we investigated ARG dissemination via mobile AMR plasmids. Mice were pre-treated with streptomycin, ampicillin, or sulfamethazine to induce varying levels of gut dysbiosis. They were then inoculated with beta-lactam-resistant Salmonella Heidelberg (donor) and beta-lactam-susceptible Salmonella Typhimurium (recipient) with or without additional food matrix microbes. Fecal samples were cultured to detect ARG transfer among Salmonella, E. coli, and other gut bacteria, which were confirmed using whole-genome sequencing. Changes in gut microbiota were assessed using 16S rRNA sequencing.

Results: Without background food matrix microbes, streptomycin caused severe gut dysbiosis, enhancing AMR plasmid transfer from S. Heidelberg to S. Typhimurium and E. coli. Ampicillin induced moderate dysbiosis, allowing S. Heidelberg colonization and plasmid transfer to E. coli. Sulfamethazine caused mild dysbiosis, hindering both Salmonella colonization and plasmid transfer. In contrast, food matrix microbes reduced AMR plasmid transfer, except in streptomycin pre-treated mice, where Enterobacteriaceae enrichment enabled AMR plasmid transfer to Escherichia, Enterobacter, Citrobacter, and Proteus.

Conclusions: Pre-existing gut microbiome disturbances from antibiotics significantly affect ARG dissemination. These findings support more judicious antibiotic use to mitigate resistance spread.

3.17. Potential Sustainable Antagonistic Biocontrol Strategy Against Xanthomonas vesicatoria

Belén Álvarez ^1,2, Isabel Salas ¹, Thais Castellón ¹, Elena G. Biosca ¹

¹ 

Departamento de Microbiología y Ecología, Universitat de València, 46100 Valencia, Spain

² 

Área de Investigación Aplicada y Extensión Agraria, IMIDRA, 28805 Madrid, Spain

The use of microorganisms with antagonistic activity against one or more pathogens can be an alternative to chemical control in agroecosystems. In this work, the potential for biocontrol by antagonism of a collection of environmental bacterial isolates against a phytopathogenic bacterium of Solanaceae, Xanthomonas vesicatoria, which causes bacterial spot in tomato and pepper, was evaluated. Control of this disease is usually limited by the lack of resistant hosts, the threat to the environment and the global health risks posed by the continued use of chemical agents.

Bacteria were isolated from different environmental samples from disease-free areas and tested for their in vitro antagonistic capacity against a reference strain of X. vesicatoria. The isolates with the greatest antagonistic activity were selected for initial ex vivo assays to determine this activity in plant material against the reference strain, using leaves from tomato plants of a variety sensitive to bacterial spot. The isolates with the greatest activity were further characterized for their capacity to produce hydrolytic enzymes, such as proteases, lipases, amylases and DNases, and for their nitrogen fixation capacity.

Overall, the results revealed the presence of bacterial isolates able to inhibit the growth of X. vesicatoria in vitro and reduce the severity of bacterial spot symptoms ex vivo, in addition to producing different hydrolases related to biological control and biotechnological use, and fixing molecular nitrogen. The prospects for improving biological control strategies against bacterial spot in tomatoes and peppers caused by X. vesicatoria are promising, thus promoting sustainable agricultural production that favors ecosystem balance.

Funding: This work is part of the R+D+i project PID2021-123600OR-C44, funded by MICIU/AEI/10.13039/501100011033 and by ERDF A way to do Europe, ERDF/EU.

3.18. RecA Inhibitor Mitigates Bacterial Antibiotic Resistance

Jin Ma, Keke Shang, Liwen Xu, Qing-Yu He, Gong Zhang

• Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes and MOE Key Laboratory of Tumor Molecular Biology, Institute of Life and Health Engi-neering, Jinan University, Guangzhou, China

Bacterial antibiotic resistance (AR) has become a critical global health threat. AR is mainly driven by adaptive resistance mutations and the horizontal gene transfer of re-sistance genes, both of which are enhanced by genome recombination. We have previously discovered that genome recombination-mediated tRNA up-regulation is important for AR especially in the early stages. RecA is the most important genome recombination factor. Therefore, RecA inhibitors should be effective in reducing AR. In this study, we found that BRITE338733 (BR), a RecA inhibitor, can prevent ciprofloxacin (CIP) resistance in subcul-turing Escherichia coli strain BW25113 in the early stages (up to the 7th generation). In the presence of BR, the tRNA decreased, so the bacteria cannot evolve resistance via the tRNA up-regulation-mediated AR mechanism. The RecA expression level was also not increased when treated with BR. Transcriptome sequencing revealed that BR causes a down-regulation of ATPase activity and therefore suppresses the energy state of bacteria. Also, the effective concentrations of BR do not harm human cell viability, indicating its clinical safety. These findings demonstrate that BR effectively delays the emergence of spontaneous antibiotic resistance by targeting RecA-mediated pathways. Our findings shed light on a new strategy to counteract the clinical AR: applying BR with the antibiotics together at the beginning.

3.19. Silent Mupirocin Resistance in Methicillin-Resistant Staphylococcus aureus

Martyna Kasela ¹, Weronika Słotwińska ², Sylwia Andrzejczuk ¹, Agnieszka Grzegorczyk ¹

¹ 

Department of Pharmaceutical Microbiology, Medical University of Lublin, 20-093 Lublin, Poland

² 

Student’s Scientific Circle at the Department of Pharmaceutical Microbiology, Medical University of Lublin, 20-093 Lublin, Poland

Staphylococcus aureus, including methicillin-resistant strains (MRSA), is a bacterium responsible for multiple types of human infections. The discrepancy between phenotypic and genotypic resistance is called silencing of antibiotic resistance by mutation (SARM), while the genes are called silent or cryptic. The presence of SARM bacteria carries a risk of implementing an ineffective antimicrobial drug in therapy, as the gene would not be activated until therapy. This study aimed to determine the frequency of SARM and its genetic mechanisms. The virulence and sequence type of the SARM strains were also characterized.

In total, 334 S. aureus strains were investigated for phenotypic resistance to cefoxitin, erythromycin, tetracycline, gentamycin, and mupirocin. PCR was used to screen for the presence of corresponding antibiotic-resistance genes. SARM isolates were further characterized by the detection of genes encoding toxins, adhesins, types of SCCmec cassette, and genotyping with MLST. Silenced genes were sequenced to determine the genetic defect and its consequences for protein translation.

The analysis showed the presence of SARM in 0.6% of S. aureus strains (2/334). In both cases, they were strains harboring the mupA gene (resistance to mupirocin). Sequencing showed the presence of a deletion, resulting in the incorrect translation of the nucleotide into an amino acid sequence, shortening the amino acid chain and inhibiting the synthesis of the protein responsible for mupirocin resistance.

Mupirocin is an antibiotic applied in the treatment of staphylococcal infection of the skin, including the eradication (the removal of a microorganism from the body) of S. aureus from the nasal cavity. Despite the fact that the analysis showed a low share of SARM (1%), there is still a risk of antimicrobial therapy failure and reinfection. Further studies should also focus on determining factors that increase the probability of activation of silent genes responsible for resistance to mupirocin.

3.20. Targeting the Resistant F533L Mutant in PBP3 of Pseudomonas aeruginosa: A Computational Approach Using ZnO and CuO Nanoparticles

Badr-Edine Sadoq ¹, Mohamed Sadoq ², Yassir Boulaamane ¹, Mohammed Reda Britel ¹, Adel Bouajaj ¹, Amal Maurady ^1,3

¹ 

Laboratory of Innovative Technologies, National School of Applied Sciences of Tangier, Abdelmalek Essaadi University, Tetouan, Morocco

² 

Laboratory of Chemistry and Biology Applied to the Environment, URL-CNRST-N◦13, Faculty of Sciences, Moulay Ismail University, 50050 Meknes, Morocco

³ 

Faculty of Sciences and Techniques of Tangier, Abdelmalek Essaadi University, Tetouan, Morocco

Introduction: Pseudomonas aeruginosa (P. aeruginosa) is a multidrug-resistant opportunistic pathogen frequently encountered in clinical settings, posing substantial challenges for effective treatment. Its resistance often arises from mutations in critical drug targets, such as penicillin-binding protein-3 (PBP3), a key β-lactam target essential for bacterial survival. The F533L mutation is linked to resistance against multiple antibiotics, underscoring the need for alternative therapeutic strategies. Methods: This study employed molecular docking to analyze the interactions between zinc oxide (ZnO) and copper oxide (CuO) nanoparticles (NPs) and the mutated PBP3 (F533L) of P. aeruginosa. Additionally, computational approaches were used to assess the impact of the F533L mutation on the structural stability of PBP3. Binding energy and interaction analyses were conducted to evaluate the PBP3-nanoparticle complexes. Results: The F533L mutation was found to alter the stability of PBP3, potentially contributing to resistance mechanisms. Molecular docking revealed that ZnONPs and CuONPs demonstrated binding energies of −2.15 kcal/mol and −1.48 kcal/mol, respectively. Both nanoparticles formed hydrogen bonding interactions with the mutated residue 533L, highlighting their potential to mitigate resistance. Conclusions: The findings suggest that ZnO and CuO nanoparticles could serve as promising therapeutic agents against antibiotic resistance in P. aeruginosa, particularly targeting the F533L mutation in PBP3. Further experimental and in vitro studies are essential to validate their therapeutic efficacy and broader applications.

3.21. The Use of Metal Ions as Potential Inhibitors of Ferrochelatase

Anna Zdubek, Irena Maliszewska

• Department of Organic and Medicinal Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland

Introduction

The need for alternative techniques of microbial control has been determined by the rapid spread of antibiotic-resistant bacteria. One approach that is gaining popularity is antimicrobial photodynamic therapy (aPDT). This method involves the use of a chemical compound (photosensitizer), which is activated by light in the presence of molecular oxygen. The activated photosensitizer induces the production of reactive oxygen species (ROS), which leads to cell destruction. Significant attention has been directed toward the application of 5-aminolevulinic acid (5-ALA) in aPDT. This compound occurs naturally in cells, where it is converted mainly to the intracellular photosensitizer protoporphyrin IX (PpIX). Over time, PpIX is transformed into heme (which does not possess photosensitizing properties) by the ferrochelatase enzyme [1]. An increased concentration of PpIX in cells can be achieved by inhibiting ferrochelatase with divalent metal ions (e.g., Mn²⁺, Zn²⁺) [2].

Methods

The aim of this study is to evaluate the combined effect of 5-ALA (2.5 mM), light, and metal ions on the photosensitization of bacteria. The microorganism tested was antibiotic-resistant Proteus mirabilis (PCM 543). Irradiation was performed with a 404 nm diode laser (the light dose was 23.5 J cm⁻²). For the research, ions of manganese (1 mM), zinc (1 mM), calcium (10 mM), and magnesium (10 mM) were used.

Results

The most effective approach was found to involve the use of 5-ALA with zinc ions, resulting in the destruction of almost 99% of bacterial cells, while the use of 5-ALA alone led to the eradication of approximately 90% of cells after the same exposure time.

Conclusions

The results obtained suggest that metal ions, especially zinc, may compete with iron ions for the ferrochelatase binding site, leading to the inhibition of the enzyme and, at the same time, the accumulation of PpIX in cells.

3.22. Thymoquinone as a Potential Antimicrobial Agent Against Resistant Pseudomonas aeruginosa: A Computational Chemistry Approach

Goran Slivšek ^1,2,3, Sandra Mijač ^3,4, Nives Matković ⁵, Marija Fabijanec ³, Ivan Dolanc ¹, Marin Marinović ², Silvija Petković ², Renato Mautner ², Donatella Verbanac ³, Miran Čoklo ¹

¹ 

Institute for Anthropological Research, Zagreb, Croatia

² 

Faculty of Medicine, University of Rijeka, Rijeka, Croatia

³ 

Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

⁴ 

Children’s Hospital Srebrnjak, Zagreb, Croatia

⁵ 

University Hospital of Split, Split, Croatia

Introduction: Antimicrobial resistance is a significant public health challenge worldwide, especially in Gram-negative bacteria such as Pseudomonas aeruginosa. In recent years, P. aeruginosa has shown increasing multi- and pan-drug resistance, even to colistin, which is considered to be the last line of defense against resistant bacteria. Black cumin or Nigella sativa L., globally used as a spice, contains the bioactive compound thymoquinone, which is promising as a natural antibacterial agent due to its chemical properties. The high cost and complexity of developing new antibiotics is a significant obstacle and often leads to high attrition rates. However, computational chemistry is a promising approach that optimizes the selection of candidates and supports the development of targeted antibacterial agents. This can speed up the discovery process and reduce costs in the ongoing fight against antimicrobial resistance.

