Applied Microbiology doi: 10.3390/applmicrobiol6050062

Authors: Frank Fernandez-Rosillo Carlos Culqui-Arce Eliana Milagros Cabrejos-Barrios Katia Karlita Rodríguez Frias Jhuly Vanessa Pérez Gonzáles Nestor A. Sánchez-Goycochea Nilthon Arce Fernández Ralph Rivera Botanares Fredy Velayarce-Vallejos Diner Mori-Mestanza César R. Balcázar-Zumaeta

Temperature abuse during storage represents a critical factor influencing microbial behavior in meat products, particularly in non-conventional matrices such as guinea pig meat. This study aimed to characterize and compare the primary growth kinetics of Aerobic mesophilic bacteria and Staphylococcus aureus (S. aureus) in guinea pig meat burgers under controlled temperature abuse conditions (30, 35, and 40 °C). Microbial growth was monitored over 96 h and described using the modified Gompertz model to estimate key kinetic parameters, including maximum specific growth rate (µmax) and lag phase duration (λ). Aerobic mesophilic bacteria exhibited increasing µmax values with temperature, indicating enhanced metabolic activity under elevated thermal conditions. In contrast, S. aureus showed reduced µmax and prolonged λ at 40 °C, suggesting stress-induced modulation of growth dynamics. These findings demonstrate that temperature increases do not uniformly accelerate microbial proliferation across different populations within the same food matrix. The contrasting kinetic responses indicate that Aerobic mesophilic bacteria and S. aureus respond differently to temperature abuse conditions, highlighting that total aerobic counts alone may not reliably predict pathogen behavior in guinea pig meat burgers.

Applied Microbiology doi: 10.3390/applmicrobiol6050061

Authors: Jekaterina Martynova Kristiana Rozensteina Janis Liepins Agnese Kokina Armands Vigants

Kluyveromyces marxianus is a non-conventional yeast capable of efficiently metabolizing lactose, but acetate can inhibit its growth. Because K. marxianus strains differ physiologically, their tolerance to acetate also varies. Acetate tolerance was investigated in four K. marxianus strains grown on glucose, lactose, and an equimolar mixture of glucose and galactose. The inhibitory effect of 40 mM acetate on growth was evaluated at pH 4.5–6.0 by using acetate and citrate buffer systems. In lactose-containing media at pH 4.5, the strongest inhibition was observed in strain NCYC 2791, whose specific growth rate decreased from 0.51 ± 0.01 h−1 to 0.13 ± 0.01 h−1, while lag-phase duration increased from 10.11 ± 0.35 h to 21.09 ± 1.95 h. In contrast, DSM 5422 showed a smaller decrease in specific growth rate, from 0.54 ± 0.03 h−1 to 0.31 ± 0.06 h−1. NCYC 2791 also reached only OD600 = 0.18 after 45 h in acetate-containing lactose media, whereas the other three strains reached approximately OD600 = 0.6. The distribution of cytosolic and non-cytosolic β-galactosidase activity differed among strains, with the highest proportion of cytosolic activity in NCYC 2791 (80% of total activity). A significant positive correlation was found between the proportion of cytosolic β-galactosidase and the degree of growth inhibition by acetate at pH 4.5 (Pearson r = 0.9967, p = 0.0033). These findings suggest that strain-dependent β-galactosidase localization may be related to acetate tolerance, although this association should be interpreted cautiously because localization was not measured under acetate stress conditions.

Applied Microbiology doi: 10.3390/applmicrobiol6050060

Authors: Adrian Heckel Berke Ovat Jan Reichinger Nico Hanenkamp Andreas Burkovski

Water-miscible metalworking fluids are widely used in industrial processes. Despite the fact that they typically contain biocides, they are almost always colonized by microorganisms, which degrade different components of the liquid, may clog machines due to biofilm formation, and might pose a health risk to workers. In this study, samples from four metalworking machines operated with the same metalworking concentrate were analyzed with respect to microbial growth. Twenty-seven bacterial species and one fungus were identified. From these, twenty species were not observed before as colonizers of metalworking fluids. Growth of microorganisms, putative contamination sources, metabolic pathways involved in biodegradation, and resulting health risks are analyzed and discussed in this study.

Applied Microbiology doi: 10.3390/applmicrobiol6050059

Authors: Sujit Shah Biva Shah Gajanan Mane Mukti Ram Paudel Bijaya Pant Rohit Sharma Deepak Kumar

Background: Endophytic fungi are known for diverse bioactive compounds with immense potential for agriculture and medicinal applications. Coniochaeta dendrobiicola isolated from the roots of Dedrobium longicornu was investigated for its antioxidant and metabolite composition. The present study compares the antioxidant properties, flavonoid and phenolic contents and metabolic profiles of broth and mycelium extracts. The broth and mycelium extracts were tested for their antioxidant potential using DPPH, while the total flavonoid and phenolic contents were measured using a UV–VIS spectrophotometer. High-resolution mass spectrometry (HRMS) revealed a markedly richer and more diverse metabolite profile of putatively annotated compounds in the broth extract compared with the mycelium fraction. The broth extract exhibited significantly higher antioxidant activity and flavonoid and phenolic contents, correlating with the presence of diverse bioactive compounds, including indole derivatives, flavonoids, phenolic acids, quinoline derivatives, and antifungal metabolites. Notably, several indole-related and phenolic compounds detected predominantly in the broth are known for antioxidant, antimicrobial, and plant growth-promoting properties. These findings indicate that C. dendrobiicola actively secretes biologically relevant secondary metabolites into the extracellular medium, highlighting its potential for agricultural and pharmaceutical applications.

Applied Microbiology doi: 10.3390/applmicrobiol6050058

Authors: Nguyen Quoc Khuong Chau Ly An Nguyen Duc Trong Le Thanh Quang Le Thi My Thu Nguyen Phuong Van Do Thi Xuan

Diseases caused by Fusarium spp. vary around the world. It is important to determine the causals agents and indigenous antagonists against these pathogens. Thus, this study aimed to (i) determine the pathogens of root rot and yellow leaf disease (RRYLD), (ii) select Trichoderma spp. strains to control the pathogens, and (iii) evaluate methods for preparing the antagonistic fungi. Diseased soil samples were collected from pomelo orchards in Ben Tre province, Vietnam. The experiment isolated 08 Fusarium spp. strains, with the fastest growth in PDA in FP-C16, FP-B18, FP-B16, and FP-B03 (8.33–17.3 mm) on day 4 of culture. They were identified as Fusarium fujikuroi FP-C16, F. verticillioides FP-B18, F. verticillioides FP-B16, and F. incarnatum FP-B03. On the other hand, 25 Trichoderma spp. strains were isolated from the pomelo rhizosphere. Among them, 13 Trichoderma spp. strains showed rapid growth and strong antagonistic activity against two Fusarium spp. strains under laboratory conditions. The two Trichoderma spp. strains TP-C40 and TP-G50 had antagonistic efficiencies against FP-C16 and FP-B16 at 47.7–63.5%. The two selected Trichoderma spp. strains were identified as Trichoderma asperellum TP-C40 and T. yunnanense TP-G50. The two Trichoderma spp. strains TP-C40 and TP-G50 reduced the number of leaves and roots infected by Fusarium spp.

Applied Microbiology doi: 10.3390/applmicrobiol6050057

Authors: Kseniia V. Galkina Arina M. Adamovich Dmitry A. Knorre

Yeast harbour more than ten different multiple drug resistance (MDR) genes encoding transporters that extrude xenobiotics from the cytoplasm into the environment. These transporters, belonging to the ATP-binding cassette (ABC) or major facilitator superfamily (MFS), exhibit broad and significantly overlapping substrate specificities, though the precise boundaries of their individual substrate ranges remain undefined. During evolution, genes with overlapping functions tend either to specialize or to degenerate into pseudogenes. Here, we propose several explanations for how this apparent redundancy of MDR efflux pumps benefits cells, and we discuss the potential individual roles of the full MDR efflux pump repertoire in the model organism Saccharomyces cerevisiae. We posit that individual MDR transporters may vary in stability under challenging environmental conditions, in the energetic cost of their synthesis and maintenance, and in their degree of specialization toward particular classes of xenobiotics. Furthermore, given that ABC transporters and MFS transporters exploit distinct driving forces for xenobiotic efflux, each class may have its own vulnerabilities. We argue that deciphering the distinct roles of MDR proteins will reveal critical weaknesses in the MDR system and guide the development of strategies to overcome multidrug resistance in pathogenic fungi.

Applied Microbiology doi: 10.3390/applmicrobiol6050056

Authors: Phuoc-Vinh Nguyen Gia Phong Vu Luyen Tien Vu Luong Hieu Ngan Minh-Tri Le Thu-Hoai Le Nhat-Thong Le Linh X. T. Tran Bac V. G. Nguyen

The rapid emergence of multi-drug resistant (MDR) bacteria has become a major health concern, driving the need to identify new antimicrobial resources. Recently, endophytes, inhabiting in internal tissues of medicinal plants, have drew important interest from the scientific community, as reservoirs of bioactive metabolites. Numerous studies highlight the symbiotic relationship between plants and their endophytes, in which these microorganisms produce antimicrobial compounds, helping the host plant’s defense against pathogens. Plantago major (commonly known as plantain) is widely recognized for its therapeutic properties, especially for its antimicrobial properties. In this study, endophytic fungi were isolated from Plantago major, morphologically characterized and identified using ITS sequencing. Their antibacterial activity was assessed using the agar diffusion assay. In total, 21 endophytic fungal isolates were obtained from different plant tissues, including leaves, stems, roots, and flowers. Antibacterial assays against methicillin-resistant Staphylococcus aureus (MRSA) were investigated on PDA, SDA, and CDA media. Amongst the isolates, nine strains (MD-H1, MD-L1, MD-L2, MD-L3, MD-L4, MD-L5, MD-R1, MD-T1, MD-T2, and MD-T10) showed medium to strong antibacterial effects, with inhibition zones exceeding 15 mm. The result suggests that endophytic fungi associated with Plantago is a valuable source of anti-MRSA compounds. Further work will focus on identifying the secondary metabolites responsible for this activity and elucidating their chemical structures, providing a basis for the development of new potent antibiotic agents.