Methods: The pharmacokinetic properties of thymoquinone and its pharmacological potential and bioavailability were computationally evaluated using the SwissADME tool. The topologies of the receptor proteins were analyzed using the CASTp web server, and triple molecular docking simulations were performed using AutoDock Vina 1.1.2. Molecular visualization and analysis were performed with PyMol and DS Visualizer

Results: Thymoquinone fulfills all of Lipinski’s rules of five criteria, which indicates favorable pharmacokinetic properties for potential therapeutic use. Molecular docking simulations show a strong binding affinity to the MvfR protein in P. aeruginosa, which is an important regulator of virulence and quorum sensing. Seven (out of nine) models showed consistent interactions with a root mean square deviation (RMSD) of less than 5 Å, often involving the amino acid Ile236.

Conclusions: Computational chemistry can enhance the development of new antibiotics. Thymoquinone shows favorable pharmacokinetics and a strong binding affinity to the MvfR protein in P. aeruginosa. Thymoquinone shows potential as an antimicrobial agent against P. aeruginosa, but further in vitro and in vivo tests, including toxicological studies, are needed to evaluate its cytotoxicity.

3.23. Use of Medicinal Plants for Treatment and Prophylaxis of Canine Parasitic Infections: A Literature Review

Patrícia Santos ¹, Mariana Sousa ^1,2, Agostinho Cruz ²

¹ 

Escola Superior de Saúde, Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal

² 

LAQV|REQUIMTE, Escola Superior de Saúde, Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal

Introduction: Dogs have been perceived as providing companionship to all members of the family, yet they can act as a vector for the transmission of parasitic agents to humans, particularly those who own or care for them. Traditional medicine seem to demonstrate effects in the treatment of diseases in animals; however, it is necessary to prove their effectiveness using more recent scientific evidence.

Objective: The objective is to identify medicinal plants commonly used in the treatment and prophylaxis of parasitic infections in dogs.

Methods: A literature review was conducted by gathering data from the MEDLINE database, using the following search equations: “(“Plants”[Mesh] OR (“Plants/parasitology”[Mesh]) AND (“Phytotherapy/veterinary”[Mesh])”; “Medicinal Plants Treatment for Infested Dogs”; “Parasitic Infections in Dogs Treatment with Plants”. The selection criteria established ensured that only articles published between 2000 and 2023, which referenced the utilization of medicinal plants in the management and prevention of parasitic infections in canines, were included. Furthermore, review articles and articles that referenced pharmacological treatments or treatments for other comorbidities were excluded.

Discussion and Conclusions: A total of 26 articles were selected, resulting in the identification of 112 species from 16 countries that can be used to treat or prevent infections and parasitic infestations. The most commonly used medicinal plants were: Azadirachta indica, Juniperus communis L., Melissa officinalis, Clibadium surinamense L., Olea europaea L., Juglans regia L., Allium sativum L., Coriandrum sativum L., Artemisia cina Berg ex Poljakov, Calendula officinalis L., Mentha piperita L., Cocos nucífera L., Ricinus communis L., Andrographis paniculata (Burm.f.) Wall. ex Nees and Juglans nigra L. The species in question contain a variety of compounds that may confer medicinal properties; however, the anthelmintic and anti-parasitic activity may be attributed to the presence of phenolic compounds, which are known to possess a range of beneficial properties, including anti-parasitic and anthelmintic effects.

4. Session: Emerging Infectious Diseases

4.1. Influence of Non-Treatment of Partners on the Rate of Congenital syphilis: A Comparative Analysis Between Demographic Regions of Brazil (2014–2024)

Maria Fernanda Valentim De Paula, Pedro Henrique Paesi Dutra, Gabriela Gerevini Dal Alba, Camilly Vitória Cansan Loss

• Academic of Medicine, School of Medicine Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre 90050-170, Brazil

Introduction: Congenital syphilis continues to be a serious public health problem in Brazil, with significant impacts on neonatal morbidity and mortality. One of the main factors associated with the high rate of this condition is the lack of adequate treatment for pregnant women’s partners, which facilitates the vertical transmission of the infection.

Methods: Secondary data were collected from the Department of Informatics of the Unified Health System (DATASUS) regarding Congenital syphilis cases in Brazil from 2014 to 2024. The analysis focused on comparing rates of Congenital syphilis across demographic regions, considering the influence of untreated partners on these rates.

Results: Between 2014 and 2024, 250.310 cases of Congenital syphilis were reported in Brazil. Of these, 135.693 cases (54.2%) were from untreated partners, 46.096 (18.4%) were from treated partners, and 68.521 (27.3%) were classified as ignored/blank. The southeast region has the highest rate with 108.874 (43.5%) cases and untreated partners represent 57.762 (53%) cases in this region. This is followed by the northeast region with 72.113 (28.8%) total cases and 39,608 (54.9%) cases of untreated parents. The highest rate of untreated parents occurs in the northern region with 12,599 (58.9%) cases.

Conclusions: The data indicate that Congenital syphilis remains a critical public health issue in Brazil, with a significant proportion of cases linked to untreated partners. The southeast and northeast regions exhibit the highest absolute numbers, while the northern region shows the highest percentage of cases from untreated partners. These findings highlight the urgent need for targeted interventions, including comprehensive prenatal screening and effective partner treatment strategies. Strengthening public health policies and awareness campaigns is essential to reducing vertical transmission rates and mitigating neonatal morbidity and mortality associated with Congenital syphilis.

4.2. Design and Pilot Scale Production of an Alternative Non-Live Attenuated BTV Vaccine in Yeast

Ikram Joubair ¹, Jesus Zueco ², Ismail MOUKADIRI ¹, sergi Maicas ³, Salim Bounou ¹

¹ 

Biomedical & Biotechnology School of Engineering, Euro-Mediterranean University of Fez, Eco-Campus UEMF, Route de Meknès (RN6, Rond-Point Bensouda), 30070 Fez, Morocco

² 

Department of Microbiology and Ecology, Faculty of Pharmacy and Food Science. Universitat de València, Burjassot, Spain

³ 

Department of Microbiology and Ecology, Faculty of Biology, Universitat de València. Burjassot, Spain

Bluetongue (BT) is an infectious disease of domestic and wild ruminants caused by Bluetongue virus (BTV), an arbovirus of the Orbivirus genus within Sedoreoviridae family. It is primarily transmitted by biting midges of the Culicoides genus. BTV poses a significant threat to the livestock industry, particularly sheep. To date, a total of 36 serotypes of BTV have been characterized worldwide, causing periodic outbreaks, occurring most frequently in the Mediterranean basin. The control of BT disease is based on vaccination and several vaccines have been developed. Live attenuated vaccines and inactivated vaccines are widely used to prevent BT. However, despite their demonstrated efficacy, those vaccines have several limitations, including safety concerns and incomplete cross-protection among BTV serotypes. Recombinant subunit BT vaccines based on BTV structural proteins or virus-like particle vaccines (VLPs) may solve some of these limitations. In this study, we aim to design and develop vaccines that constitute an alternative to conventional attenuated vaccines. For this, we intend to express the VP2 and VP5 BTV antigens in Saccharomyces cerevisiae, a GRAS microorganism, by fusing the antigens to the secretion and cell wall retention signals of the Pir4 yeast cell wall protein. The VP2 and VP5 BTV structural proteins will be expressed in S. cerevisiae, targeting them to the yeast cell surface, or to the culture medium. The possibility of obtaining virus-like particles (VLPs), mimicking the virus, will also be studied.

Given that the research is currently underway, cloning, gene confirmation, sequencing and transformation of the constructs into the yeast expression system have been successfully completed. The confirmation of VP2, VP5, VLPs expression and assembly is currently under investigation, using immunofluorescence and Western blot analysis. The assessment of antigenicity and evaluation of the potential immunogenicity will be performed to validate the activity of the vaccine candidate in animals.

4.3. Epidemiological Profile of Gestational syphilis Cases in Brazil Between 2020 and 2023: Insights for Public Health Improvement

Yasmin Marques Loureiro, Maria Fernanda Valentim de Paula, Enrico Lázaro Guidugli, Lucas Conzatti Rodrigues

• Academic of Medicine, School of Medicine, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre 90050-170, Brazil

Introduction: Gestational syphilis remains a significant public health issue due to its severe maternal and neonatal complications. Understanding its epidemiological profile in Brazil is essential for guiding public health interventions and reducing its impact on vulnerable populations. Methods: A cross-sectional, descriptive, retrospective, and quantitative study was conducted on cases of Gestational syphilis across Brazil’s five regions between January 2020 and December 2023. Data were collected through the Department of Informatics of the Brazilian Unified Health System (DATASUS), as well as from the 2022 census conducted by the Brazilian Institute of Geography and Statistics (IBGE). The analysis included variables such as age groups, patient race, sex, and regional distribution. Results: Between 2020 and 2023, 246,265 cases of Gestational syphilis were reported in Brazil. The Northern region of the country presented the highest incidence rate, with approximately 157 cases per 100,000 inhabitants. The most affected age group was women aged 20 to 39 years, accounting for approximately 76% of the cases, followed by adolescents aged 15 to 19 years (21%). Furthermore, brown women (pardas) comprised about 53% of the reported cases, while white women represented 28%. Conclusions: The elevated number of Gestational syphilis cases in Brazil from 2020 to 2023 underscores persistent regional disparities in healthcare access. The disproportionately high rates in the North may be linked to limited healthcare infrastructure and socioeconomic vulnerabilities. These findings highlight the urgent need for targeted public health interventions to address the unique challenges faced by this region.

4.4. Exploring Methodologies from Isolation to Excystation for Giardia lamblia: A Systematic Review

Susie Sequeira ¹, Mariana Sousa ², Agostinho Cruz ²

¹ 

ESS, Polytechnic of Porto, rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal

² 

REQUIMTE/LAQV, ESS, Polytechnic of Porto, rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal

Introduction: Giardia lamblia is a flagellated protozoan and the etiological agent of giardiasis, a leading cause of epidemic and sporadic diarrhea globally. The clinical significance and public health impact of giardiasis underscores the need for robust methodologies to investigate and manage this pathogen. This study systematically reviews the main methodologies described in the literature for studying the life cycle of G. lamblia, focusing on isolation, purification, axenization, excystation, and encystation processes. Methods: A systematic literature review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) statement. The search was performed in the MEDLINE, ScienceDirect, and Web of Science databases using the following search equation: “(“axenization” OR “isolation” OR “excystation” OR “encystation” OR “purification”) AND (“method”) AND (“giardia”)”. Results: A total of 39 studies were included in the review, revealing 56 methods for isolation and purification, 7 methods for excystation, and 3 methods for axenization and encystation. Isolation and purification methods exhibited significant variability, often involving two phases: an initial separation using simple techniques such as filtration and centrifugation, followed by a purification phase using a density gradient for fecal samples and immunomagnetic separation for water samples or nucleic acid extraction. The effectiveness of these methods differed depending on the source and sample type, highlighting the need for standardized protocols to ensure consistent and reliable results. Conclusions: Methods for the isolation and purification of G. lamblia exhibit notable variability and lack uniformity compared with the more consistent methods that re used for other life cycle stages. These findings underscore the urgent need for the development of standardized methodologies to enhance the reproducibility and reliability of research outcomes in this field.

4.5. Gestational Toxoplasmosis: An Analysis of Cases over the Last 5 Years in Brazil According to Clinical Evolution

Isabela Karina Vilas Boas, Andihel Dionísio Ramos, Gabriela Gerevini Dal Alba, Maria Fernanda Valentim de Paula

• Academic of Medicine, School of Medicine, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre 90050-170, Brazil

Introduction: Gestational toxoplasmosis (GT) results from Toxoplasma gondii infection during pregnancy, potentially causing severe fetal complications. Studying GT in Brazil is crucial to understanding clinical outcomes and gaps in epidemiological records.

Methods: A descriptive analysis of GT cases recorded in DATASUS from 2019 to 2024 was conducted. Data were extracted from the notification module in SINAN. Cases were classified into four outcomes: blank, cure, death due to GT, and death from other causes.

Results: The data showed 58,912 reported cases. Of these, 36,182 (61.4%) recovered, while 22,654 (38.4%) had unknown outcomes. Deaths totaled 76, with 39 attributed to GT and 37 to other causes.

Annual analysis revealed the highest notifications in 2022 (12,447 cases, 24.6%), followed by 2021 (11,093 cases, 22%) and 2023 (10,849 cases, 21.5%). The lowest numbers were in 2019 (8,436 cases, 14.3%) and 2024 (6,961 cases, 13.8%). Comparing pre-pandemic (2019) and post-pandemic years (2020–2024), the average number of reported cases remained stable, suggesting little impact from the pandemic.

Conclusions: GT remains a significant concern in Brazil, with 58,912 cases between 2019 and 2024. Most cases (61.4%) recovered, but 38.4% had unknown outcomes, reflecting gaps in reporting. The 76 deaths, including 39 from GT, highlight its severity. The 2022 peak may reflect improved surveillance or outbreaks, while the 2024 decline suggests control or underreporting. The stable case average pre- and post-pandemic suggests minimal pandemic impact. However, limitations include unexamined confounders and high missing data rates, which may affect accuracy. Strengthening reporting, monitoring pregnant women, and investing in prevention are key to reducing GT’s impact.