Applied Microbiology doi: 10.3390/applmicrobiol6040055

Authors: Nokhanyo G. Mbewana-Ntshanka Titus A. M. Msagati Thabo I. Nkambule Bhekie Mamba Rian. R. E. Pierneef Awelani Mutshembele

Antimicrobial resistance (AMR) has become one of the top ten global public health threats. Many countries have recognized the societal and economic burden of AMR. AMR has reduced the effectiveness of antimicrobial therapies, and this results in high mortality, morbidity, and health care expenditure. Like all the other developing countries, South Africa (SA) falls under the same ambiguous management system of antimicrobials. A lot of research focused on the global public health threat “AMR”. However, studies on AMR in wastewater are not yet enough, even though they are beginning to gain momentum. This paper highlights the imperatives of surveying AMR pathogens in wastewater since wastewaters are consecrated as hotspots for the dissemination and propagation of AMR genes. RNA was extracted from the untreated wastewater samples collected from the Tshwane district in Gauteng province, SA. Metatranscriptomics analysis was proposed for the analysis and profiling of AMR genes present in the wastewater. A total of 39 AMR gene families and 39 AMR drug classes were detected across 17 samples. The Metatranscriptomics approach discussed in this paper demonstrates the importance of wastewater surveillance, as it can be used as an early detecting system for communicable diseases and for monitoring wastewater.

Applied Microbiology doi: 10.3390/applmicrobiol6040054

Authors: Martine Denis Stéphanie Bougeard Virginie Allain Mélanie Guy Emmanuelle Houard Arnaud Felten Jean Lagarde Benoit Gassilloud Evelyne Boscher Pierre-Emmanuel Douarre

This study evaluated the performance of Fourier-transform infrared (FTIR) spectroscopy for identifying spectral signatures associated with two key traits of Listeria monocytogenes: serogroup classification and biofilm-forming capacity. A total of 100 strains, previously serogrouped by PCR and categorized as high, intermediate, or low biofilm producers, were analyzed. Whole-genome sequencing was performed, and comparative genomics was conducted at core-genome, pangenome, and whole-genome (k-mer) levels to determine which genomic representation best reflected the phenotypes. Strains were typed using Fourier-Transform Infrared (FTIR Biotyper® system from Bruker Daltonics GmbH and Co., Bremen, Germany) with five technical replicates. Spectral data from the polysaccharide region (1300–800 cm−1) were extracted and used to train twelve statistical models within a machine learning pipeline combined with cross-validation to predict four serogroups and three biofilm clusters from 501 spectral variables. Genomic analyses showed strong concordance between population structure and serogroup, whereas biofilm formation displayed only weak genomic association, explaining less than 0.1% of genomic variance (PERMANOVA R2 ≤ 0.001). Penalized discriminant analysis achieved the highest performance for serogroup prediction (overall accuracy 97.2%), while the k-nearest neighbor model performed best for biofilm prediction (74.8%). Two dedicated R Shiny applications were developed to facilitate model use. Overall, FTIR spectroscopy coupled with machine learning can provide a rapid and cost-effective alternative to PCR, genomic analyses, and in vitro assays for phenotypic trait prediction.

Applied Microbiology doi: 10.3390/applmicrobiol6040053

Authors: Maja Šikić Pogačar Mia Pogačar Sabina Fijan

Clostridium butyricum is a well-known Gram-positive, spore-forming, obligate anaerobic, and butyrate-producing bacterium with a few species of next-generation probiotic strains. By far, the most well-known strain is Clostridium butyricum CBM588 (also known as MIYAIRI 588). This strain has gained significant attention for its therapeutic potential across a variety of human health conditions. Preclinical studies have shown its ability to stabilize gut microbiota, enhance short-chain fatty acid (SCFA) production, and modulate immune responses, which contribute to its therapeutic effects in conditions such as ulcerative colitis, allergies, and cancer. We examined 28 interventional clinical trials and 7 observational studies investigating the effect of Clostridium butyricum strains. These studies have supported the findings of preclinical trials and demonstrated symptom improvement and immune modulation in diverse conditions. Clostridium butyricum CBM588 has shown efficacy in managing gastrointestinal diseases, such as acute gastroenteritis and inflammatory bowel disease, and has also proven beneficial in immune modulation, as evidenced by its positive effects in allergic rhinitis and cancer immunotherapy. Additionally, CBM588 has been reported to have a favorable safety and tolerability profile in various patient populations, including children, adults, and critically ill patients. Despite these promising results, clinical studies face limitations such as small sample sizes, varied protocols, and short study durations. Future well-designed, large-scale trials are necessary to further validate the long-term safety and efficacy of Clostridium butyricum in clinical practice.

Applied Microbiology doi: 10.3390/applmicrobiol6040052

Authors: Fatima Hamadeh Thibaut Armel Chérif Gnimadi Mano Joseph Mathew Charbel Al-Bayssari Mounir Kassir Rana El Hajj Dalia El Badan

Synthetic dyes, such as methylene blue (MB), constitute a major category of environmental pollutants due to their toxicity, persistence, and resistance to standard treatment methods. In this study, Bacillus cereus BC WW Saida was isolated from the heavily polluted Saida dumpsite in Lebanon and evaluated for its MB degradation efficiency. The isolate was identified through whole-genome sequencing, which revealed the presence of key enzymatic systems involved in azo dye degradation. Under optimized conditions, the strain achieved 82% decolorization, as determined by optical density measurements using a microplate reader. The process was further examined using High-Performance Liquid Chromatography (HPLC), which revealed a significant reduction in the original dye peak and the emergence of new intermediate products. These findings suggest the strong biodegradation capability of B. cereus BC WW Saida isolated from contaminated environments and highlight its potential application in the eco-friendly treatment of azo dye-contaminated wastewater.

Applied Microbiology doi: 10.3390/applmicrobiol6040051

Authors: Afrah I. Waheeb Saleem Obaid Gatia Almawla Mayada Abdullah Shehan Sameer Ahmed Awad Mohammed Mukhles Ahmed Saja Saddallah Abduljaleel

Background: The ability of synthetic plastics to persist in the environment and the accumulation of microplastics has intensified the need to explore biological mechanisms capable of interacting with, and possibly degrading, polymeric materials. Microbial enzymes that have extensive catalytic flexibility represent promising candidates in this context. Aim: This study set out to examine the molecular interaction patterns and dynamical stability of Pseudomonas aeruginosa elastase (LasB) with representative structural fragments of typical synthetic plastics to assess the suitability of the enzyme to polymer-derived substrates. Methods: The crystallographic structure of LasB (PDB ID: 1EZM) was retrieved from the Protein Data Bank and pre-prepared with the help of AutoDock4.2.6 Tools. Those polymer-derived ligands that were associated with the major industrial plastics such as polyamide (PA), polyvinyl chloride (PVC), polycarbonate (PC), poly-ethylene terephthalate (PET), polymethyl methacrylate (PMMA), and polyurethane (PUR) were retrieved in the PubChem database and geometrically optimized with the help of the MMFF94 force field. AutoDock Vina, with a specific grid box around the catalytic pocket, including Zn2+ ion, was used to perform molecular docking simulations. PyMOL and BIOVIA Discovery Studio software were used to analyze binding conformations, interaction residues and types of intermolecular contacts. Phosphoramidon, a known metalloprotease inhibitor, served as a positive control to confirm the docking protocol. Additional assessment of the structural stability and conformational behavior of the enzyme–ligand complexes was conducted by molecular dynamics (MD) simulations with the Desmond engine and explicit solvent model in a 50 ns trajectory using the OPLS4 force field. RMSD, RMSF, radius of gyration, hydrogen bonding analysis and solvent accessibility parameters were used to measure structural stability. Results: The docking experiment showed varying binding affinities with the test polymers. Polycarbonate (−5.774 kcal/mol) and polyurethane (−5.707 kcal/mol) had the highest in-teractions with the LasB catalytic pocket, polyamide (−5.277 kcal/mol) and PET (−4.483 kcal/mol) followed PMMA and PVC, which had weaker affinities. The following were the important residues involved in interaction networks: Glu141, His140, Val137, Arg198, Tyr114, and Trp115 that were implicated in interaction networks with hydrophobic interactions, π-cation interactions and van der Waals forces that were the major stabilization forces. MD simulations had stabilized complexes, and RMSD values were found to be within acceptable ranges of stability, and ligand-specific changes (around 1.0-3.2 A), which is also in line with stable protein-ligand systems. Phosphoramidon used as a positive control had an RMSD of 1.205 A which is within this stability range. PCA determined various ligand-bound conformational states of LasB with PA in com-pact state, PC and PVC in intermediate states and PUR, PMMA and PET in ex-panded conformations, indicating structur-al stability and adaptability of the binding pocket. Conclusion: These findings show that LasB has a structurally flexible catalytic pocket that can accommodate a wide range of polymer-derived ligands. These results offer an insight into the recognition of enzymes with polymers at the molecular level and also indicate that LasB might help in the interaction of microorganisms with synthetic plastics in environmental systems.

Applied Microbiology doi: 10.3390/applmicrobiol6040050

Authors: Valery M. Dembitsky Alexander O. Terent’ev Sergey V. Baranin

Siderophores are high-affinity iron-chelating metabolites that underpin microbial survival in iron-limited environments and play central roles in metal homeostasis, ecological competition, and pathogenesis. Traditionally viewed as dedicated Fe(III) scavengers, siderophores are now recognized as structurally and functionally versatile coordination agents whose donor-set architectures—particularly catecholate and α-hydroxycarboxylate motifs—permit conditional interactions beyond iron. In iron- and boron-rich niches, especially marine and mildly alkaline systems where borate availability increases, certain siderophores are chemically capable of forming reversible borate complexes through cis-diol coordination. Although Fe(III) exhibits substantially higher thermodynamic affinity and remains the primary biological target, boron binding represents a predictable secondary property arising from shared oxygen-donor chemistry. This dynamic interplay allows siderophores to cycle between iron-bound, boron-bound, and apo states depending on local redox conditions, pH, and metal availability. Here, we synthesize current knowledge on the structural classes of microbial siderophores, their transport and regulatory mechanisms, and emerging evidence for boron coordination within catecholate and carboxylate systems. By integrating coordination chemistry with microbial ecology, we propose an expanded model in which siderophores function not only as iron acquisition molecules but also as modulators of boron speciation and environmental sensing. This functional plasticity positions siderophores at the intersection of iron and boron biogeochemical cycles and highlights new directions for understanding microbial adaptation in complex metal-rich environments.

Applied Microbiology doi: 10.3390/applmicrobiol6040049

Authors: Sinethemba H. Yakobi Uchechukwu U. Nwodo

Quorum sensing (QS) represents a promising target for anti-virulence therapy; however, effective pharmacological intervention requires a detailed understanding of regulatory network architecture and environmental context. In Klebsiella pneumoniae, the orphan LuxR-type receptor SdiA lacks a cognate LuxI synthase and instead detects exogenous acyl-homoserine lactones (AHLs), positioning it as an inter-species signal integrator. Here, we demonstrate that SdiA functions as a context-dependent regulator whose impact on biofilm formation and virulence gene expression is gated by environmental AHL availability. Using isogenic ΔluxS, ΔsdiA, and ΔluxSΔsdiA mutants in a clinical bloodstream isolate, we show that under AHL-limited conditions, SdiA promotes baseline biofilm development, whereas in the presence of exogenous C6-HSL, it restrains excessive biofilm maturation. Two-way ANOVA confirmed significant genotype, treatment, and interaction effects, establishing that SdiA-mediated regulation is signal contingent. We further investigated the halogenated thiolactone meta-bromo-thiolactone (mBTL), previously described as a QS inhibitor in Pseudomonas aeruginosa. In K. pneumoniae, mBTL acts as a context-selective modulator rather than a simple inhibitor. Under AHL-limited conditions, mBTL phenocopied ΔsdiA, reducing biofilm formation and inducing overlapping transcriptional profiles. In contrast, under AHL-replete conditions, mBTL opposed SdiA-dependent gene expression, consistent with competitive antagonism of ligand-bound receptor. RNA-seq analysis revealed substantial concordance between ΔsdiA and WT + mBTL under AHL-free conditions, with the inversion of transcriptional directionality in the presence of C6-HSL. The findings redefine SdiA as a conditional quorum-sensing integrator and identify mBTL as a ligand-context-dependent modulator of LuxR-type signaling. Our results highlight the necessity of evaluating anti-virulence compounds across relevant signal environments and introduce receptor state-selective modulation as a strategic framework for targeting hybrid quorum-sensing systems in polymicrobial pathogens.

Applied Microbiology doi: 10.3390/applmicrobiol6040048

Authors: Henry Paniagua González Guadalupe Guzmán Barboza José Bolaños Jiménez Melissa Moya Granados Vanessa Bagnarello Madrigal

Helicobacter pylori is a highly prevalent pathogen associated with chronic gastritis, peptic ulcers, and gastric cancer. Treatment is increasingly challenging due to antibiotic resistance and adverse effects that can reduce adherence. These limitations have encouraged the exploration of complementary strategies. This study evaluated the in vitro antibacterial activity of selected probiotic strains and synbiotic formulations containing inulin against clinical isolates of H. pylori. Isolates obtained from gastric biopsies were identified by MALDI-TOF. Four probiotic strains (Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, and Streptococcus thermophilus) were tested individually and as a mixed culture, both alone and combined with inulin. Antibacterial activity was assessed using the agar well diffusion method under microaerophilic conditions after 72 h of incubation at 37 °C. Variable inhibitory effects were observed, with L. fermentum (8.08 ± 1.98 mm) and the probiotic mixture (7.92 ± 0.90 mm) showing greater activity, while S. thermophilus exhibited limited inhibition. The addition of low-dose inulin (3 mg/mL) was associated with increased inhibition by the probiotic mixture (9.58 ± 1.51 mm), whereas higher concentrations did not enhance this effect. These findings indicate that certain probiotic and synbiotic formulations exhibit in vitro activity against H. pylori and warrant further investigation as complementary approaches.

Applied Microbiology doi: 10.3390/applmicrobiol6030047

Authors: Ayaz Ahmad Mian Muhammad Ahmed Aadab Akhtar Chen Shuihong Zeeshan Zafar Rehmat Ullah Muhammad Asim Zhenli He Muhammad Bilal Khan

Fungal endophytes are microorganisms that inhabit plant tissues without causing disease and emerge as critical mediators of plant stress tolerance, nutrient acquisition, and ecosystem resilience under diverse climate change scenarios. Their unique position within the host allows them to modulate physiological responses more closely than external microbiota. This review explores how endophytic fungi contribute to plant adaptation under climate-induced stresses such as heat, salinity, drought, pollution, and nutrient limitation, with a focus on molecular crosstalk, functional trait modules, and metabolic trade-offs. Key findings emphasize multilayered signaling systems, including MAMP/DAMP recognition, phytohormone regulation, immune tuning, ROS dynamics, and effector deployment, while emerging mechanisms such as cross-kingdom RNA and extracellular vesicle (EV)-mediated exchange are discussed as promising but currently limited in empirical validation within many endophytic systems. Endophytes also enhance nutrient exchange through conditional carbon-for-benefit trade and may shape rhizosphere microbiota and soil activities through plant-mediated inputs. Integrative multi-omics approaches provide predominantly correlational insights into the mechanistic basis of these effects, linking molecular function to ecosystem and community outcomes. These insights have potential applications in climate-resilient agriculture, phytoremediation, and ecosystem restoration; however, their large-scale implementation requires further field-based validation and context-specific assessment. Future priorities should focus on trait-based selection, ecological modeling, and biosafety evaluation to translate microbial functions into reliable field-level strategies that support sustainable crop performance under accelerating environmental stress.

Applied Microbiology doi: 10.3390/applmicrobiol6030046

Authors: Tom E. Schierling Ralf T. Voegele Abbas El-Hasan

Neopestalotiopsis rosae is an emerging fungal pathogen that causes leaf blight and fruit rot on strawberry. Due to limited fungicide availability and the small number of substances confirmed to be effective against this pathogen, alternative disease control strategies have become a focus of current research. This study aimed to assess, quantify, and compare the efficacy of extracts and inocula of Trichoderma spp. with the conventional fungicide Switch in controlling N. rosae. In the presence of T. harzianum T16 and T. asperellum T23 extracts, conidia production of N. rosae was reduced by 45.0% and 62.7%, respectively. Extracts of T. koningiopsis T10 strongly inhibited both mycelial growth and conidia production (>92.0%), demonstrating efficacy comparable to that of the reference fungicide. Furthermore, T. koningiopsis T10 extracts were able to inhibit N. rosae conidia viability by 55.6%. Under greenhouse conditions, strawberry plants treated with extracts from T. koningiopsis T10 showed protection from N. rosae leaf spots at levels similar to Switch. These findings highlight T. koningiopsis T10 extracts as a promising alternative to chemical fungicides in the integrated management of N. rosae on strawberry.

Applied Microbiology doi: 10.3390/applmicrobiol6030045

Authors: Abdelhak Menasri Rosario Lucas Javier Rodríguez López Antonio Gálvez Mª José Grande Rubén Pérez Pulido

Celiac disease (CD) is an autoimmune disorder triggered by immunogenic gluten peptides that resist gastrointestinal digestion. The only current treatment is a strict gluten-free diet, which is challenging to maintain. Lactic acid bacteria (LAB) with specific proteolytic systems offer a promising strategy for gluten hydrolysis and potential reduction of immunogenicity. This study aims to isolate and characterize gluten-degrading LAB from traditional Spanish and Algerian dairy products. A total of 27 artisanal dairy samples were collected. LAB were isolated on MRS and Elliker agar. Gluten-degrading activity was screened using a well diffusion assay with cell-free supernatants and a spot assay with live cultures. Active isolates were identified by 16S rRNA gene sequencing. Out of 123 isolates, 40 (32.5%) were positive in the well assay, while 67 (54.5%) were positive in the spot assay, indicating the latter’s higher sensitivity for detecting cell-associated proteases. Halo diameters ranged from 6 to 16 mm. Algerian isolates exhibited significantly stronger activity (mean halo: 12.6 ± 2.1 mm) compared to Spanish isolates (10.2 ± 2.0 mm; p < 0.001). Molecular identification of the 32 most active isolates revealed the following dominant species: Lactiplantibacillus plantarum, L. pentosus, Levilactobacillus brevis, and Enterococcus faecium. This study confirms that artisanal dairy fermentations are rich sources of LAB with robust gluten-degrading potential. The superior activity of Lactiplantibacillus spp. aligns with their complex peptidase systems. The geographical variation highlights the influence of local fermentation practices. Selected strains represent excellent candidates for developing adjunct cultures to produce gluten-reduced foods and warrant further investigation as potential probiotics, pending safety and efficacy validation in vivo and in clinical studies.