4.6. Impact of the 2024 Floods on the Increase in Leptospirosis Cases in the City of Canoas, Southern Brazil: A Comparison with 2023

Letícia Souza ¹, Lucas Conzatti Rodrigues ², Enrico Lázaro Guidugli ², Gabriela Pereira Macelaro ²

¹ 

Universidade Federal do Rio Grande do Sul

² 

Universidade Federal de Ciências da Saúde de Porto Alegre

Introduction: Leptospirosis, a zoonosis caused by Leptospira bacteria, is transmitted through contact with infected animals’ urine, often in flooded, unsanitary environments. In 2024, Rio Grande do Sul experienced severe floods due to heavy rains, inundating cities like Canoas and creating ideal conditions for leptospirosis spread through contaminated water exposure.

Methods: Data on confirmed leptospirosis cases in Canoas for 2023 and 2024 were sourced from the Department of Informatics of the Unified Health System (DATASUS). Monthly notifications were analyzed using time-series analysis to detect trends and descriptive statistics to highlight peak incidence periods. Historical data from 2020 to 2022 were reviewed to place recent findings in a multi-year context.

Results: In 2023, 14 cases were recorded, while 27 cases were reported in the first half of 2024. From March to June, aligned with the 2024 flooding, cases increased from 7 in 2023 to 27 in 2024, with May 2024 representing 52% of the year’s total. Historical data (2020–2022) show an annual average of 12 to 15 cases, suggesting the 2024 surge exceeds typical fluctuations, particularly during flood-affected months. This indicates a significant deviation linked to environmental conditions.

Conclusions: The 2024 floods in Rio Grande do Sul likely contributed to the notable rise in leptospirosis cases in Canoas, as floodwaters heightened infection risk. Compared to historical averages, this increase appears anomalous, reinforcing the influence of extreme weather on disease incidence. These findings emphasize the importance of tailored public health responses in flood-prone regions, such as improved sanitation and early warning systems, to mitigate future outbreaks and protect vulnerable populations.

4.7. Increase in Dengue Cases in Rio Grande do Sul in 2024 and Its Relation to Floods

Lucas Conzatti Rodrigues, Gabriela Pereira Macelaro, Guilherme Ferreira Cruz, Andihel Dionísio Ramos

• Academic of Medicine, School of Medicine, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre 90050-170, Brazil

Introduction: Dengue is a public health challenge in Brazil, characterized by its mosquito-borne nature and potential for severe clinical outcomes. The distribution and dynamics of this disease can be influenced by various factors, including environmental, social, and economic changes. During periods of heavy rainfall and subsequent flooding, like those experienced in Rio Grande do Sul in May of 2024, there is often an increase in reports of Dengue cases. Understanding the epidemiological trends of Dengue is essential for guiding prevention and control efforts. Methods: A cross-sectional, descriptive, retrospective, and quantitative study was conducted on cases of dengue in the Brazilian state of Rio Grande do Sul during 2023 and 2024. The data were obtained from the Department of Informatics of the Unified Health System (DATASUS). Results: In May and June 2024, following the flooding in Rio Grande do Sul, a total of 57,899 dengue cases were reported (47,733 in May and 10,166 in June). In comparison, during the same period in 2023, 18,195 cases were recorded (13,494 in May and 4701 in June), representing a 218% increase in total cases between the two years. This surge was particularly significant in May, which saw more than triple the number of cases compared to the previous year. Conclusions: The significant increase in Dengue cases in Rio Grande do Sul in 2024, particularly following the severe flooding in May, underscores the influence of environmental factors on disease transmission. The 218% surge in cases compared to 2023 highlights the vulnerability of affected regions to vector proliferation during and after extreme weather events. These findings emphasize the urgent need for integrated public health strategies, including enhanced surveillance, community education, and rapid response measures, to mitigate the impact of environmental changes on Dengue outbreaks.

4.8. Demographic Analysis of Zika Virus Cases in Brazil from 2020 to 2024

Enrico Lázaro Guidugli ¹, Letícia De Paula e Souza ², Theodora Garcia Furtado ¹, Pedro Henrique Paesi Dutra ¹

¹ 

Academic of Medicine, School of Medicine, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre 90050-170, Brazil

² 

Academic of Medicine, School of Medicine, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre 90035-003, Brazil

Introduction: The Zika virus is a flavivirus transmitted through the bite of the Aedes aegypti mosquito, which led to an epidemic in Brazil in 2016. In the majority of adult cases, infection is asymptomatic. However, in pregnant women, Zika virus infection can result in fetal abnormalities such as microcephaly. This study intends to analyze Zika virus cases in Brazil during the period from 2020 to 2024. Methods: A cross-sectional, descriptive, retrospective, and quantitative study on the number of Zika Virus cases in Brazil from 2020 to 2024 is described. Data from the Notification of Diseases Information System available in the DATASUS database were utilized. Among the analyzed variables, age group, education level, and sex were particularly highlighted. Results: In the analyzed period, 133,202 cases were recorded, with women being the most affected, accounting for 62% of the cases. Regarding age, the highest number of registered cases (52,423) was in the 20–39 years age group, followed by the 40-59 age group, computing 31,412 cases. Furthermore, illiterate people recorded the lowest incidence of Zika (1077), as the highest incidence was recorded in the group that completed high school (22,030). Conclusions: The data from 2020 to 2024 demonstrate that Zika virus infection in Brazil predominantly affected women, particularly those in their reproductive and working-age groups (20–39 years), highlighting the continued vulnerability of this population and their possible children. The higher incidence observed among individuals with higher educational levels, particularly those who completed high school, suggests that exposure and reporting factors may play a role in case distribution. These findings emphasize the importance of tailored public health interventions, with a focus on reproductive-age women, as well as the need for enhanced education and outreach strategies to address at-risk populations effectively.

4.9. Fungal Pathogenesis in Bovine (Bos Taurus Indicus) Mastitis: Therapeutic Protocol with Ethno Veterinary Formulation

Amruthavarshini S N, Senthilkumar Rajagopal

• Department of Biotechnology School of Applied Sciences, REVA University

Bovine are susceptible to various infections caused by a variety of pathogens. Due to their low immunity or poor hygiene conditions, they succumb to diseases, contributing to an important set of problems. These include animal welfare, productivity losses, uncertain food security, and loss of income and health. Fungal diseases with respect to their correlation to infection have not been explored.

In this study, we aimed to understand the effects of fungi in bovine by extracting and analyzing blood and milk samples from infected mastitis, as well as evaluating the rate of growth, general topography, reverse pigmentation, and microscopic examination for mycelial studies and slide culture. Mycelial studies will be performed to further identify these fungi. The fungal culture slide will be prepared by the tease mount technique using methylene blue stain. The slides will then be analyzed to characterize the fungi. Identification will be performed according to the guidelines recommended by Larone.

The research methodology used will benefit the farmers by reducing the cost on herbal medicine and the treatment of fungal pathogenesis by lowering the price of treatment and medication. Comparatively, in regular treatment checks, it takes 5–7 days for treatment and 7 days for recovery, whereas ethnoveterinary formulations take 5 days and recovery takes 3 days.

This study shows that when bovine are infected with diseases such as mastitis, they can also succumb to opportunistic pathogens that are commensal. Due to their weakened immune system, during an infection, these fungal pathogens can take advantage and cause pathogenesis in bovine. This is the first study on the opportunistic pathogenesis of fungal pathogens during an infection in bovine. Further studies have to be carried out in order to study the mechanisms and roles involved in pathogenesis.

4.10. Fungi and Wildlife: The Case of Avian Aspergillosis

Andreia Manuela Vieira Garces ¹, Isabel Pires ²

¹ 

Universidade de Trás os Montes e Alto Douro, Vila Real, Portugal

² 

Animal and Veterinary Research Centre (CECAV), Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), University of Trás-os-Montes e Alto Douro (UTAD), 5000-801 Vila Real, Portugal

Avian aspergillosis is a fungal disease caused predominantly by Aspergillus fumigatus, affecting the respiratory system of birds. It occurs in both wild and domesticated avian species, often leading to significant health and conservation concerns. The disease typically arises from the inhalation of fungal spores, which proliferate in damp or poorly ventilated environments, such as contaminated feed, litter, or nesting material. Clinically, aspergillosis manifests in acute or chronic forms. Acute cases are common in young or immunocompromised birds, presenting with respiratory distress, lethargy, and sudden death. Chronic aspergillosis, frequently seen in adult birds, leads to granuloma formation in the air sacs and lungs, causing progressive respiratory issues, anorexia, and weight loss. Diagnosis relies on clinical signs, imaging, fungal cultures, or histopathology. This study aims to describe the epidemiology and the macroscopic, histological, and microbiological lesions from wild birds during postmortem exams. Samples were collected for histology and Sabouraud agar. The necropsy and histological examination established the aspergillosis diagnosis in 10 animals examined over 7 years. The main species affected was Buteo buteo. Macroscopic alterations in the animals with aspergillosis were varied. All animals presented thickened air sacs with abundant caseous and necrotic debris and grayish-green fungal colonies and nodules in the air sacs and lungs. One animal presented dissemination to the bone and muscle. The main lesions observed were on the lungs and air sacs as expected. This work shows that aspergillosis is an important disease that causes mortality in wild birds, particularly during the recovery process of rehabilitation of these animals in the recovery centers. This work shows that the part of mycological identification is still often overlooked during the necropsy, and the diagnosis is based only on the macroscopic diagnosis. Avian aspergillosis remains a critical issue in veterinary medicine and wildlife conservation, requiring continued research for improved diagnostic and therapeutic approaches.

4.11. Harnessing the Intrinsic Disorder in Human Metapneumovirus for Therapeutic Development

Deepak Chaurasiya

• Biomedical Informatics Lab, Indian Institute of Information Technology, Allahabad 211002, India

Human metapneumovirus (hMPV) is a significant respiratory pathogen affecting populations worldwide, particularly young children, the elderly, and immunocompromised individuals. Despite its clinical importance, the intrinsic disorder status of hMPV proteins and their roles in the pathogenic mechanism remain largely unexplored. Intrinsically disordered proteins (IDPs) and disordered protein regions (IDPRs) are members of the “dark proteome,” characterized by the absence of a stable three-dimensional structure but with critical biological functions, such as host–virus interactions and immune evasion. In this study, we evaluated the prevalence and functionality of IDPs/IDPRs in the hMPV proteome using computational tools, including PONDR^®, IUPred2A, DISOPRED, and ESpritz, validated with NMR, X-ray crystallography, and DisProt data. Our findings revealed significant levels of intrinsic disorder in key hMPV proteins, such as the fusion (F), nucleocapsid (N), and glycoprotein (G), which are implicated in viral replication, host entry, and immune modulation. Proteins SPQ8B9Q8 and SPQ6WB94 showed 100% intrinsic disorder, highlighting their potential roles in immune evasion, while structural proteins like SPQ6WB99 displayed minimal disorder. The hMPV proteome exhibited a mean disorder percentage of 19.19%, with nearly half of the proteins containing disordered regions exceeding 30 amino acids. These findings emphasize the critical role of intrinsic disorder in the hMPV life cycle and identify IDPs/IDPRs as promising targets for disorder-based drug design.

4.12. Impact of the COVID-19 Pandemic on Visceral Leishmaniasis Reporting in Brazil: A Retrospective Analysis of Epidemiological Trends

Gabriela Pereira Macelaro, Yasmin Marques Loureiro, Guilhherme Ferreira Cruz, Isabela Karina Vilas Boas

• UFCSPA—Universidade Federal de Ciências da Saúde

Introduction: Visceral Leishmaniasis represents a significant public health challenge in Brazil, characterized by its zoonotic nature and potential for severe clinical outcomes. The distribution and dynamics of this disease can be influenced by various factors, including environmental, social, and economic changes. Understanding its epidemiological trends is essential for guiding effective prevention and control efforts. Methods: A cross-sectional, descriptive, retrospective, and quantitative study was conducted on confirmed cases of Visceral Leishmaniasis in the five regions of Brazil during the pre-pandemic period (2016 to 2019) and the pandemic period (2020 to 2023). The data were obtained from the Department of Informatics of the Unified Health System (DATASUS), specifically from the “Notifiable Diseases and Conditions” section. Results: During the pre-pandemic period (2016–2019), a total of 14,589 cases of Visceral Leishmaniasis were reported in Brazil, with a 46% overall reduction during the pandemic period (2020–2023). The decrease in the number of cases varied across the country’s regions, with the North region showing the largest decline of 56%. The Northeast experienced a 44% reduction, followed by the Southeast with a 52% decrease. In the South, the reduction was 16.9%, and in the Central West, it was 6.1%. Conclusions: Based on data analysis, it is possible to infer that the COVID-19 pandemic had an impact on the reporting of Visceral Leishmaniasis cases in Brazil. The sharp decline in notifications during the pandemic, particularly in the Northern region, suggests the possibility of an overburdened healthcare system, reduced demand for diagnosis and treatment due to social isolation, and a decrease in control programs and epidemiological surveillance. Therefore, it can be proposed that the reduction in Visceral Leishmaniasis cases is associated with lower case detection and reporting.