Applied Microbiology doi: 10.3390/applmicrobiol6030044

Authors: Benedict Ndou Beauty-Ben Baloyi Nokufa Morrieson Mabona Charity Masilela Bonisiwe Bhiya Matsobane Godfrey Tlou

Bacterial endophytes isolated from medicinal and wild plant species have recently gained significant attention for their medicinal properties, often closely linked to those of their plant hosts. This study identified two endophytic Bacillus isolates using 16S rRNA sequencing-based phylogeny. The impact of sublethal concentrations (0.5 mg/mL) of cadmium and hydrogen peroxide on metabolite production and bioactivity was also investigated. Phytochemical testing and antimicrobial and antioxidant assays revealed shifts in metabolite production under stress conditions. According to the phylogenetic analysis, Bacillus sp. NV35 and NV1 are respectively related to Bacillus cereus and B. mycoides. Phytochemical screening of methanolic crude extracts from both isolates tested positive for alkaloids, flavonoids, and saponins. Notably, tannins were detected only after cadmium treatment, while steroids were present following exposure to both cadmium and H2O2. LC-MS fingerprinting confirmed the presence of several tannins and steroids in treated samples. The untreated crude extracts exhibited an IC50 of ~3 mg/mL with the DPPH assay, which decreased to ~1.5 mg/mL after treatment with cadmium or H2O2, demonstrating enhanced antioxidant potential under stress conditions. Additionally, extracts from both treated and untreated bacteria displayed antimicrobial activity against selected bacterial pathogens, with MIC values ranging from 62.5 μg/mL to 125 μg/mL. LC-MS analysis identified various antimicrobial and antioxidant metabolites, including phenoxymethylpenicilloyl, maculosin, (S,R,S)-alpha-tocopherol, 3-indoleacrylate, procyanidin A2, cis-11-eicosenamide, 3-hydroxy-3-phenacyloxindole, and 9-octadecenamide.

Applied Microbiology doi: 10.3390/applmicrobiol6030043

Authors: Baby Lyn T. De Guzman Maria Luisa T. Mason Pariyanuj Chulaka Pechrada Pinjai

Microbial biofertilizers offer a sustainable alternative to reduce inorganic fertilizer inputs in intensive vegetable production. While rhizobia are traditionally associated with legumes, their co-inoculation with native rhizobacteria for non-leguminous crops like tomatoes remains under-explored. This study aimed to isolate native rhizobacteria compatible with Bradyrhizobium diazoefficiens NE1-65 and evaluate their combined effect on the tomato plant (var. max F1) under reduced inorganic fertilizer rates. From the initial eighteen isolates screened on nitrogen-free media, and solubilization assays of phosphorus and potassium, three isolates (RM-8, RM-17, RM-18) were found compatible with B. diazoefficiens NE1-65. Isolate RM-17 (tentatively identified as Aureimonas sp. based on 16S rRNA gene sequence) was selected for its high K-solubilizing capacity (KSI = 8.60). Then, a 90-day growth trial compared various fertilizer application rates (0, 25, 50, 75, and 100%) with and without the bacterial consortia. The 75% fertilizer rate plus the consortia significantly outperformed the 100% fertilizer rate alone. Specifically, it increased plant height (11.57%), fruit diameter (9.23%), fruit number (53.90%), and fruit weight (16.15%). These findings demonstrate that the RM-17 and B. diazoefficiens NE1-65 consortia can partially substitute inorganic fertilizers while significantly enhancing tomato growth and yield, highlighting its potential application for sustainable tomato production systems.

Applied Microbiology doi: 10.3390/applmicrobiol6030042

Authors: Gabriel Martín

Well established long ago for bacterial and fungal detection, Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) technique is not so well established in the virology field, and taking care of its advantages (speed, precision and low cost), this can be a powerful method for viral identification. To explore the feasibility and potential of MALDI-TOF for viral detection, this study shows the development of an in-house spectral library including several uninfected cell cultures and cultures infected with different clinically relevant viruses, such as SARS-CoV-2. This library was applied to the identification of viral infections directly on cell cultures, assessing the ability of the technique to discriminate between infected and non-infected profiles. Additionally, bioinformatic analyses were conducted to evaluate the structure, specificity, and reproducibility of the in-house library, and to understand its strengths and limitations. Sensitivity and specificity of the method were estimated by testing multiple culture batches from selected viruses included in the library. Together, these results provide a deeper understanding of the performance and applicability of MALDI-TOF in the virological context, highlighting its potential as a valuable research platform and a prospective tool for clinical viral detection.

Applied Microbiology doi: 10.3390/applmicrobiol6030041

Authors: Muhammad Usman Nadeem Najam Naveed Fatima Murtaza Maryam Ali Izzah Shahid

The excessive use of chemical fertilizers has depleted agricultural soils, necessitating a paradigm shift toward eco-friendly alternatives such as plant-beneficial microbes. However, the integration of plant-beneficial bacteria into global agroecosystems requires strategic and comprehensive analyses, as well as the development of optimally designed bioinocula to maximize their benefits. In this study, twenty-one rhizobacteria isolated from the maize rhizosphere were systematically screened for plant-beneficial traits, including phosphate and zinc solubilization, indole-3-acetic acid (IAA) production, and the synthesis of extracellular hydrolytic enzymes, followed by their evaluation for plant growth promotion. Among all bacterial isolates, Pseudomonas sp. NCR2 displayed the most comprehensive plant growth-promoting traits. In a pot-scale experiment, maize plants inoculated with multifaceted Pseudomonas sp. NCR2 showed significantly increased root growth, chlorophyll, soluble proteins, and phenolic contents as compared to untreated plants. This study underscores the significance of systematic screening of host-adaptive rhizobacteria for developing promising and tailored bioinocula. Furthermore, the results of this study also demonstrate the use of multifunctional biofertilizing inoculum for the systematic decrease of chemical inputs while simultaneously maintaining the crop productivity.

Applied Microbiology doi: 10.3390/applmicrobiol6030040

Authors: Kiran Gurung Bregje Wertheim

Pest insect-associated microbes display great phenotypic and genotypic diversity, with many members inhabiting broader ecological niche. Several of these bacteria are ubiquitous in nature and contribute to fruit spoilage. When microbes occur in both environmental niches and insect hosts, their ability to adapt to diverse substrates may facilitate their ecological success. This study focuses on characterization of the metabolic capability of three bacterial isolates belonging to the genera Acetobacter and Pantoea associated with Drosophila suzukii collected in the Netherlands. Carbon utilization patterns and tolerance to environmental stressors were assessed under varying conditions of salinity, pH, and antibiotics. The isolates differed in their metabolic profiles but collectively demonstrated the capacity to utilize a wide range of carbon sources. In addition, they exhibited tolerance towards different chemicals including salt and antibiotics. The metabolic flexibility of bacteria associated with D. suzukii may facilitate their persistence within fruit environments and contribute to host ecology. Overall, this study provides functional insight into insect-associated bacteria and underscores the importance of metabolic characterization in understanding their ecological significance.

Applied Microbiology doi: 10.3390/applmicrobiol6030039

Authors: Jack W. Sample Matteo Redaelli Jun Chen Tanya L. Hoskin Stephen Johnson Marina Walther-Antonio Tina J. Hieken

(1) Background: Mammographic breast density (MBD) is a well-established predictor of breast cancer risk, yet the biological mechanisms underlying this association remain incompletely understood. MBD is characterized by alterations in breast stromal architecture, including increased collagen deposition and changes in immune cell composition. Given emerging evidence that the breast harbors a resident microbiome, we investigated whether the breast tissue microbiome correlates with MBD. (2) Methods: Adjacent normal breast tissue was collected under sterile conditions from 33 women undergoing surgery for benign or malignant breast disease. DNA was extracted and subjected to 16S rRNA gene sequencing (Illumina MiSeq). (3) Results: We observed a non-significant trend toward lower α-diversity in high-MBD samples compared to low-MBD samples, p = 0.13. β-Diversity analyses identified a modest association between MBD and microbial community composition (MiRKAT p = 0.049). A random forest-based model incorporating genus-level relative abundances improved prediction of MBD over clinical characteristics alone, identifying Corynebacterium (Actinobacteria) and other genera as key predictors. (4) Conclusions: Breast tissue microbial features vary with mammographic breast density, suggesting a potential association with density-associated breast cancer risk. These exploratory findings warrant validation in larger cohorts to better elucidate biological mechanisms and clinical relevance.

Applied Microbiology doi: 10.3390/applmicrobiol6030038

Authors: Varsha Bohra Wang-Hei Wong Chun Au-Yeung Kit-Ling Lam Emily Sze-Wan Wong Steven Jing-Liang Xu Fred Wang-Fat Lee Wing-Yin Mo

Prophylactic antibiotic use in high-density ornamental aquaculture aims to mitigate infections, yet it is hypothesized to induce severe gut microbiome dysbiosis, contributing to high post-purchase mortality of goldfish purchased from retail stores by end consumers. This study utilized 16S rRNA gene amplicon sequencing, a rapid and high-resolution tool to characterize gut bacterial communities in six goldfish (Carassius auratus) sourced from antibiotic-intensive retail market in Hong Kong SAR, China. Diversity metrics were compared to unexposed reference controls and experimentally antibiotic-exposed cyprinid groups from published datasets. Market-sourced goldfish showed a profound collapse in alpha diversity (mean Shannon index 0.107 ± 0.141), far lower than controls (typically 2.0–4.5) and experimental groups (1.06–4.34). The microbiota exhibited extreme oligodominance by Cetobacterium and Vibrio, with near-total loss of beneficial commensal taxa. Principal coordinates analysis (PCoA) revealed distinct clustering, indicating fundamental and likely irreversible microbial restructuring. These findings show that chronic antibiotic exposure in ornamental supply chains induces a depauperate microbiome state, compromising host resilience and physiological homeostasis during environmental transitions. This dysbiosis provides a microbiological explanation for widespread post-purchase die-off, highlighting a major animal welfare and biosecurity concern. High-throughput sequencing offers quick, in-depth microbiome health assessment, essential for developing interventions to improve husbandry and reduce antimicrobial reliance in the global ornamental fish trade.

Applied Microbiology doi: 10.3390/applmicrobiol6030037

Authors: Nikola Atanasov Denis Borisov Yana Evstatieva Dilyana Nikolova

The human oral cavity contains a variety of habitats, all of which are colonized by microorganisms. Oral bacteria form multi-genera communities, exhibiting adhesive properties, both to oral tissue surfaces and to each other. In certain conditions, these properties represent the first step towards the development of oral diseases. The oral microbiome undergoes changes in its composition, which can alter the balance between health and disease and is dynamically interconnected with the host. Probiotics with a targeted effect on the oral cavity can successfully compete with pathobionts and increase the presence of beneficial bacteria, thus contributing positively mainly to the prevention of oral diseases. The application of probiotics to maintain balance of oral microbiota has been a subject of intensive research. Oral health products containing lactic acid bacteria represent a modern approach to prevent or reduce the level of infections in the oral cavity. The application of these products is an alternative and promising way to prevent diseases through competitive interactions of beneficial microorganisms with pathobionts.

Applied Microbiology doi: 10.3390/applmicrobiol6030036

Authors: Anastasia S. Tugbaeva Olga V. Voropaeva Gregory I. Shiryaev Alexander A. Ermoshin Irina S. Kiseleva

In a changing climate, sustainable agriculture urgently requires environmentally friendly solutions. Increasing soil salinity severely limits crop productivity, as excess salts induce osmotic and ion-specific toxicity in plants. A promising strategy for mitigating these effects and enhancing plant salt tolerance involves the use of biofertilizers based on plant growth-promoting (PGP) rhizobacteria. In this study, novel salt-tolerant PGP strains were isolated and characterized from the rhizosphere of the halophyte Atriplex prostrata grown in soils with varying salinity levels. Twelve isolates were screened for key PGP traits, including indole-3-acetic acid (IAA) production, phosphate solubilization, siderophore synthesis, and NaCl tolerance. Two strains, AP9 and AP12, demonstrated the most comprehensive PGP potential. Based on 16S rRNA gene sequencing, they were identified as members of the genus Serratia. In an experiment under salt stress (75, 150, and 225 mM NaCl), inoculation of wheat (Triticum aestivum L.) seeds with these strains significantly improved germination rates and stimulated root and shoot development. The treated plants also exhibited reduced levels of key oxidative stress markers—malondialdehyde (MDA) and proline. Thus, the Serratia sp. AP9 and AP12 strains exhibit pronounced PGP activity and efficacy in enhancing the salt tolerance of wheat. These results indicate that these isolates are promising candidates for the development of novel biofertilizers for sustainable agriculture on saline soils.

Applied Microbiology doi: 10.3390/applmicrobiol6030035

Authors: Shota Torigoe Kentaro Yamamoto Manabu Ato

Mycobacteria form cellulose-containing biofilms and exhibit drug resistance at infection sites. However, the function of mycobacterial biofilms in host defense remains unclear. Herein, we demonstrate that reductive stress-induced mycobacterial biofilms evade macrophage capture and protect bacilli from nitric oxide stress. We first determined the optimal conditions for biofilm formation by mycobacteria. Using green fluorescent protein (GFP)-labeled mycobacteria, we then examined the protective effect of biofilms on the capture of bacilli by macrophages and found that macrophage capture was inhibited in the presence of biofilms. Furthermore, we constructed GFP-expressing mycobacteria that respond to acidic pH and nitric oxide stress, both of which are bactericidal factors. The results showed that mycobacterial biofilms protect bacilli from nitric oxide-mediated stress, but not from acidification. Finally, the removal of biofilms by cellulase enhanced the capture of mycobacteria by macrophages and the exposure of mycobacteria to nitric oxide. These findings highlight the protective roles of mycobacterial biofilms in innate immunity, particularly against macrophage capture and nitric oxide-induced stress.

Applied Microbiology doi: 10.3390/applmicrobiol6020034

Authors: Nivethika Ajeethan Lord Abbey Svetlana N. Yurgel

Plants are sessile organisms and are constantly subjected to varying environmental stressors. However, they can mitigate the effects of these stresses by deploying plant growth-promoting (PGP) microbes for their protection. PGP microbes can boost plant growth and enhance plant protection from biotic and abiotic stresses through a wide variety of mechanisms. PGP mechanisms such as biological fixation of nitrogen in soil and plant roots, phosphate solubilization, siderophore production, ACC (1-aminocyclopropane-1-carboxylic acid) deaminase enzyme activity, and production of plant hormones to promote nutrient acquisition and mitigate stresses. Therefore, this review aims to document studies that reported on the role of PGP microbes in plant growth and development and how PGP traits mentioned above and a novel trait flavins (FLs) secretion help plants against biotic and abiotic stress. Several important PGP functions, and the bacterial strains involved in these functions, that can potentially improve plant growth, development, and plant health are reviewed. This review will help to identify gaps for future studies and guide the development of an alternative strategy to use PGP microbes as biofertilizers and biocontrol agents to support eco-friendly agriculture by reducing the indiscriminate use of synthetic agrochemicals.

Applied Microbiology doi: 10.3390/applmicrobiol6020033

Authors: Servando H. Cantú-Bernal Alonso A. Orozco-Flores Víctor E. Aguirre-Arzola Ricardo Gomez-Flores Orquídea Pérez-González Patricia Tamez-Guerra

Agriculture is an essential activity in Mexico, representing the main source of income of numerous families. Crops are negatively affected by many diseases, particularly caused by phytopathogenic fungi, whose control by biological agents emerges as an advantageous alternative. The aim of the present study was to evaluate the antagonistic activity of microorganisms isolated from Larrea tridentata L. (Sessé & Moc. Ex DC.) Coville leaves, stems, roots, and rhizospheric soil against Fusarium spp. and other phytopathogen fungi. We identified 54 microorganisms: 30 bacteria species and 24 actinobacteriota. Initial dual-confrontation experiments with phytopathogenic fungi determined the bacillus and actinobacteriota inhibited growth from 57 to 100% and 42 to 83%, respectively. Based on our initial results, selected isolates were confronted with Rhizoctonia sp. and two Fusarium spp. isolates (orchid and garlic isolates). All microorganisms inhibited Rhizoctonia, but only 13 bacillary bacteria and eight actinobacteriota isolates inhibited Fusarium and were selected for the third confrontation, in which firmicutes –Bacilli:Bacilliales- and actinobacteriota isolates inhibited Fusarium spp. growth from 55 to 92% and 14 to 74%, respectively. In addition, supernatant fluids from six selected actinobacteriota were evaluated, and the results determined that the strains OP-AGsD3, OP-AGsM4R7, and OP-AGsM1R5 possessed the highest antagonist activity against all Fusarium spp. isolates. Molecular identification analysis indicated that actinobacteriota belonged to the Streptomyces genus. Our results revealed the potential of native Streptomyces spp. from L. tridentata rhizosphere soil as biocontrol agents against phytopathogenic Fusarium spp.

Applied Microbiology doi: 10.3390/applmicrobiol6020032

Authors: B. D. Meisinger E. G. Olson C. D. Coufal S. C. Ricke

Hatching eggs possess multiple physical and chemical barriers that limit microbial invasion; however, the role of the eggshell membrane in shaping late-stage embryonic and early post-hatch gastrointestinal (GI) microbiota remains poorly understood. This study aimed to (i) validate a reproducible eggshell membrane extraction method, (ii) assess whether microbial loads differ between nest- and floor-laid eggs, (iii) examine relationships between eggshell membrane-associated microbiota and embryonic intestinal microbiota, and (iv) determine whether microbial blooms align with key stages of the hatching process. In a preliminary experiment using White Leghorn hatching eggs, no significant differences were observed in aerobic, anaerobic, or fungal membrane counts between nest- and floor-laid eggs. In a commercial hatchery study using Ross 708 broiler eggs, membrane and GI microbial populations were evaluated across days 18–20 of incubation, corresponding to pre-pipping, internal pipping, and external pipping/post-hatch stages. Significant, day-dependent shifts in microbial counts were observed, with strong interactions between sampling day and location (membrane vs. GI) for most bacterial groups. Enterococci and anaerobic bacteria were enriched in the GI tract prior to hatch, whereas aerobic, Gram-negative, and Staphylococcus populations were more abundant on membranes during late incubation. Post-hatch chicks exhibited markedly higher GI microbial loads compared to embryos, indicating rapid colonization during the hatch transition. Collectively, these findings demonstrate that the pipping and hatching process represents a critical window for microbial redistribution from eggshell membranes to the developing chick gut, highlighting the hatchery as a key control point for early-life microbial exposure and intervention strategies.

Applied Microbiology doi: 10.3390/applmicrobiol6020031

Authors: Natalia G. Bednarska Marta A. Kisiel

MicroRNAs (miRNAs) are key regulators of host–microbiome interactions. They influence diverse physiological processes through post-transcriptional gene regulation. Growing evidence indicates that host-derived miRNAs and microbially encoded miRNA-like molecules contribute to bidirectional signaling between the gut microbiota and the central nervous system. These interactions play a role in gut–brain axis communication. This review summarizes current findings on how host miRNAs shape microbial composition and function. It also examines emerging evidence that microbial miRNA-like molecules can modulate host gene expression. Particular attention is given to pathways involved in metabolic regulation, immune signaling, and neuroinflammatory processes relevant to gut–brain communication. In addition, we discuss the role of extracellular vesicles in miRNA transport and signaling. We critically assess the translational potential of miRNA-based biomarkers and therapeutic strategies, highlighting both their promise and current limitations. Overall, this review provides an integrated overview of miRNA-mediated host–microbiome interactions within the gut–brain axis and outlines key conceptual and experimental challenges that remain unresolved.

Applied Microbiology doi: 10.3390/applmicrobiol6020030

Authors: Pablo Sánchez-Rey Fernando Moro Cordobés Alina Greslebin Alejandra L. Pérez Francisco Kuhar

Hyphal systems have been essential for the morphoanatomical characterization of basidiomes and mycelia of aphyllophoroid fungi for taxonomic purposes. They have also been shown to influence the consistency of basidiomes. Recent developments in areas such as mycelium composite production as sustainable materials have redirected scientists’ attention to these structures, particularly regarding their material resistance, where complex hyphal systems enhance the properties of these composites. Compounds such as farnesol and octenol trigger growth and differentiation processes in many fungal groups, and laccases have been proposed as enzymes involved in these processes, given their roles in the synthesis of cell wall pigments and other cell wall components. Given the easily quantifiable differences in hyphal knots and dimitic mycelium between Fuscoporia torulosa and Inocutis tamaricis, we employed them as models to study their responses to these compounds, thereby helping fill the knowledge gap in the modulation of macrofungal mycelial differentiation. A variable effect was observed on laccase induction, while radial growth was reduced by octenol by up to 83% in F. torulosa and 65% in I. tamaricis, and by farnesol by up to 80% in I. tamaricis, showing slight effects on F. torulosa. Reductions of up to 100% were observed in the combination of high doses of both chemicals.