4.13. Molecular Analyses and Sindbis Virus Pathogenesis in Human Neuroblastoma Cell Line

Kornélia Bodó ¹, Viktória Nyári ¹, Zoltán Kopasz ¹, Péter Engelmann ², Krisztina Leiner ¹, Mónika Madai ¹, Brigitta Zana ¹, Zita Potzné-Árvai ¹, Gábor Kemenesi ^1,3, Anett Kuczmog ^1,3

¹ 

National Laboratory of Virology, Szentágothai Research Center, University of Pécs, H-7624 Pécs, Hungary

² 

Department of Immunology and Biotechnology, Medical School, Clinical Center, University of Pécs, H-7624 Pécs, Hungary

³ 

Department of Microbiology and Molecular Biology, Institute of Biology, Faculty of Sciences, University of Pécs, H-7624 Pécs, Hungary

Alphaviruses cause mosquito-borne erythrogenic diseases. Sindvis virus (SINV) circulates in an enzootic cycle between its vectors and birds. In humans, SINV triggers musculoskeletal syndromes characterized by fever, rash, acute and chronic polyarthritis, etc. Few studies examining the virus’s impact on the nervous/immune systems are available. SINV often causes cerebral infection, so understanding the brain’s response is crucial. The SH-SY5Y cell line, from neuroblastoma, is used in many neurobiological and virological studies. This study aimed to understand viral infection in vitro and examine immune markers using SINV infection in SH-SY5Y cells.

Due to the lack of adequate knowledge of SINV, optimization was necessary for all experimental designs. After the virus stock preparation, the median tissue culture infectious dose was used to determine the multiplicity of infection (MOI). Taqman-probe-based RT-qPCR was designed for the SINV nucleic acid, and immunofluorescence-based ds-RNA antibody staining was used to reinforce the virus replication. Apoptotic markers were also investigated by means of immunofluorescence. To monitor specific immune gene expressions (pattern recognition receptors (PRRs), regulator gene, cytokines) in a time-dependent manner (3/6/12/16/24/30 hr), RT-qPCR was also performed.

SINV infected the cells, and after 24 hr, a cytopathogenic effect and virus replication could be observed. SINV induced the caspase-dependent apoptotic pathway. Most of the investigated genes, including PRRs, (TLR-3/7, RIG1/MDA5), a regulator (b-catenin), and inflammatory (IL-1b, IL-6, TNFa), and antiviral (IL-10, IFNβ) genes, exhibited a consistent induction up to 12/16 hr; by the end of the 30 hr, their expression mainly (e.g., RIG1/MDA5, IFNβ) decreased compared with those of our controls. We can conclude that in vitro, SINV can be transmitted even in small quantities, and an inflammatory environment can be developed, which later leads to cell death.

These preliminary results allow us to gain insights into SINV–SH-SY5Y interactions, which are better understood by examining them in much finer molecular detail.

4.14. Surge in Dengue Cases in Rio Grande do Sul in 2024: Epidemiological Trends and Contributing Factors

João Paulo Farezin Fortti, Gabriela Pereira Macelaro

• UFCSPA—Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Brazil

Introduction: Dengue is a viral disease transmitted by Aedes mosquitoes; endemic in over 100 countries; including Brazil. It represents a significant public health challenge due to its high incidence and the simultaneous circulation of all four virus serotypes, creating a hyperendemic scenario. This situation complicates efforts for prevention and control, making it a persistent concern for healthcare systems. The state of Rio Grande do Sul has seen a notable rise in dengue cases in 2024; highlighting the need to understand its epidemiological trends; impact on healthcare infrastructure; and contributing factors. Methods: Data were collected from the Department of Informatics of the Unified Health System (DATASUS) on dengue cases in Rio Grande do Sul from January 2021 to December 2024. The data were analyzed based on the following variables: epidemiological profile; hospitalization rates; and clinical outcomes. Results: From 2021 to 2024; 329,185 dengue cases were reported in Rio Grande do Sul; with 212,321 (64%) occurring in 2024; peaking in April (84,513 cases). A 400% increase was observed in 2024 compared to 2023; 200% compared to 2022; and 1800% compared to 2020. The epidemiological profile remained consistent; with 54% of cases in females and 60% in patients aged 20-49. Most patients were of White ethnicity (82%). Regarding outcomes, 73% did not require hospitalization; and 75% recovered, following trends from previous years. Conclusions: The significant rise in dengue cases in Rio Grande do Sul in 2024, particularly with a 400% increase compared to 2023, which highlights the influence of favorable climate conditions, including increased rainfall and higher temperatures; which promote mosquito proliferation. Additionally, factors such as urban water accumulation and insufficient mosquito control measures contribute to the surge. Despite this, the epidemiological profile and clinical outcomes remained consistent; with most cases not requiring hospitalization and the majority of patients recovering.

4.15. Zika Virus Surveillance in Brazil (2010–2024): Epidemiological Insights

Gabriela Gerevini Dal Alba ¹, Yasmin Marques Loureiro ¹, Andihel Dionísio Ramos ¹, Camilly Vitória Cansan Loss ², Isabela Karina Vilas Boas ¹

¹ 

Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Brazil

² 

Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

Introduction: The Zika virus emerged as a significant public health challenge in Brazil, particularly after its epidemic peak in 2016. Initially identified in Africa, Zika spread to the Americas in the mid-2010s, causing widespread concern due to its association with severe neurological complications, including microcephaly in newborns and Guillain–Barré syndrome in adults.

Methods: Secondary data were collected from the Department of Informatics of the Unified Health System (DATASUS) regarding Zika virus notifications in Brazil between January 2010 and December 2024, analyzing the number of cases by year of occurrence.

Results: Between 2010 and 2024, 497,550 cases of Zika were reported in Brazil. The peak occurred in 2016, with 277,072 cases (55.7%). In 2017 and 2018, the numbers dropped to 32,225 (6.5%) and 20,092 (4%), respectively. After an increase in 2019, with 30,800 cases (6.2%), there was another decline in 2020 and 2021, with 20,637 (4.1%) and 19,112 (3.8%) cases. In 2022, notifications rose to 34,786 (7%), followed by 22,778 (4.6%) in 2023 and 35,146 (7.1%) in 2024. The initial period (2010–2014) recorded fewer than 100 cases annually, reflecting the absence of outbreaks.

Conclusions: The data reveal a significant epidemiological dynamic of the Zika virus in Brazil. After an initial period of low notification numbers (2010–2014), the 2016 outbreak marked the peak of the epidemic, accounting for more than half of the cases recorded over the 14 years investigated. A sharp decline was observed in the following years, until a slight increase in 2019. New fluctuations occurred, with rises in 2022 and 2024. The post-2016 reduction may be associated with population immunity and public health interventions, while the occasional increases suggest seasonal or regional factors. Continuous surveillance is essential to monitor and prevent new outbreaks.

5. Session: Microbiome and Soil Science

5.1. The Microbial and Physicochemical Interactions of Soils in the Degradation of Buried Polyethylene Pipes in Colombia

Maria Andrea Reyes Reyes ^1,2, Brayan Danilo Vergel ¹, Jenny Andrea Oviedo ², Clara Inés Sánchez ¹, Jorge Hernando Panqueva ²

¹ 

Escuela de Microbiología, Universidad Industrial de Santander (UIS), Bucaramanga 680002, Colombia

² 

Corrosión, Corporación para la Investigación de la Corrosión (CIC), Piedecuesta 681011, Colombia

The widespread use of polyethylene (PE) in industries such as agriculture and construction poses environmental challenges due to its recalcitrance in natural ecosystems. This study focuses on the microbial and physicochemical interactions of soils from Colombia, in the degradation of buried polyethylene pipes, combining molecular, microbiological, and physicochemical approaches to understand soil–microbe–plastic dynamics. Soil samples were collected from sites with buried polyethylene pipes exhibiting degradation signs. Microbial communities were characterized using 16S rRNA and ITS sequencing, with Nanopore technology, to identify bacterial and fungal taxa. Additionally, microbiological analysis was performed by taking swabs from the surface of degraded pipes to directly observe microbial colonization. Fungal isolates were cultured on Potato Dextrose Agar (PDA) to correlate traditional isolation techniques with molecular findings. Soil physicochemical properties, including pH, moisture, redox potential, particle size distribution, and others, were analyzed. Polyethylene degradation was assessed through surface analysis by scanning electron microscopy (SEM). Bacterial phylum Proteobacteria and genera such as Pseudomonas, along with fungal genera such as Aspergillus and Fusarium, were identified as key players in soils with higher degradation rates. Microbiological analysis of pipe surfaces confirmed microbial colonization, consistent with soil sequencing data. Fungal isolates grown on PDA matched molecular findings, validating the integration of traditional and molecular approaches. SEM imaging revealed significant surface erosion and microbial colonization on the polyethylene pipes. Physicochemical analyses showed that soils with neutral pH and higher moisture content supported more active and diverse microbial communities. This study demonstrates the critical role of microbial activity and soil physicochemical properties in polyethylene degradation. The integration of microbiological, molecular, and physicochemical methods offers a comprehensive framework to advance sustainable strategies for managing plastic pollution in soil environments.

5.2. Analysis of Soil Biological Activity in the Surroundings of Sfântu Gheorghe in Covasna

Éva Abod ¹, László Veres ², Levente Csiszér ¹

¹ 

Department of Life Sciences, Faculty of Life Sciences and Sports, Sfântu Gheorghe, Sapientia Hungarian University of Transylvania, 520036 Sfântu Gheorghe, Romania

² 

Master’s student between 2019 and 2021, Sapientia Hungarian University of Transylvania, Faculty of Technical and Human Sciences—Târgu-Mureș, Department of Horticulture, 540485 Târgu-Mureş, Romania

Covasna County in Romania is regarded as an agricultural region due to the exceptionally high proportion of arable land. One of the most significant indicators of agricultural productivity is soil fertility, which is influenced by numerous factors. Among these, the most important is the biological condition of the soil.

In our research, we analyzed the biological activity of soils in the surroundings of Sfântu Gheorghe. Soil samples were collected from five different soil types (preluvosol, faeoziom, cambic faeoziom, aluviosol and gleysol) in autumn 2018 and spring 2019. For each field, 20 samples were collected and homogenized, and the biological activity was measured in three replications using the fluorescein diacetate (FDA) hydrolysis method.

The faeoziom soil type showed an enzymatic activity of 3.9631 ± 0.1818 µg/g·h in autumn and 2.9788 ± 0.3277 µg/g·h in spring; the cambic faeoziom soil type showed an activity of 4.0072 ± 0.1145 µg/g·h in autumn and 3.0548 ± 0.1621 µg/g·h in spring; the gleysol type showed an activity of 3.2869 ± 0.1817 µg/g*h in autumn and 2.9988 ± 0.4679 µg/g·h in spring; the aluviosol soil type showed an activity of 2.4186 ± 0.0616 µg/g·h in autumn and 1.3421 ± 0.3779 µg/g·h in spring; and the preluvosol type recorded the highest values, with 5.4517 ± 0.2609 µg/g·h in autumn and 5.3677 ± 0.1548 µg/g·h in spring. For the faeoziom, cambic faeoziom, and aluviosol soil types, an approximately one-unit decrease was observed in spring due to the lower temperatures during the spring sampling period. For the gleysol soil type, the decrease was much smaller, and the differences between the values were not statistically significant. We attribute this to the application of manure by farmers after the autumn sampling, as manure contains a considerable number of microorganisms. For the preluvosol soil type, enzymatic activity also decreased slightly, which can be explained by the fact that this area is a pastureland with continuous vegetation cover and no soil disturbance, providing a stable habitat for microorganisms, and, therefore, stable microbial activity.

5.3. Microbial Diversity in Composting: Identification and Analysis of Fungus-Related and Bacterial Communities for Enhanced Organic Waste Recycling

Patrícia Branco ^1,2, Elisabete Muchagato Maurício ^1,3

¹ 

BIORG—Bioengineering and Sustainability Research Group, Faculdade de Engenharia, Universidade Lusófona, Av. Campo Grande 376, 1749-024 Lisbon, Portugal

² 

Linking Landscape, Environment, Agriculture and Food (LEAF), Associated Laboratory TERRA, Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017 Lisboa, Portugal

³ 

CBIOS—Research Center for Biosciences & Health Technologies, Universidade Lusófona, Campo Grande 376, 1749-024 Lisbon, Portugal

Composting is an ancient and sustainable method of recycling organic waste into humus, a nutrient-rich substance that enhances soil productivity and reduces the environmental impact of waste disposal. Microorganisms, including fungi and bacteria, are essential to this process, as they drive the decomposition of organic matter.

This research aimed to identify fungal and bacterial species involved in composting and optimize their roles in decomposition. Compost samples were collected from Lusófona University’s composting system after six months of processing organic waste, primarily fruit peels and vegetable scraps mixed with dry plant material. Microbiological analyses, including DNA extraction, PCR amplification, and Sanger sequencing, were used to identify the microorganisms present.

The results showed that the most abundant microorganisms were filamentous fungi, with the most predominant species being Aspergillus heyangensis, Aspergillus creber, Cladosporium asperulatum, Pestalotiopsis lespedezae, and especially Penicillium brevicompactum, which was the most prevalent. These species dominate the decomposition of the complete residue and have potential applications in industry and composting. The bacterial species identified were predominantly represented by the genera Bacillus (e.g., B. halotolerans, B. subtilis, B. amyloliquefaciens, B. mojavensis), as well as species from Sphingobacterium (S. kitahiroshimense), Serratia (S. fonticola), and Pseudomonas (P. fluorescens). The collective contribution of these microorganisms to the breakdown of organic material during composting is significant.

This study highlights the diversity and significance of fungal and bacterial communities in composting, emphasizing their potential to optimize organic waste recycling processes. Understanding the microbiota involved in composting opens avenues for developing biotechnologies, such as microbial inoculants, to enhance process efficiency and sustainability. Future research should focus on optimizing environmental conditions to support beneficial microorganisms, further advancing composting practices and producing higher-quality humus.

5.4. Phage-Based Biocontrol of Nitrification in Agricultural Soil

Laura Sala-Comorera ¹, Pedro Blanco-Picazo ¹, Gloria Vique ¹, Pablo Quirós ², María Dolores Ramos-Barbero ¹, Clara Gómez-Gómez ¹, Lorena Rodriguez-Rubio ¹, Tula Yance-Chávez ², Ignasi Salaet ², Maite Muniesa ¹

¹ 

Departament de Genètica, Microbiologia i Estadística, Universitat de Barcelona, Barcelona, Spain

² 

Departamento de I+D+i de Fertinagro Biotech S.L., Teruel, Spain

Introduction

Nitrogen fertilization boosts crop yields but is inefficient due to the rapid activity of nitrifying bacteria, which leads to the loss of useful nitrogen forms. This requires the use of large amounts of fertilizers, leading to environmental pollution from compounds like NO₃ and N₂O. Although chemical inhibitors can improve fertilization, their impact on the environment and human health remains uncertain. A new strategy involves the use of bacteriophages (phages) that specifically target nitrifying bacteria.

Methods

Bacteriophages were isolated from sewage samples. The inhibition of nitrifying activity was tested in AOB medium and soil to monitor nitrite production with a colorimetric assay. The increase in bacteriophage particles during infection was assessed with qPCR. Controls without the addition of bacteriophages or bacteria were included for in vitro and soil samples. The persistence of infectious bacteriophages was analyzed in water suspensions and soil under different pH levels (3, 5, 6, 7, 8) and temperatures (4, 15, 22, 37 °C) over 6 months.

Results

We isolated a bacteriophage (FNF-1) capable of infecting four species of Nitrosomonas. FNF-1 inhibited bacterial growth, resulting in a reduction in nitrite formation in phage-treated samples, both in vitro and in the soil. Meanwhile, FNF-1 increased by 2-log₁₀ units within 48 h. Infectious FNF-1 remained stable in water suspensions for 6 months at 4, 15, 22, and 37 °C and at pHs 6, 7, and 8 but was inactivated when exposed to pHs 3 and 5. In soils, FNF-1 infectivity showed a 2-log₁₀ reduction over 6 months.

Conclusions

This study presents a novel approach using bacteriophages to eliminate nitrifying bacteria. Bacteriophages as biocontrol agents offer an effective and environmentally friendly alternative to inhibit nitrification while overcoming the limitations of chemical products. While their application shows potential for improving agricultural fertilization, further research is required to scale up and optimize a product.

5.5. Preliminary Data on Biodiversity Effects of Replacing Plastic Row Covers with Legume and Non-Leguminous Covers in Organic Artichoke Cropping

Savanah Senn ¹, Brianna Zimmerman ¹, Arianna Bozzolo ², Ray A. Enke ³, Maxim Recke ¹, Jordan Schoonover ¹, Julia Perederiy ⁴, Daila Melendez ^1,5, Les Vion ¹

¹ 

Los Angeles Pierce College Department of Agriculture Sciences, Plant Science program, Woodland Hills, CA 91371, USA

² 

Rodale Organic Institute, California Organic Center, Camarillo, CA 93010, USA

³ 

James Madison University, Biology Department, Harrisonburg, VA 22807, USA

⁴ 

OmBiome, Torrance, CA 90502, USA

⁵ 

Oregon State University, Department of Horticulture, Corvalis, OR 97331, USA

The purpose of this study was to investigate how replacing polyethylene row covers with other types of cover influences soil properties, microbial diversity, the composition of soil communities, and carbon metabolism in artichoke cropping. We carried out five treatments in four replicates, including plastic film, buckwheat, kurapia, crimson clover, and white clover. The samples were collected after rainfall events, totaling 2 in. of precipitation during February 2024.

In the cross-sectional data, results were comparable between treatments with regard to physical and chemical properties, but there were significant differences in soil moisture and pH, with plastic having the lowest moisture and lowest pH. Higher CFUs in plastic at a 10⁻⁴ dilution were noted.

For the composition and abundance of bacteria, the results indicated the following differences between white clover vs. plastic: higher Adhaeribacter sp. and Phycicoccus sp. abundance in white clover treatments; higher Rhodobacter sp. and Sphingobium sp. abundance in plastic treatments. The following differences were observed between the legume treatment vs. the control: Phycicoccus sp. and Stenotrophomonas sp. abundance was higher in the legume treatment. Results with low fold change values 1.33 were omitted.

The Chi-square test was negative for differences in proportions of carbon sources that could be metabolized in Biolog Ecoplate trials by each group (p = 0.16). There was a gap in the mosaic plot between the proportion of carbon sources metabolized by white clover communities and other treatments. For the artichokes, the GLM logit model indicated a difference between white clover treatments when compared with other treatments (p = 7.76 × 10⁻⁵). The carbon sources that differed significantly in the microbes’ ability to metabolize themwere delta-glucosaminic acid, L-alpha-glycerol phosphate, glucose-1-phosphate, i-erythritol, itaconic acid, phenylalanine, serine, and threonine.

Further studies should include microbial biomass analyses and nutrient analyses and consider the effects of the treatments on yield. Longitudinal analysis of soil physical and chemical properties will confirm the effects of the treatments on physical and chemical properties of the soil.

6. Session: Microbial Characterization and Bioprocess

6.1. Evaluation of In Vivo and In Vitro Pathogenicity of Selected Staphylococcus aureus Strains Isolated from Humans and Animals

Filip Jacek Kot, Marta Książczyk, Gabriela Bugla-Płoskońska

• Department of Microbiology, Faculty of Biological Sciences, University of Wrocław, Wrocław, Poland

Staphylococcus aureus is a major pathogen responsible for numerous human and animal diseases, including Staphylococcal scalded skin syndrome, folliculitis, and mastitis. Known for its virulence factors, it can evade host immune responses and enhance colonization. Recent research has focused on alternative in vivo models, such as the Galleria mellonella larval model, which offers a cost-effective, less harmful, and accessible approach to studying pathogens and testing therapies. This model is compatible with the 3R principle (replacement, reduction, and refinement).

In this study, the virulence of 35 S. aureus strains isolated from humans, dogs, and cats was assessed both in vivo and in vitro. The bactericidal effect of a commercial human serum reagent on these strains was evaluated, and selected isolates were tested in the G. mellonella model. Biofilm formation was also analyzed using the crystal violet assay. Most strains were resistant to the human serum reagent, with only three being susceptible. In the larval model, varying levels of virulence were observed. Biofilm formation was detected in only five out of thirty-five strains.

No significant differences in serum resistance were observed between strains from different hosts. The Gram-positive structure of S. aureus likely explains this. Strains exhibiting the MRS (Methicillin-resistant Staphylococcus) phenotype caused higher mortality in the larvae, and those capable of forming biofilms displayed both low and high virulence levels in vivo on the larval model.

This study was partially financed by an internal grant of the University of Wrocław IDUB titled “Galleria mellonella—the use of larvae in the optimization and improvement of the in vivo insect model for bacterial pathogenicity studies.”, No. BPIDUB.7.2024

The tested strains were graciously provided by the Wrocław University of Environmental and Life Sciences, Department of Epizootiology and Clinic of Birds and Exotic Animals.

6.2. Antifungal Activity of Some Rhizospheric Actinomycetes

Ratchanon Thongpheak, Prapanrat Manota, Anillada Nettakul, Ekachai Chukeatirote

• School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand

Rhizosphere soil is one of the unique microbial habitats, especially the actinobacteria. This study was carried out to screen and isolate actinomycetes exhibiting antifungal activity. Initially, the soil samples collected from Allium ascalonicum L., Alpinia galangal, Amorphophallus sp., Clausena harmandiana, and Cymbopogon citratus were pretreated by air-drying and subsequent heat incubation. By using three different media (international Streptomyces project 1 and 2 (ISP-1 and ISP-2) and starch casein agar (SCA)), 79 isolates were obtained and identified as actinomycetes by morphology. Ten actinomycete isolates were then randomly selected to assess their antifungal activity against five pathogenic fungi: Aspergillus sp., Colletotrichum sp., C. acutatum, C. gloeosporioides, and Lasiodiplodia theobromae. Initial screening showed that there were three most active strains, namely CN1, CN4, and LG5, exhibiting a strong and broad spectrum. The bacterial culture supernatants were then prepared and evaluated for their stability under various pH and temperature conditions. The data obtained showed that the antifungal activity of the three culture supernatants was varied depending on the bacterial source and testing fungal strains. The present work implies that rhizospheric soil samples are an attractive source for the discovery of numerous actinomycetes with activity against phytopathogenic fungi. Identification of the bacterial species and determination of the active compounds are essential for further analysis for use in fungal control.

6.3. Assessing the Potential Association Between Pigeons and the Presence of Acanthamoeba spp. in the River Soar and Public Parks in Leicester, England

Umar Anjum, Antonio Peña-Fernández

¹ 

Leicester School of Allied Health Sciences, De Montfort University, Leicester LE1 9BH, UK

² 

Department of Surgery, Medical and Social Sciences, Faculty of Medicine and Health Sciences, University of Alcalá, Ctra. Madrid-Barcelona, Km. 33.600, 28871 Alcalá de Henares, Madrid, Spain

Acanthamoeba spp. are free-living amoebae that can cause life-threatening brain infections and can also infect the eyes and skin. Amoebas/cysts of this species have previously been isolated from the River Soar, which runs through the city of Leicester, England, and is frequently used by the public. It has also been found in grass samples collected from public parks and green spaces across the city. Acanthamoeba can host endosymbionts, which can include important human microbes, acting as Trojan horses. Although their zoonotic role is poorly understood, cysts of Acanthamoeba spp. and other free-living amoebae have been found in the feces and/or the gastrointestinal tract of various animals, including domestic livestock (pigs, cows) and wildlife (squirrels, pigeons), suggesting a potential source of transmission. To investigate their zoonotic potential in pigeons, we analyzed fresh droppings from 628 pigeons collected from various urban parks and green spaces in the city of Leicester and surrounding rural areas as follows: 136 samples in summer 2017, 140 in autumn, 124 in winter 2017/18, 135 in spring, and 94 in summer 2018. After appropriate pre-concentration, thin smears were stained with trichrome and examined by light microscopy. DNA was extracted using a Fast DNA^® Spin Kit. PCR inhibitors were removed using the QIAamp^® micro DNA extraction kit. The extracts were screened for Acanthamoeba spp. using a triplex real-time TaqMan PCR assay with an appropriate positive control. All samples tested microscopically and molecularly were negative. Although their zoonotic role is not well understood, a potential and unknown animal reservoir could explain the moderate to high distribution found in both the grass samples and the river, posing a potential risk to people involved in sports such as rowing, football, or rugby. Culture techniques should be used to better investigate the potential interaction of Acanthamoeba with urban pigeons.