Applied Microbiology doi: 10.3390/applmicrobiol6020029

Authors: Maja Mijač Sunčanica Ljubin-Sternak Marin Bajek Tajana Balaban Lucija Vlahek Tatjana Marijan Jasna Knežević Jasmina Vraneš

The rise in antimicrobial-resistant Neisseria gonorrhoeae (NG) strains poses major challenges to gonorrhea treatment worldwide. Ceftriaxone remains the first-line antibiotic therapy; however, emerging resistance, particularly driven by the mosaic penA 60.001 allele, necessitates vigilant surveillance. This study assesses the antimicrobial susceptibility patterns of NG isolates in the northwestern region of Croatia and evaluates the correlation between phenotypic susceptibility testing for extended-spectrum cephalosporins (ESC) and genotypic detection of the penA 60.001 allele. A total of 39 clinical NG-positive specimens by a multiplex PCR panel for urogenital infections were collected between 1 July 2022, and 30 June 2024. Phenotypic antimicrobial susceptibility testing was performed using the Etest method. Genotypic detection of ceftriaxone resistance determinants was performed using a multiplex nested PCR assay. All NG isolates were susceptible to ceftriaxone and cefixime. High resistance rates were observed for ciprofloxacin (70.6%), tetracycline (44.1%), and azithromycin (20.6%). Mutations in the penA gene associated with decreased susceptibility to ceftriaxone were detected in three samples, although phenotypic resistance was not observed. The high resistance rates to ciprofloxacin, tetracycline, and azithromycin limit their use for empirical therapy in Croatia. While ceftriaxone remains effective, the detection of penA mutations highlights the need for ongoing surveillance.

Applied Microbiology doi: 10.3390/applmicrobiol6020028

Authors: Silverio Andrés Quintana María Magdalena Sarmiento Andrea Arrúa Alvarenga Rosa Morel Andreas Ries Gilberto Benitez Rodas

Tannery effluents constitute highly complex chemical and biological matrices that can affect ecosystem integrity and public health. In Paraguay, metagenomic information on industrial discharge remains limited. In this context, the aim of this study was to characterize microbiome diversity and detect antibiotic resistance genes (ARGs) via metagenomic sequencing complemented by chemical analyses. Total DNA was sequenced using Oxford Nanopore technologies and analyzed with Kraken2 for taxonomic assignment and CARD for ARG detection. The results revealed a hypersaline, metal-containing effluent with a high organic load and measurable nitrogen and phosphorus concentrations. Microbiome profiles were dominated by Pseudomonadota (77.2%), primarily Thiocapsa (27.8%) and Francisella (23.0%). The phototrophic and sulfur-oxidizing metabolism characteristic of Thiocapsa may explain the distinctive coloration of the effluent, while the predominance of Francisella is consistent with tolerance to hostile environmental conditions. DNA sequences assigned to taxa of clinical relevance, including Pseudomonas aeruginosa, Salmonella enterica, and Klebsiella pneumoniae, were also detected, along with a range of ARGs associated with resistance to tetracyclines, β-lactams, and aminoglycosides. These findings demonstrate that treated tannery effluent can retain clinically relevant genetic material and ARGs, underscoring the need to integrate metagenomic surveillance into environmental monitoring frameworks to better understand and mitigate emerging resistance determinants in aquatic systems. This study provides one of the first metagenomic characterizations of a tannery effluent in the country and contributes novel insights at a regional scale.

Applied Microbiology doi: 10.3390/applmicrobiol6020027

Authors: Rafail Fokas Nikolaos P. Kostopoulos Maria-Eleni Dimitrakopoulou Apostolos Vantarakis

This study investigated the effectiveness of ethanol (70%, 3 min), Ultraviolet-C irradiation (6 and 12 min), and ultrasound (37 kHz, 15 min) for decreasing Candida albicans, Aspergillus brasiliensis, and Listeria innocua on Greek Kalamon table olives before brining. Ethanol demonstrated the greatest decreases (>2.80 log10 for C. albicans, >2.09 log10 for A. brasiliensis, and >3.79 log10 for L. innocua). UV-C had a time-dependent impact, with 12 min producing more inactivation than 6 min (1.30, 1.05, and 1.57 log10, respectively, for C. albicans, A. brasiliensis, and L. innocua). Ultrasound alone produced minimal reductions (<0.60 log10). Overall, ethanol outperformed Ultraviolet-C and ultrasound in the test settings, with Ultraviolet-C providing moderate, exposure-dependent decreases. These findings stimulate additional research into non-thermal therapies and their practical use in table olive processing.

Applied Microbiology doi: 10.3390/applmicrobiol6020026

Authors: Marilena Bazzano Anna Rita Attili Vincenzo Cuteri Fulvio Laus Andrea Spaterna Andrea Marchegiani

Research on the reproductive tract microbiology of broodmares has primarily focused on the uterus, with a limited set of tools for the rapid detection of pathogenic bacteria in the perineal region. Accurate, real-time identification of bacterial contamination could improve the diagnosis and management of post-breeding infectious endometritis. In this proof-of-concept study, we evaluated the ability of a portable MolecuLight i:X fluorescence imaging device for the rapid, non-invasive detection of potentially pathogenic perineal bacteria in healthy broodmares, comparing results with microbiological culture as the gold standard. Using ultraviolet-induced fluorescence imaging guided for swabbing and microbiological culture, the device demonstrated 80% sensitivity, 96% specificity, and 91% accuracy in differentiating potential pathogenic from commensal bacteria in clinically healthy broodmares. These preliminary findings may represent the basis for further assessment of the real-time, fluorescence-based technology in diseased or symptomatic broodmares, potentially aiding timely clinical decision-making. Further multicentred studies with larger inclusion of mares with confirmed endometritis are needed to strengthen the relevance of this technology and to expand the device’s application in equine reproductive health.

Applied Microbiology doi: 10.3390/applmicrobiol6020025

Authors: Euclides Ticona Chayña Pompeyo Ferro Eli Morales-Rojas Guzman Saucedo Jorge Bautista Lizbeth Córdova-Rojas Antony Guevara Yshoner Antonio Silva-Diaz Romel Guevara Edwaldo Villanueva Pedraza Polan Ferro-Gonzales

Antimicrobial resistance is a growing threat to public health and the environment, especially in vulnerable ecosystems such as the Amazon. The confluence of the Marañón, Utcubamba, and Chinchipe rivers, known as the Pongo de Rentema, is a strategic area where water pollution could facilitate the spread of antibiotic resistance genes. This study aims to assess water quality in this region under the “One Health” approach by analyzing physicochemical parameters, heavy metals, and the presence of antimicrobial resistance genes. Water samples were collected from five sampling points during September and October 2024. Physicochemical parameters were analyzed in situ, and heavy metal concentrations were determined using atomic emission spectrophotometry. The presence of Escherichia coli and Pseudomonas aeruginosa was evaluated through selective culture, and the detection of resistance genes (marA, ermC, amp, QEP, and qEmarA) was performed using conventional PCR. Physicochemical parameters were within the limits established by Peruvian regulations, except for total dissolved solids in the Utcubamba River. Elevated levels of lead and chromium were detected at some points. Additionally, resistance genes were identified in E. coli and P. aeruginosa, providing evidence of antimicrobial resistance dissemination in the water. Water pollution in the Pongo de Rentema poses an environmental and public health risk due to the presence of heavy metals and antimicrobial resistance genes. Continuous monitoring and environmental management strategies under the “One Health” approach are recommended to mitigate these risks.

Applied Microbiology doi: 10.3390/applmicrobiol6020024

Authors: Hayet Aouimeur Faiza Boublenza Grégori Gerald Aude Barani Yasmina Makhlouf

The first step to selecting interesting lactic acid bacteria for commercial use is testing their resistance to different physicochemical stresses. In this study, we evaluated the viability of Enterococcus faecium and Enterococcus durans, obtained from two traditional fermented cheeses, subjected to several stresses (thermal, osmotic, acidic, alkaline, oxidative, detergent, and alcoholic). The assessment of cell viability was conducted via flow cytometry (FCM) combined with nucleic-acid double staining (NADS) and was compared to the conventional plate count method (CFU). The findings from the two approaches indicated that Enterococcus faecium and Enterococcus durans demonstrated a substantial proportion of viable cells following exposure to osmotic, thermal, and acidic stress. The alkaline stress treatment does not diminish the proportion of viable cells. Both strains exhibited extensive sensitivity to SDS, oxidative stress, and experienced total cell death under alcoholic stress. We observed a satisfactory correlation between cell viability as measured by FCM and CFU under all stress conditions. These data demonstrate the existence of indigenous strains of Enterococcus spp. that exhibit notable stress resistance. FCM for viability enumeration is better than the conventional plate counting method due to its rapid results and precision, which offer an effective evaluation of live, dead, and permeabilised cells. This technique holds promise for physiological state research in dairy applications to evaluate the quality of fermented products and the viable cell count for probiotic manufacturing.

Applied Microbiology doi: 10.3390/applmicrobiol6020023

Authors: Angelica Beatriz Condori Mamani Anthony Brayan Rivera Prado Kelly Geraldine Yparraguirre Salcedo Luis Lloja Lozano Vicente Freddy Chambilla Quispe Claudio Willbert Ramirez Atencio

Giardiasis, caused by the protozoan parasite Giardia lamblia, remains a prevalent intestinal infection worldwide and a growing concern due to increasing resistance to nitroimidazole drugs. This study proposes an alternative therapeutic strategy by targeting fructose-1,6-bisphosphate aldolase (FBPA), a key glycolytic enzyme of the parasite, through structure-based virtual screening. A curated library of microbiome-derived metabolites was computationally evaluated and compared with clinically used antigiardial drugs. Several indole-based compounds exhibited favorable binding affinities and stable interactions within the catalytic pocket of FBPA. These findings suggest that microbiome metabolites could serve as promising scaffolds for the rational design of new antiparasitic agents. Overall, the study highlights the potential of integrating metabolic and computational approaches to identify next-generation therapeutics against giardiasis.

Applied Microbiology doi: 10.3390/applmicrobiol6020022

Authors: Nivethika Ajeethan Lord Abbey Svetlana N. Yurgel

Plant growth-promoting (PGP) bacteria are beneficial microbes that can help plants mitigate various biotic and abiotic stresses through different PGP functions. Flavins (FLs) are involved in flavoprotein-mediated reactions essential for plant metabolism and could act as PGP molecules. The aim of this study was to isolate and characterize potential FLs secreting bacteria from apple (Malus domestica [Suckow] Borkh) roots based on their fluorescence and to evaluate their PGP properties, including FLs secretion. A total of 26 bacteria with increased fluorescence in liquid culture were isolated from the apple roots. Based on 16S rRNA sequencing analysis, 11 genetically different strains mostly from Burkholderia and Rhizobia spp. were identified. All isolates secreted considerable amounts of riboflavin. In vitro plant assays showed that under nitrogen (N) limitation, inoculated alfalfa (Medicago sativa) plants yielded at least 25% more dry mass than non-inoculated plants, and inoculation with AK7 and FL112 enriched plant tissue N content compared to non-inoculated plants. This improved N acquisition was not linked to symbiotic N fixation. Additionally, the isolates exhibited some other PGP properties. However, no specific PGP functions were linked to improved plant N acquisition but could potentially be linked to the FLs secretion. For future investigation, the mechanisms underlying improved plant N uptake should be assessed to gain a more in-depth understanding.

Applied Microbiology doi: 10.3390/applmicrobiol6020021

Authors: Samantha Nestel Robert Wagner Mareike Meister Thomas Weihe Uta Schnabel

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Applied Microbiology doi: 10.3390/applmicrobiol6010019

Authors: Louise Davison Zoë Alice Bell Hong Gao

Cloning large biosynthetic gene clusters (BGCs) is fundamental to unlocking microbial natural product potential for drug discovery and biotechnology. These clusters encode diverse bioactive compounds, but their size, high GC content, and complex architecture pose significant technical challenges. This review scrutinises recent advances in BGC cloning strategies, categorising them into three major groups: (1) direct release-and-capture methods, (2) genome-integrated preconditioning systems, and (3) CRISPR-assisted hybrid platforms. This review compares the strengths, limitations, and reported efficiencies of BGC cloning strategies, highlighting trade-offs in precision, scalability, and workflow complexity. Emerging trends, such as AI-driven genome mining, modular synthetic biology toolkits, and high-throughput automation, are reshaping the cloning landscape, enabling predictive design and streamlined assembly of clusters exceeding 100 kb. By integrating comparative analysis with future perspectives, this review provides outlines on how next-generation strategies will accelerate heterologous expression, natural product discovery, and sustainable biomanufacturing.

Applied Microbiology doi: 10.3390/applmicrobiol6010020

Authors: Vaishali Poswal Sanjeev Anand Jose L. Gonzalez-Hernandez Brian Kraus

Listeria monocytogenes is a persistent foodborne pathogen capable of surviving in food processing environments, often in association with diverse environmental microflora. This study examines genomic determinants of persistence, specifically stress adaptation and biofilm-associated traits, in environmental Listeria species and other environmental microflora from a dairy processing facility by analyzing whole-genome sequences of 6 environmental Listeria isolates, 4 ATCC reference strains, and 22 air and floor swab cultures, annotated using the RAST platform. Subsystem analysis revealed that Listeria isolates carried a defined set of genes linked to biofilm formation, antimicrobial resistance, and stress response, though in lower abundance than environmental cultures. Listeria exhibited fewer flagellar genes but greater consistency in core stress-related genes, including those for disinfectant and osmotic stress resistance, with SigB operon and RpoN genes highlighting strong stress tolerance. In contrast, environmental cultures exhibited broader transcriptional regulators (RpoE, RpoH) and greater diversity in acid and heat shock response genes, indicating distinct survival strategies. All examined Listeria species harbor biofilm and stress-resistance genes enabling independent survival, while environmental microbiota show greater genetic diversity that may promote persistence and multispecies biofilm formation. This study underscores the complex genetic landscape that may contribute to the persistence of Listeria and environmental microbiota in dairy processing environments, providing foundational insights for environmental cross contamination control strategies.

Applied Microbiology doi: 10.3390/applmicrobiol6010018

Authors: José Cantaro Segura Héctor Cántaro-Segura Raul Blas

Diet plays a pivotal role in shaping the gut microbiota, influencing host physiology, immune function, and nutrient metabolism. In this study, we evaluated the impact of three distinct feeding systems—Forage only, Balanced feed only, and Mixed system—on the cecal microbiota of guinea pigs (Cavia porcellus) using 16S rRNA gene amplicon sequencing in a randomized allocation of 18 males across the three diets (n = 6 per group) over 7 weeks. A total of 2,135,852 high-quality reads were obtained, with rarefaction curves and Good’s coverage confirming sufficient sequencing depth. Alpha diversity indices revealed significantly higher microbial richness and evenness in the mixed group, while beta diversity analyses demonstrated distinct microbial community structures across diets. Taxonomic profiling showed that forage-based diets enriched fiber-degrading genera such as Fibrobacter and Treponema, whereas the Balanced feed group favored mucin- and protein-degrading bacteria like Akkermansia and Bacteroides. LEfSe and t-test analyses identified several biomarkers and diet-specific genera, suggesting functional divergence in microbial metabolism. Forage-fed animals showed microbiota associated with short-chain fatty acid production and enhanced fiber utilization, while the Balanced feed group showed microbial traits linked to mucin degradation and potential gut barrier disruption. These findings highlight the strong influence of dietary composition on gut microbial ecology and suggest that fiber-rich diets promote a more diverse and functionally beneficial cecal microbiome in guinea pigs.

Applied Microbiology doi: 10.3390/applmicrobiol6010017

Authors: Victor Eduardo Zamudio-Sosa Luis Angel Cabanillas-Bojórquez Evangelina García-Armenta Marilyn Shomara Criollo-Mendoza José Andrés Medrano-Felix Alma Haydee Astorga-Gaxiola José Basilio Heredia Laura Aracely Contreras-Angulo Erick Paul Gutiérrez-Grijalva

Microbial fermentation is a key biotechnological tool for producing bioactive metabolites such as alkaloids, carotenoids, essential oils, and phenolic compounds, among others, with applications in human health, agriculture, and food industries. This review comprehensively reviews recent information on the synthesis of valuable compounds and enzymes through fermentation processes. Here, we discuss the advantages of the different types of fermentation, such as submerged and solid-state fermentation, in optimizing metabolite production by bacteria, fungi, and yeast. The role of microbial metabolism, enzymatic activity, and fermentation conditions in enhancing the bioavailability and functionality of these compounds is discussed. Integrating fermentation with emerging biotechnologies, including metabolic engineering, further enhances yields and specificity. The potential of microbial-derived bioactive compounds in developing functional foods, pharmaceuticals, and eco-friendly agricultural solutions positions fermentation as a pivotal strategy for future biotechnological advancements. Therefore, microbial fermentation is a sustainable tool to obtain high-quality metabolites from different sources that can be used in agriculture, animal, and human health.

Applied Microbiology doi: 10.3390/applmicrobiol6010016

Authors: Yara Loforte Mariem Zanzan André Martinho de Almeida Vasco Cadavez Ursula Gonzales-Barron

Previous research showed that a strain of Leuconostoc mesenteroides, isolated from goat’s raw milk cheese, was effective in slowing down the growth and reducing the maximum concentration of L. monocytogenes when evaluated in a milk model; furthermore, the extent of inhibition was dependent on the milk’s initial pH. The objectives of this study were as follows: (1) to determine whether the growth of L. monocytogenes in goat’s pasteurized milk cheese during maturation could be approximated from growth data obtained in the milk model medium, either in monoculture or in coculture with L. mesenteroides, and if so, (2) to model a milk-to-cheese conversion factor (Cf) for L. monocytogenes growth rate. Challenge tests were conducted by inoculating L. monocytogenes in monoculture and in coculture with L. mesenteroides in goat’s pasteurized milk adjusted at initial pH levels of 5.5, 6.0, and 6.5. The process of cheesemaking continued, and cheeses were ripened at 12 °C for 12 days. Each experimental growth curve was adjusted to a pH-driven dynamic model where the microbial maximum growth rate is a function of pH. As observed in the milk model medium, in coculture with L. mesenteroides, the optimum growth rate (μopt) of L. monocytogenes in maturing cheese was affected by the initial pH of milk: the lowest rate of 0.863 ± 0.042 day−1 was obtained at the initial pH 5.5, in comparison to 1.239 ± 0.208 and 1.038 ± 0.308 day−1 at pH 6.0 and 6.5, respectively. Regardless of the milk’s initial pH, L. mesenteroides did not reduce the maximum load of L. monocytogenes in maturing cheeses, as it did in the milk medium. On the contrary, at the milk’s initial pH of 5.5, 6.0, and 6.5, L. mesenteroides was able to decrease, on average, 2.2-fold, 1.5-fold, and 1.9-fold the μopt of L. monocytogenes in both milk medium and cheese, without significant differences between matrices. Following such validation in goat’s cheese, the square root of milk-to-cheese Cf for L. monocytogenes was estimated as 0.751 (SE = 0.0108), and the type of culture (monoculture and coculture) was not found to affect Cf (p = 0.320). In conclusion, this work validated the pre-acidification of milk as an efficient strategy that, when combined with the use of a protective culture, can synergically enhance the control of L. monocytogenes in cheese.