6.4. Characteristics of Biofilms Formed by Bacillus subtilis subsp. Spizizenii Growing in a Simple Culture Medium with Two Different Carbon Sources

Gabriela Cristina Sarti ^1,2, Josefina Ana Eva Cristóbal-Miguez ¹, Antonio Paz-González ², Ivanna Lorena Avram ¹, Elizabeth García-Guzmán ¹, Andrea Belén Alegre ¹, Ana Rosa García ¹, Mirta Esther Galelli ³

¹ 

Inorganic and Analytical Chemistry Cathedra, Department of Natural Resources and Environment, Faculty of Agronomy, University of Buenos Aires, Av. San Martín 4453, Buenos Aires C1417DSE, Argentina

² 

AQUATERRA Research Group, Interdisciplinary Center for Chemistry and Biology, CICA, As Carballeiras, s/n Campus de Elviña, University of A Coruna, 15008 Coruna, Spain

³ 

Agrofood Area, Department of Applied Biology and Food, Faculty of Agronomy, University of Buenos Aires, Av. San Martín 4453, Buenos Aires C1417DSE, Argentina

Biofilms represent a key survival strategy, providing bacteria with effective protection against abiotic and biotic stress. In the laboratory, Bacillus subtilis subsp. Spizizenii forms a biofilm at the liquid–air interface, the matrix of which is primarily composed of polysaccharides, proteins, and nucleic acids. The aims of this study are as follows: (1) to evaluate how the chemical composition of the matrix and the stability of the biofilm are modified when the bacteria grow using either glucose or glycerol as carbon sources; (2) to study the effect of temperature on biofilm production and planktonic growth. The bacteria were cultured in a saline medium with 55 mM L-glutamic acid and 1% glucose or glycerol at different temperatures. Biofilm was produced under static conditions and planktonic cells by shaking at 150 rpm. Biofilms were analyzed by high-resolution chromatography. In the presence of glucose, polysaccharide monomers at the biofilm matrix showed substitutions that conferred acidic properties. This biofilm disintegrated after 5 days at 20 °C and 15 days at 4 °C. In contrast, when cultured with glycerol, the polysaccharide sugars were neutral, and the biofilm disintegrated after 20 days at 20 °C and 60 days at 4 °C; visually, this biofilm was more compact. Planktonic growth of B. subtilis was highest at 45 °C, whereas the maximum biofilm formation (1.2 mg/mL of culture) was achieved between 30 °C and 37 °C. In the static cultures, the planktonic form of B. subtillis disappeared within 24 h after biofilm synthesis. In conclusion, the biofilms obtained from different carbon sources show differences, suggesting that the substituents of the polysaccharide monomers might be crucial for their structural properties. Moreover, in cases where the biofilm was formed at the liquid–air interface, the planktonic cells developed in this culture migrated toward the biofilm and as a result, the medium appeared clear; this was likely due to nutrient depletion, which does not support planktonic growth.

6.5. Development of New Tools for Sustainable Management Olive Trees Crop

Clara Izquierdo Jiménez ¹, Cecilia Recuero García ¹, Sergi Maicas Prieto ², Inmaculada del Castillo Madrigal ¹

¹ 

R&B Department; Microbiology Area; SEIPASA. L’Alcúdia (Valencia). 46250. Spain

² 

Department of Microbiology and Ecology, Faculty of Biological Sciences, Universitat de València, Valencia, Spain

1. Introduction

Climate change significantly impacts agrarian systems, compromising the quality of agricultural production. This situation has led farmers to demand sustainable tools to enhance crop performance while maintaining soil fertility and product quality. Microorganisms capable of interacting with plants represent an opportunity for cultivating healthy crops through growth promotion and biostimulation. Numerous studies point to native microbiomes in agricultural soils as a primary source of well-adapted microorganisms to develop new tools for crop treatment. This study was focused on the olive tree, a high-value Mediterranean crop. Microbial diversity was analyzed at two different phenological stages of the crop, and both the growth promotion and inhibitory capability against phytopathogens of isolated microorganisms were also evaluated.

2. Material and Methods

A metagenomic analysis was performed using high-throughput sequencing. Additionally, culturable microorganisms were selected based on morphological criteria. Diversity data, combining molecular analysis and variability on isolated microorganisms, were studied to determine the effect of the crop stage on the soil microbiome structure.

The isolated strains were characterized by their plant growth-promoting properties (PGPR), such as nitrogen fixation, phosphate solubilization, siderophores, and phytohormones production, to identify and select candidates to be used for the development of biotechnological tools.

3. Results

The number of morphotypes isolated during the fruit development stage was twice that obtained during the flowering stage. This increment is associated with greater root activity and the presence of exudates richer in organic compounds. Furthermore, microorganisms with remarkable PGPR characteristics were found, granting their strong potential as biocontrol and biofertilization agents.

4. Conclusions

This study demonstrates that soil is a valuable reservoir of microorganisms, that can be used to create bio-tools for sustainable agriculture.

6.6. Evaluation of Antioxidant Stability of Jinicuil (Inga paterno) Probiotic Beverages Fermented by Lactobocillus delbrueckii ssp. Bulgaricus and Streptococus thermophilus During Refrigeration Storage

Lizbeth Rosas-Ordoñez ¹, Oscar Jiménez-González ¹, Taisa S.S. Pereira ²

¹ 

Department of Chemical, Food and Environmental Engineering, Universidad de las Américas Puebla, Puebla 72810, Mexico

² 

Department of Health Sciences, Universidad de las Américas Puebla, Puebla, 72810 Mexico

Inga paterno produces seeds (Se) covered by a sarcotesta (Sa), both of which remain understudied. Meanwhile, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (L-S) are commonly used in fermented products. This study investigated the potential of Se and Sa as substrates for probiotic fermentation using L-S.

Three formulations were prepared: 5% (w/v) Sa, 5% (w/v) Se, and a 1:1 mixture (Ae). These were sterilized, inoculated with L-S (7.67 × 10⁶ CFU/mL), fermented (72 h at 35°C), and stored at refrigeration temperature (5°C). Antioxidant activity (AA), total phenolic content (TPC), condensed tannins (CT), flavonoids (FC), and phytic acid content (PAC) were measured at 0, 4, 11, and 25 days of storage. One-way ANOVA and Tukey’s test (p 0.05) were used to analyze the data.

L-S counts ranged from 6.69 to 9.41 log CFU/mL, with higher stability in Se (8.39 to 7.62 log CFU/mL) and Sa (8.44 to 8.01 log CFU/mL). AA ranged from 27.50 to 115.92 μg Trolox equivalents/mL, with Sa showing the highest and stable levels (84.07 to 113.46 log CFU/mL). PAC increased in Sa (from 34.22 to 42.54 μg of phytic acid/mL) but decreased in Se (from 40.13 to 37.73 μg of phytic acid/mL). CT decreased in all formulations (ranging from 1.53 to 0.70 mg of catechin equivalents/mL), and Sa presented a higher amount of FC (from 0.53 to 1.04 mg of quercetin equivalents/mL) than Se (from 0.03 to 0.08 mg of quercetin equivalents/mL) and Ea (from 0.09 to 0.21 mg of quercetin equivalents/mL). AA was mainly correlated with FC (R² = 0.6). L-S remained viable throughout storage in all formulations, while Sa provided a rich source of TPC and FC. These findings suggest that I. paterno sarcotesta can serve as promising substrates to produce a functional fermented beverage with enhanced antioxidant properties.

6.7. Evaluation of the Sensitivity of Sulfate-Reducing Bacteria and Indicators of the Microbiologically Influenced Corrosion of Steel Under the Influence of Dimethyl Sulfoxide

Nataliia Tkachuk ¹, Liubov Zelena ², Yaroslav Novikov ³

¹ 

T.H. Shevchenko National University “Chernihiv Colehium”

² 

Department of Virus Reproduction, Danylo Zabolotny Institute Microbiology and Virology NAS of Ukraine, Kyiv, Ukraine

³ 

Department of Biology, T.H. Shevchenko National University “Chernihiv Colehium”

Anaerobic sulfate-reducing bacteria (SRB) play an important role in the process of microbiologically influenced corrosion (MIC). In the study of corrosion processes without bacteria with the participation of DMSO, an increase in the rate of corrosion of metals was shown. However, the bactericidal properties of DMSO against SRB, the indicators of MIC of steel in the presence of SRB and DMSO, have not been investigated; thus, this forms the aim of this study. The sensitivity of SRB Desulfovibrio oryzae strain NUChC SRB1 (accession number in GenBank is MT102713.1) to DMSO at concentrations of 1–100% (v/v) was investigated by the method of dilution in liquid Postgate’s “C” medium. The corrosion activity of D. oryzae strain NUChC SRB1 against steel 3 with the addition DMSO (final concentration of 45%) was investigated by the biofilm formation ability based on biofilm biomass on the surface of steel samples (crystal violet method) and the effect on corrosion rate (gravimetric method). SRB and DMSO were not added in the control. Corrosion research took place for 35 days in Eppendorf-type tubes (50 mL) under anaerobic conditions and at a temperature of 29 ± 2 °С. Statistical analysis was carried out. It was found that DMSO with a concentration of 10% to 100% exhibits antibacterial properties against the studied SRB. Adding DMSO to the medium with bacteria ensured a significant reduction in biofilm biomass. Therefore, DMSO, despite its bactericidal properties and ability to inhibit biofilm formation, does not demonstrate inhibiting activity against microbiologically influenced corrosion in the presence of SRB.

6.8. Identification of a Strain of the Genus Janibacter Isolated in a Pharmaceutical Industry Using Molecular and Proteomic Techniques

Andressa Sbano S. Ferreira, Maria Luiza Souza Galvão Dacio, Maria Giovanna de Araújo Ferreira, Pamella de Sousa Silva, Talita Bernardo Valadão, Rebeca Vitória da Silva Lage de Miranda, Joyce Modesto de Andrade, Marcelo Luiz Lima Brandão, Luciana Veloso da Costa

• DEQUA, Bio-Manguinhos, Rio de Janeiro 21040-900, Brazil

The production of medicines and vaccines must comply with strict Good Manufacturing Practice standards to ensure the safety of the manufactured products. Consequently, environmental monitoring of pharmaceutical production areas is essential to ensure compliance with recommended limits for microbiological contamination, and identifying microorganisms isolated from these areas supports preventive and corrective actions. This study aimed to identify at the species level strain B1373/24, which was isolated during air monitoring in a viral vaccine production area of a pharmaceutical industry in Rio de Janeiro. The strain was initially analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the MALDI Biotyper^® (Bruker); however, it could not be identified. Therefore, the 16S rRNA gene of B1373/24 was sequenced, and the resulting data were compared with the EzBioCloud database, using the ≥98.7% threshold for species-level identification. The following species were proposed: Janibacter anophelis (100.0%), Janibacter hoylei (98.82%), and Janibacter cremeus (98.75%). The species was confirmed as Janibacter anophelis through phylogenetic analysis using the following software: MegaX, jModelTest, Mafft-online, and FigTree. This confirmation allowed the spectrum of strain B1373/24 to be incorporated into the MALDI Biotyper^® system, contributing to the expansion of its database (DB). A subsequent reanalysis of B1373/24 using MALDI-TOF MS successfully identified it as Janibacter anophelis, confirming the validity of the newly added spectrum within the system’s DB. The identification of B1373/24 as Janibacter anophelis highlights the importance of combining molecular tools to expand the MALDI-TOF MS database, thereby improving its capacity to identify environmental microorganisms. This study underscores its significant technological and scientific value for the pharmaceutical industry.

6.9. Interplay of Symbiotic Bacteria in Wolbachia Density Modulation Among Naupactini weevils (Coleoptera, Curculionidae)

Lucia Fernandez Goya ¹, Alejandra Carla Scannapieco ², Panagiota Stathopoulou ³, Naima Bel Mokhtar ³, Silvia Beatriz Lanzavecchia ², Claudia Alejandra Conte ², Diego Fernando Segura ², George Tsiamis ³, Marcela Silvina Rodriguero ^1,4

¹ 

Instituto de Ecología, Genética y Evolución de Buenos Aires (CONICET-UBA), Argentina

² 

Instituto de Genética “E. A. Favret”, Instituto Nacional de Tecnología Agropecuaria (INTA)—Grupo vinculado al Instituto de Agrobiotecnología y Biología Molecular (IABIMO)—Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Hurlingham

³ 

Laboratory of Systems Microbiology and Applied Genomics, Department of Sustainable Agriculture, University of Patras, Agrinio, Greece

⁴ 

Departamento de Ecología, Genética y Evolución de la Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

Wolbachia pipientis is the most widespread endosymbiotic bacterium, known for its ability to alter host reproduction. In Naupactini weevils, their reproductive mode appears to be linked to Wolbachia density, whereby species with low levels or an absence of Wolbachia reproduce sexually, while high-density infections would promote parthenogenesis. Although the factors driving high Wolbachia loads remain unclear, evidence suggests that the diversity of the host’s microbiota may influence Wolbachia abundance. This study explored the bacterial microbiota of Naupactini species with contrasting reproductive modes and Wolbachia densities using targeted metagenomics. The species analyzed included Naupactus dissimulator and Naupactus xanthographus (sexually reproducing), as well as Naupactus versatilis and Pantomorus postfasciatus (parthenogenetic), all of which were infected with the dominant strain within the tribe, i.e., wNau1. Notably, P. postfasciatus comprises both parthenogenetic (PpP) and sexually reproducing populations (PpS), providing unique insights into factors influencing Wolbachia colonization.

A total of 1,630,618 high-quality reads yielded 923 operational taxonomic units (OTUs) across 106 bacterial genera. The four most abundant genera, Wolbachia (26.47%), Spiroplasma (20.35%), Enterobacter (13.88%), and Rickettsia (11.49%), accounted for 72% of all reads. Parthenogenetic populations showed a marked predominance of Wolbachia and Rickettsia, whereas Spiroplasma and Enterobacter were the most prevalent symbionts in sexual populations, which also exhibited a richer microbiota in terms of the genera identified. Principal Coordinate Analysis based on Bray–Curtis distances clearly distinguished populations by reproductive mode. PpS’ bacterial microbiota differed significantly from both PpP and other sexually reproducing species, exhibiting a profile that combined features of both groups. The low relative abundance of Wolbachia, coupled with the increased levels of Enterobacter and Spiroplasma, distinguished it from parthenogenetic populations, while elevated Rickettsia levels set it apart from other sexually reproducing species. These observations suggest that Enterobacter and Spiroplasma may inhibit Wolbachia proliferation, whereas Rickettsia might promote its growth.

6.10. Photobioreactors for Atmospheric Carbon Dioxide Fixation (Liquid Trees): A Conceptual Approach Using Chlorella vulgaris Cultivation

Henry Adolfo Lambis Miranda ¹, Juliana Puello-Mendez ², Ildefonso Baldiris-Navarro ³, Jorgelina C. Pasqualino ⁴, Miguel Cuesta-Peña ⁵, Shirley Osorio-Herrera ⁵

¹ 

Processes and Systems Engineering, Fundación Universitaria Tecnológico Comfenalco, CIPTEC Research Group, Cartagena, Colombia

² 

GICI Research Group, Chemical Engineering Department, Universidad de San Buenaventura Cartagena, Cartagena, Colombia

³ 

Universidad de Cartagena, Departamento de Ing. Química, Campus Piedra de Bolívar, Cartagena, Colombia

⁴ 

GISAH Research Group, Environmental Engineering Program, Universidad Tecnológica de Bolívar, Campus Tecnológico, km 1 vía Turbaco Cartagena, Cartagena, Colombia

⁵ 

Processes and Systems program, Fundación Universitaria Tecnológico Comfenalco, Cartagena, Colombia

Liquid trees are photobioreactors cultivating microalgae such as Chlorella vulgaris and represent a promising innovation in the fight against environmental pollution. These systems leverage the photosynthetic capacity of microalgae to capture atmospheric carbon dioxide (CO₂), thereby contributing to the mitigation of climate change effects. Furthermore, the produced algal biomass can be utilized for diverse applications, ranging from biofuel production to animal feed.

Construction Methodology:

• Reactor: The core of the system is a reactor, typically a transparent glass or plastic vessel, which provides a controlled environment for microalgae cultivation, including adequate illumination, mixing, and aeration of the culture.
• Light Source: A solar panel serves as the primary energy source for the system. During daylight hours, the solar panel provides the necessary energy for microalgae photosynthesis. Additionally, a battery can store excess energy generated during the day to power the system at night.
• Aeration System: A compressor, powered by the solar panel, supplies CO₂-enriched air to the reactor. CO₂ is essential for the photosynthetic process and enables efficient microalgae growth.
• Control System: A control system, which can be electronic or mechanical, monitors and regulates culture parameters, such as temperature, pH, nutrient concentration, and light intensity.
• Recirculation Pump: A recirculation pump ensures a homogeneous distribution of nutrients and oxygen within the culture.

Expected Outcomes:

• Fix Large Amounts of CO₂: Microalgae, due to their high growth rate and photosynthetic efficiency, can capture significant amounts of atmospheric CO₂.
• Produce Algal Biomass: The produced algal biomass can be used for various applications, such as the production of biofuels, animal feed, fertilizers, and chemical products.
• Improve Air Quality: By removing CO₂ from the atmosphere, liquid trees improve air quality.
• Be Energy Self-Sufficient: Through the use of solar panels, liquid trees can operate autonomously, without requiring connection to the electricity grid.

6.11. Preliminary Genome Assembly of the Wolbachia Endosymbiont of the South American Fruit Fly Anastrepha fraterculus sp.

Claudia Alejandra Conte ¹, Máximo Rivarola ², Sergio Gonzalez ², María Romina Russo ¹, Alejandra Scannapieco ¹, Juan Pedro Wulff ³, Alfred M. Handler ⁴, Ioannis Ragoussis ⁵, Konstantinos Bourtzis ⁶, Silvia Beatriz Lanzavecchia ¹

¹ 

Instituto de Genética “E. A. Favret”, Instituto Nacional de Tecnología Agropecuaria (INTA)—Grupo vinculado al Instituto de Agrobiotecnología y Biología Molecular (IABIMO), Hurlingham, Buenos Aires, Argentina

² 

Instituto de Biotecnología IABIMO-CONICET, Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Buenos Aires, Argentina

³ 

North Carolina State University. NCSU, USA

⁴ 

U.S. Department of Agriculture, Agricultural Research Service, Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL 32608, USA

⁵ 

McGill Centre, Montreal, Canada

⁶ 

Insect Pest Control Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Seibersdorf, Austria

Wolbachia sp., an alphaproteobacterium associated with reproductive phenotypes, has previously been characterized by the fruit fly pest Anastrepha fraterculus (Diptera: Tephritidae). Two Wolbachia strains (wAfraCast1_A and wAfraCast2_A) have been identified using MLST and HVR as molecular markers. Mating assays have revealed a female-biased sex ratio in flies carrying wAfraCast1_A, while behavioral tests have suggested a protective effect against parasitoids in A. fraterculus larvae harboring wAfraCast2_A. Here, we assembled the wAfraCast2_A genome from a whole-genome project of A. fraterculus morphotype 1 (Argentina).

High-molecular-weight DNA was extracted from a single adult female, and Oxford Nanopore high-throughput genome sequencing was performed. After quality control and filtering of raw reads, a metagenomic approach was followed to obtain de novo assemblies of A. fraterculus and Wolbachia sp. genomes, and a Wolbachia-assigned contig was further analyzed. Quast and BUSCO were used to check the integrity and completeness of a preliminary Wolbachia genome assembly. Annotation was carried out with Prokka and compared to the reference genome of the Wolbachia endosymbiont of Drosophila melanogaster (wMel).

A total of 15,810,325 reads were obtained after adapter removal and quality filtering (10% of the reads were discarded). From these, a total of 1360 contigs were generated de novo, and 1 contig was assigned to the Wolbachia sp. taxon. A preliminary assembly of the wAfraCast2_A genome revealed a circularized contig of 1,463,312 bp with 93.1% completeness, 0% duplicated genes, and 6.9% missing genes (rickettsiales_odb10 database, n:364). A genome comparison with wMel showed an Average Nucleotide Identity of 99.1% and structural annotation identified 1,701 CDSs, including 633 genes, 907 hypothetical proteins, 3 rRNAs, and 34 tRNAs. These results will enhance our understanding of the Wolbachia–A. fraterculus interaction, including mechanisms influencing host behavior, physiology, and ecological functions, thereby supporting novel pest management strategies.

6.12. Proteomic Characterization of Legionella micdadei Membranes

Bożena Kowalczyk ¹, Jacek Tarasiuk ¹, Katarzyna Pastuszak ², Małgorzata Jurak ², Marta Palusińska-Szysz ¹

¹ 

Department of Genetics and Microbiology, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, 20-033 Lublin, Poland

² 

Department of Interfacial Phenomena, Institute of Chemical Sciences, Faculty of Chemistry, Maria Curie-Skłodowska University, 20-031 Lublin, Poland

Legionella spp. are pathogens of free-living protozoa that can cause a respiratory disease called legionellosis. Pneumonia caused by L. micdadei is mainly associated with hospital infections and primarily affects immunosuppressed patients. The infection of host cells by L. micdadei is facilitated by specialized mechanisms through which the bacteria modulate host cell physiology to create a replicative niche. Central to this process is the Dot/Icm Type IV secretion system (T4SS), complemented by the Type II secretion system (T2SS) and other host-manipulation strategies involving specific proteins.

This study aimed to analyze the composition of the outer (OM) and inner membrane (IM) proteins isolated from L. micdadei bacteria.

The bacterial mass collected from BCYE plates was digested with DNase and RNase and then disintegrated in a French press. A sucrose density gradient was used to isolate the outer and inner membranes of L. micdadei. Proteins isolated from these membranes were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS-MS). The L. micdadei proteome was investigated through diverse subtractive proteomics approaches, e.g., identification of pathogen-specific essential proteins, and draggability.

A detailed analysis of the L. micdadei membrane proteome allowed for the identification of 1784 proteins, including virulence factors, proteins associated with the transport of substances across cell membranes and the synthesis of cell membrane components, proteins related to bacterial mobility, and signal transduction pathways. The virulence factors present in L. micdadei membranes included protein building type IVB and II secretion systems, as well as major outer membrane protein (MOMP), macrophage infectivity potentiator (Mip), periplasmic protein EnhC, RND efflux system, outer membrane lipoprotein, the NodT family, and phosphatidylglycerol-prolipoprotein diacylglyceryl transferase.

The identification of L. micdadei membrane proteins and the elucidation of their functions provide deeper insights into the mechanisms underlying the pathogenesis of these microorganisms.

6.13. The Bacterial Microbiota of Knees Affected by Gonarthrosis

Alejandra de la Cruz Munguía ¹, Juan Manuel Vélez Ixta ¹, Tizziani Benitez Guerrero ¹, Mirna Liliana López Pérez ¹, Alberto Piña Escobedo ¹, Jossue Castañeda Garduño ², Jaime García Mena ¹

¹ 

Departamento de Genética y Biología Molecular, Cinvestav, Mexico City, Mexico

² 

Centro Médico ISSEMYM Toluca/Hospital Ángeles Lomas, Estado de México, México

Introduction: Gonarthrosis, or osteoarthritis located in the knee, is a joint disease that causes the tissues of the affected joint to progressively degenerate. When there is a wear away of the synovial membrane due to this disease, its permeability increases, causing bacteria to thrive. The objective of this study is to identify whether Gonarthrosis is associated with a microbiota profile dominated by pro-inflammatory bacteria in the synovial space.

Methods: Synovial fluid samples were extracted from 57 patients diagnosed with stage 4 Gonarthrosis and were subsequently stored at −80 C. The extraction of total DNA was carried out with a specific kit for microbiome measurement. Gene libraries were created by PCR of the V3–V4 region of the 16S ribosomal gene to subsequently characterize the diversity of the microbiota using next-generation sequencing. The analysis of the results was conducted using the QIIME version 2 pipeline together with the GreenGenes database for taxonomic assignment and the R programming environment to determine the comparison of the relative abundance of taxa.

Results: The results determined that more than half of the samples contained bacteria of the genera Blautia_A_141781, Bacteroides_H, Lacticaseibacillus, and Akkermansia, among others. This information reveals that there are bacteria in the synovial space, contributing to the field of research on microbiota in spaces that are considered sterile, such as, in this case, joints affected by the degenerative disease Gonarthrosis.

Conclusions: In conclusion, it was detected that there are bacteria in the synovial space in people with Osteoarthritis. This work was financed by CONACyT 163235 INFR-2011-01 and SECTEI/249/2019—CM-SECTEI/109/2020—CM-SECTEI/124/2021, Convocatoria 2019.

6.14. The Effect of Light and Temperature on the Accumulation of β-Carotene in Rhodotorula Yeasts

Regina Losinska-Sičiūnienė, Živilė Strazdaitė-Žielienė, Elena Servienė

• Nature Research Centre

Yeasts of the genus Rhodotorula have pink, orange, and red colors, indicating their ability to synthesize carotenoids. Due to their biological properties, carotenoids are widely used in the phytomedicine, chemical, pharmaceutical, cosmetic, food, and feed industries. One of the most important carotenoids is β-carotene, which exhibits interesting biological activities, such as antioxidant, anticancer, and antimicrobial. Rhodotorula yeasts were isolated from the natural environment and analyzed using morphological and molecular methods. Species were identified based on the restriction fragment length polymorphism analysis and sequencing of the rDNA internal transcribed spacer region (ITS). In this work, the β-carotene content of Rhodotorula yeasts was investigated when grown on a solid YEPD medium at different temperatures (4 °C and 26 °C) using various light intensities (8–10 µmol/m²s). The biomass was collected, and carotenoids were extracted using dimethylformamide (DMF). The amount of β-carotene was calculated based on absorption at 440 nm. The highest β-carotene contents were found in R. babjevae, R. glutinis, R. graminis, and R. kratovchilovae after 14 days of incubation at 26 °C in the dark. The level of β-carotene was 30 to 55% lower depending on the yeast species when the yeast culture after 2 days of cultivation at 26 °C was transferred to 4 °C, followed by 12 days of incubation in the dark. When yeasts were kept in the light at 4 °C, out of the eight yeast species tested, R. mucilaginosa and R. babjevae accumulated about 20–30% more β-carotene than under dark conditions. Our results show that yeasts that are cultured in the dark at 26 °C accumulate β-carotene better than yeasts that are held under the same conditions in the light. The synthesis and accumulation of β-carotene are decreased at a low temperature. The obtained data suggest that isolated Rhodotorula yeast species can be promising for carotenoid production under appropriate conditions.

7. Session: Microbe-Plant Interactions

7.1. Impact of Co-Inoculating Bradyrhizobium japonicum with Bacillus subtilis or Priestia megaterium on Nitrate Inhibition of Symbiotic Nitrogen Fixation in Soybean

Steven Orito, Norikuni Ohtake, Takuji Miyamoto

• Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan

Cultivated soybean is a vital source of protein and oil. Soybean obtains nitrogen (N) primarily from two sources: symbiotic nitrogen fixation (SNF) facilitated by microbes like Bradyrhizobium japonicum, and mineral nitrogen from soil or nitrate fertilizers. While nitrate fertilizers enhance plant growth, high nitrate levels inhibit nodule formation and SNF activity, reducing the proportion of nitrogen derived from SNF.

This study investigated the potential of nitrate-reducing bacteria (Bacillus subtilis and Priestia megaterium) to mitigate the nitrate-induced inhibition of SNF when co-inoculated with B. japonicum USDA 110 or USDA 6^T strains. Nitrogenase activity, nodulation, root elongation, and plant biomass were evaluated under hydroponic conditions at 1 mM and 10 mM nitrate levels, with nitrate assimilation analyzed using the ¹⁵N stable isotope. The growth responses of B. japonicum to varying nitrate concentrations were also examined. The growth of B. japonicum peaked at 7 mM nitrate but declined at 10 mM, suggesting inhibitory effects at higher concentrations.

Nitrogenase activity significantly decreased under high-nitrate conditions. However, co-inoculation with B. subtilis partially alleviated this inhibition, enhancing nitrogenase activity by 15.23% (USDA 110) and 72.62% (USDA 6^T) at 10 mM nitrate. Conversely, P. megaterium co-inoculation further reduced nitrogenase activity in some treatments. Nodulation was similarly inhibited by high nitrate levels but improved with the co-inoculation of B. subtilis under both nitrate conditions, while the effects of P. megaterium were strain-dependent. Root length and plant biomass responded positively to co-inoculation with both bacteria, although nitrate-induced reductions were observed in nodule and root N concentrations.

This study highlights the potential of B. subtilis to counteract the nitrate-induced inhibition of SNF and improve soybean growth under nitrogen-rich conditions. The findings provide insights into microbial interactions that could inform sustainable agricultural practices aimed at enhancing legume productivity in nitrogen-rich soils such as those found in intensively managed agricultural systems.

7.2. PLFA Analysis of Soil Microbial Communities: Key Insights from Organic Artichoke Cropping Systems

Savanah Senn ¹, Arianna Bozzolo ², Brianna Zimmerman ¹

¹ 

Los Angeles Pierce College Department of Agriculture Sciences, Plant Science program, Woodland Hills, CA 91371, USA

² 

Rodale Organic Institute, California Organic Center, Camarillo, CA 93010, USA

The effects of replacing polyethylene row covers in artichoke production with legume and non-legume cover crops were investigated at Rodale Organic Institute in Camarillo, CA. The treatments included plastic film mulching (PFM) and cover crops: Buckwheat, Kurapia, Crimson Clover, and White Clover with four replicates. While various studies have established the benefits of PFM versus bare soil and cover cropping versus bare soil in agriculture, few studies have directly compared plastic with legume and non-legume cover crops.

The phospholipid fatty acid (PLFA) test on soil provides a generalized community profile. Microbial biomass is measured; bacteria, fungi, and protozoans can be differentiated, along with subgroups such as Gram-positive and Gram-negative bacteria, saprophytes, and mycorrhizae. Functional Diversity Analysis is provided. The PLFA analysis was performed by Trace Genomics during year 2 post-production. Kruskal–Wallis tests were performed for the response variables to determine if cover cropping treatments were comparable to the control. A positive Kruskal–Wallis Chi-square test was followed up with the Dunn test used for multiple comparisons between groups, using the Holm correction.

There was a trend toward the plastic treatment having the lowest total bacteria biomass, the lowest fungi–bacteria ratio, lower levels of saprophytes, and less total microbial biomass in the plastic treatment. The results showed that the treatment was significant in determining the functional diversity index; plastic tended to have a lower functional diversity index than white clover (p = 0.015); the Dunn Test was negative (p-adj = 0.076). The treatment was significant in determining the mass of Arbuscular mycorrhizae in plots (p = 0.010). Arbuscular mycorrhizae biomass was different between the plastic and Kurapia treatments, according to the results (p-adjusted = 0.023).

The main result of the PLFA analysis showed that Arbuscular mycorrhizae biomass was significantly higher in the Kurapia treatments than the plastic controls. These results, combined with soil fertility analysis, will inform growers on best practices in organic artichoke cropping.

7.3. Harnessing Antioxidant-Producing Rhizobacteria to Boost Loquat Oxidative Stress Defense Under Adverse Environmental Conditions

Mian Muhammad Ahmed ^1,2, Pan Zhiyong ^3,4, Muhammad Asim ⁴, Guo Xingyu ², Hamid Syeda Maira ^1,5

¹ 

College of life Science and Technology, Tarim University, Alar, Xinjiang 843301, China

² 

National and Local Joint Engineering Laboratory for High-Efficiency and High-Quality Cultivation and Deep Processing Technology of Characteristic Fruit Trees in Southern Xinjiang China

³ 

College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, China

⁴ 

National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan 430070, China

⁵ 

Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, Alar, Xinjiang, China

Loquat (Eriobotrya japonica) is economically important, but abiotic stresses like drought, salinity, and severe temperatures are having an increasing effect on its productivity. Plant cells experience oxidative stress, which leads to the buildup of reactive oxygen species (ROS). The production of antioxidant enzymes by specific bacteria in the loquat rhizosphere can reduce ROS levels and possibly increase the plant’s resistance to these stresses. However, little is known about the processes by which these bacteria aid loquat in defending against oxidative stress. The purpose of this work is to identify and describe the bacterial strains linked to loquat that produce antioxidant enzymes and to clarify how they help loquat plants. After separating bacterial strains from the loquat rhizosphere, we examined their antioxidant enzyme activity, paying particular attention to the synthesis of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) under stress-like circumstances. To identify and examine the genes in charge of the synthesis of antioxidant enzymes and the control of stress responses, high-performing strains were chosen for in-depth characterization utilizing enzyme activity tests and molecular methods such as qPCR and genome sequencing. The chosen bacterial strains were administered to loquat seedlings, which were subsequently subjected to simulated oxidative stress, to evaluate their practical effects. To assess the effect on plant health, measurements were made of three important physiological indicators: electrolyte leakage, relative water content, and chlorophyll concentration. Furthermore, the amount of ROS in loquat tissues was measured, and qPCR was used to examine the expression of the plant’s own antioxidant genes. This study found that antioxidant-producing bacteria improved loquat’s oxidative stress tolerance, lowering ROS levels, increasing chlorophyll, and reducing electrolyte leakage, thus boosting resilience to oxidative damage. These findings pave the way for sustainable microbial inoculants to reduce oxidative stress in loquat farming, enhancing crop stability under tough conditions.

7.4. Plant–Microbial Interactions in the Phytosphere of Thyme (Thymus vulgaris L.)

Ekaterina Zharkova

• Core Shared Research Facility, Industrial Biotechnologies, Federal Research Center, Fundamentals of Biotechnology, Russian Academy of Sciences, Moscow 119071, Russian

Medicinal plants are one of the groups of economically important species and a valuable natural resource. One of these is thyme (Thymus vulgaris L.), the source of essential oil with high antimicrobial activity against microorganisms from various systematic groups. The study of thyme–microbial interactions was carried out in 2014–2024 for the collection of medicinal plants of the V. I. Edelstein Vegetable Experimental Station by cultural, chromatography, and NGS methods. The data obtained indicated the importance of the phytosphere’s physiological and biochemical features for microbial community structure and representation depending on the development stage, since these factors significantly affected the plant–microbial interaction in the thyme–soil system. In other words, the features of the studied plant–microbiological interaction depended on which microorganisms inhabited specific parts of the thyme phytosphere and at what stage of plant development this occurred. Moreover, for bacterial communities, the key roles were played by the features of the phenological stage of the plant (54.3%) and the colonized part (44.7%), while for fungi, the interaction of these two factors was the most significant (75.8%). For microbial biotechnologies, it is necessary to obtain cultured identified forms of microorganisms. So, typical representatives of microbial communities were isolated and sequenced. At least nine phytosphere microorganisms can be recommended as protective and growth-regulating agents for thyme cultivation in the Non-Chernozem zone due to the suppression of root rot pathogens that actively develop during thaws and high humidity conditions.

7.5. The Nematicide Dodecan-1-ol Has a Reduced Impact of on Plant Growth-Promoting Bacteria Compared to Conventional Pesticides

Ricardo Machado ¹, Paula Fareleira ^2,3, Jorge M. S. Faria ^2,3

¹ 

INIAV, Instituto Nacional de Investigação Agrária e Veterinária, Quinta do Marquês, 2780-159 Oeiras, Portugal

² 

INIAV, I.P., National Institute for Agrarian and Veterinarian Research, Quinta do Marquês, 2780-159 Oeiras, Portugal

³ 

GREEN-IT Bioresources for Sustainability, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB NOVA), Av. da República, 2780-157 Oeiras, Portugal

Conventional pesticides commonly used in modern agriculture pose significant risks to plant health by disrupting soil microbiota, including beneficial plant growth-promoting bacteria. These non-target effects contribute to long-term soil degradation and ecological imbalances, ultimately undermining plant productivity and sustainability. Consequently, there is an urgent need to identify safer and more sustainable pest management alternatives that support plant health. Volatile phytochemicals, known for their bioactivity against plant pests, offer a promising solution due to their reduced environmental impact and lower toxicity.

This study evaluated the toxicity of dodecan-1-ol, a volatile compound with nematicidal properties, on crop-associated bacterial species of importance for plant growth and soil health: Bacillus megaterium, Pseudomonas azotoformans, Paenibacillus zeisoli, and Burkholderia phymatum. Direct-contact bioassays in 96-well microplates were used to monitor bacterial growth spectrophotometrically. For comparison, the conventional pesticides emamectin benzoate and oxamyl were tested under identical conditions.

Dodecan-1-ol inhibited bacterial growth by a maximum of 20%, significantly lower than the 93% and 11% inhibition observed with emamectin benzoate and oxamyl, respectively. These results were obtained at a concentration of 0.02 mg/mL for all compounds. Importantly, at this concentration, dodecan-1-ol achieves its lowest maximal effective concentration (EC₁₀₀) against plant-parasitic nematodes, whereas emamectin benzoate and oxamyl exhibit limited nematicidal efficacy and require substantially higher application rates in agricultural settings.

By combining effective nematicidal activity with minimal impact on plant-beneficial bacteria, this study highlights the potential of dodecan-1-ol to support plant health while addressing pest control needs. These findings emphasize its role as a safer and more sustainable alternative to conventional pesticides, with promising applications in integrated pest and soil management strategies to promote healthy crop growth and long-term agricultural sustainability.

7.6. Unraveling the Bioremediation Potential of Arbuscular Mycorrhizal Fungi Through a Bio-Inoculation Approach

Ranjna kaundal, Vipin Parkash

• Forest Pathology Section, Forest Protection Division, ICFRE- Forest Research Institute, Dehradun, Uttarakhand-248006, India

Arbuscular Mycorrhizal Fungi (AMF) even exist in soils polluted by heavy metals and play an important role in improving plant tolerance under stress conditions. A pot culture experiment was carried out to study the effects of AMF inoculation on growth and Cadmium (Cd) uptake through soil enzymatic activities under varying levels of Cd concentrations (3, 6, and 9 mg kg⁻¹ of soil) in soil by Albizia lebbeck (L.) Benth. seedlings. AMF inoculation significantly improved both shoot and root biomass at a Cd concentration of 3 mg kg⁻¹, while a reduction in biomass was observed at 9 mg kg⁻¹ concentration of Cd. AMF also enhanced plant growth, with maximum shoot length observed at 3 mg kg⁻¹ and root length at 9 mg kg⁻¹. However, AMF spore count decreased while the root colonization rate significantly increased with Cd concentration, leading to a reduction in Cd levels in the rhizosphere of AMF-inoculated seedlings. Soil enzymatic activities like dehydrogenase, acid phosphatase, alkaline phosphatase, and catalase were significantly reduced in non-inoculated seedlings under Cd stress, whereas AMF inoculation improved these enzymatic activities. These results indicate that AMF can alleviate Cd toxicity by decreasing translocation and enhancing the bioaccumulation factor in Albizia lebbeck seedlings. Thus, AMF in combination with Albizia lebbeck offers a promising approach for the phyto-stabilization of Cd-contaminated soils. These findings also highlight the potential of the AMF–Albizia lebbeck interactions system in the bioremediation of Cd-polluted ecosystems.