Applied Microbiology doi: 10.3390/applmicrobiol6010015

Authors: Tassnapa Wongsnansilp Manoch Khamcharoen Jaran Boonrong Wipawee Dejtisakdi

This study focuses on the diversity of phytoplankton in the Koh Yaa region of Thailand and their relationship with environmental variables, aiming to assess whether human activities (primarily tourism) pose potential threats to the marine ecosystem and provide scientific support for eco-sustainable tourism management decisions in the region. In April, August, and December 2024, corresponding to peak season, off-season, and shoulder season, a total of 156 discrete samples were collected from four coastal sites to analyze water quality parameters such as temperature, pH, total nitrogen (TN), and total phosphorus (TP), along with plankton diversity and abundance. Statistical analyses including two-way ANOVA with Duncan’s Multiple Range Test (DMRT), Pearson correlation analysis, and principal component analysis (PCA) were applied. The results showed a declining trend in plankton abundance over time, peaking at 1009 × 106 cells/m3 in April and dropping to 281 × 106 cells/m3 by December. A total of 15 types of phytoplankton were identified across four phyla: Bacillariophyta, Cyanobacteria, Dinoflagellata, and Chlorophyta. Notably, Chaetoceros from Bacillariophyta accounted for 47% of phytoplankton, while Oscillatoria from Cyanobacteria made up 29.6%. The diversity index and evenness index improved from 1.34 and 0.46 in April to 1.88 and 0.64 in December, respectively. Environmental factors like pH, temperature, and TP significantly affected phytoplankton abundance (p < 0.01), with TP levels ranging from 0.27 to 0.69 mg/L. These results indicate possible pollution in this region, and changes in phytoplankton abundance were linked to seasonal climate variations—especially during peak tourist seasons—which may exacerbate eutrophication affecting community structures.

Applied Microbiology doi: 10.3390/applmicrobiol6010014

Authors: Kaoutar Boussif Ahmed Elidrissi Abdelkhaleq Elmoslih Youssef Ezzaky Mariem Zanzan Fouad Achemchem

Lactic acid bacteria (LAB) are increasingly recognized for their role in food biopreservation due to their ability to synthesize antimicrobial compounds. Milk naturally harbors a wide variety of LAB, offering a promising source for identifying strains with biopreservative potential. This study investigated the antagonistic effects, safety characteristics, and technological properties of LAB strains isolated from traditionally fermented milk. Thirty-two dairy samples were analyzed, and the resulting LAB isolates were screened for inhibitory activity against Listeria monocytogenes CECT 4032 and Staphylococcus aureus CECT 976 using agar spot and well diffusion assays. All tested strains exhibited strong antimicrobial effects, with particularly notable inhibition of L. monocytogenes. After phenotypic screening, five representative isolates were selected for molecular identification and further assessment of safety-related attributes, functional capabilities, auto- and co-aggregation properties. 16S rRNA gene sequencing revealed that four strains belonged to the genus Enterococcus, specifically, one E. faecium and three E. durans, while one was classified as a Lactococcus species. Moreover, none of the strains showed proteolytic or lipolytic activities which highlights their potential use in dairy fermentation processes.

Applied Microbiology doi: 10.3390/applmicrobiol6010013

Authors: Edwin Villegas Fernando Escobal Toribio Tejada Peter Piña Hector Cántaro-Segura Luis Diaz-Morales Daniel Matsusaka

Wheat (Triticum aestivum L.) sustains global caloric intake, but its productivity in Andean highlands is constrained by soil fertility and input reliance. This study represents one of the first field-based evaluations of biofertilizers under high-altitude, rainfed Andean conditions, addressing a major knowledge gap in low-input mountain agroecosystems. This study evaluated three seed-applied biofertilizers—Azospirillum brasilense, Trichoderma viride (Trichomax), and an anchovy (Engraulis ringens) based liquid biofertilizer, compared with an untreated control and a soil-test mineral fertilization benchmark in rainfed wheat (Triticum aestivum L.) cv. INIA 405 in the central Andes of Peru. A 5 × 5 Latin square design (25 plots) was established under farmer-realistic conditions. At physiological maturity (Zadoks 9.5), plant height, spike length, grains per spike, thousand-grain weight, test weight, root dry mass, and grain yield were recorded. Mineral fertilization achieved the highest yield (1.20 ± 0.79 t ha−1), nearly doubling the control (0.60 ± 0.47 t ha−1). Notably, A. brasilense delivered an intermediate yield of 0.90 ± 0.64 t ha−1, representing a 50% increase over the control—accompanied by a marked rise in root dry mass. T. viride and the anchovy-based input yielded 0.85 ± 0.59 and 0.81 ± 0.59 t ha−1, respectively. Grain physical quality remained stable across treatments (thousand-grain weight ≈ 42 g; test weight 68–75 kg hL−1). Trait responses were complementary: root dry mass increased with mineral fertilization and A. brasilense, whereas spike length increased with mineral fertilization and the anchovy-based input. Overall, the evidence supports biofertilizers, particularly A. brasilense, as effective complements that enable partial fertilizer substitution within integrated nutrient-management strategies for sustainable wheat production in Andean rainfed systems.

Applied Microbiology doi: 10.3390/applmicrobiol6010012

Authors: Miriam Gutierrez Eugenia Quispe-Medina Cayo García-Blásquez Morote José Antonio Quispe-Tenorio Héctor Cántaro-Segura Luis Díaz-Morales Daniel Matsusaka

Quinoa (Chenopodium quinoa Willd.) is a strategic crop for climate-smart agriculture in the Andes, yet yield gains are constrained by soil degradation and low-input systems. We tested whether synergistic bioinoculation with a plant growth-promoting rhizobacterium (Azospirillum brasilense) and an arbuscular mycorrhizal fungus (Glomus iranicum var. tenuihypharum) enhances root function and grain productivity under field conditions. A split-plot RCBD was conducted in Ayacucho, Peru (2735 m a.s.l.) using four cultivars, Blanca de Junín (BJ), INIA 441 Señor del Huerto (SH), INIA 415 Pasankalla (RP) and INIA 420 Negra Collana (NC) and four treatments: uninoculated control, Azospirillum, Glomus and co-inoculation. Vegetative, root and yield traits were quantified; ANOVA, Tukey/Dunnett contrasts, correlations and PCA were applied. Co-inoculation consistently outperformed single inoculants, increasing root diameter, length, branching, dry weight and volume dry weight, while also enlarging panicle dimensions and raising grain weight per panicle and thousand-seed weight. Grain yield reached 4.94 ± 0.59 t ha−1 under co-inoculation, almost triple that of the control (1.71 ± 0.28 t ha−1) and about 1.5 times higher than single inoculations. Genotypic effects were pronounced; BJ and SH combined superior root biomass with higher yield, RP maximized grain size and hectoliter weight, whereas NC responded weakly. Significant genotype × treatment interactions indicated cultivar-dependent microbiome benefits. Correlation and PCA linked root biomass and stem/panicle architecture to yield formation, positioning co-inoculation along trait vectors associated with belowground vigor and productivity. These results demonstrate a robust microbial synergy that translates root gains into yield, supporting co-inoculation as a scalable, low-input strategy for sustainable intensification of quinoa in highland agroecosystems.

Applied Microbiology doi: 10.3390/applmicrobiol6010011

Authors: Guadalupe Steele Andrew K. Rindsberg Hung King Tiong

Conventional and organic agriculture can both cause soil microbial community structure (SMCS) destruction, infertility, and abandonment. The application of soil productivity-improving biofertilizers is a sustainable practice that requires holistic knowledge, including complex biointeractions, diverse microbial metabolism, and culture requirements, the last of which rely on methodology and technology. In this study, a holistic culture-based and meta-analysis approach was employed to explore pristine and domesticated soils for presumptive plant growth-promoting (PGP) bacteria. Various soil samples were logistically acquired and processed using enrichment and heat alternatives. Morphologically diverse isolates were streak-purified and analyzed for 16S rRNA bacterial identification. Meta-analysis of PGP bacteria in domesticated environments was conducted using Google Search and NCBI PubMed. Soil fertility was analyzed for the pH and nitrogen/phosphorus/potassium (NPK) contents using biochemical tests. Notably, 7 genera and 15 species were differentially recovered, with Bacillus being the most prevalent and diverse in species. Conversely, Aeromonas, Lactobacillus, Lelliottia, Pseudomonas, and Staphylococcus were found only in pristine soil. While soil pH was consistent in all pristine soil samples, NPK contents ranged widely across the pristine (i.e., P/K) and domesticated samples (i.e., N/P/K). These findings could enhance biofertilizer SMCS, function, and effectiveness in the agricultural productivity needed to feed the expanding population.

Applied Microbiology doi: 10.3390/applmicrobiol6010010

Authors: Said Ezrari Abdessamad Ikken Oussama Grari Mohamed Ou-zine Mohammed Lahmer Abderrazak Saddari Adil Maleb

Many emerging and re-emerging infectious diseases have been observed over the last few decades around the globe due to population growth, international travel, environmental changes, and microbial adaptation and evolution, despite advances in the medical field. The spread of these diseases is related to complex interactions between pathogens and their hosts. Accordingly, this review summarises current knowledge on infection development and discusses methods used for detection and modeling. Recent studies have revealed the limitations of two-dimensional models and increasingly rely on 3D systems, including spheroids, organoids, and organ-on-a-chip systems, that offer more realistic tissue environments, allowing researchers to more effectively study host–pathogen interactions. Overall, the integration of complementary approaches and the development of 3D models are crucial for enhancing diagnosis, developing new therapeutic approaches, and strengthening control strategies of emerging outbreaks.

Applied Microbiology doi: 10.3390/applmicrobiol6010009

Authors: Prashansani M. D. Silva Travis R. Glare John Graham Hampton Diwakar R. W. Kandula Josefina Narciso

New Zealand’s only tea (Camellia sinensis) plantation supplies a niche market for organically produced high value tea but faces challenges from climatic conditions and the decision to use only organic production methods. Fungi from the genus Trichoderma have been commercialised in New Zealand and elsewhere as disease-suppressing and plant growth-promoting agents. However, the potential benefits of using Trichoderma as a microbial biostimulant for tea cultivation have not been investigated in New Zealand. The ability of T. atroviride application to stimulate tea plant growth at a tea plantation was investigated over one year of production. The study involved foliar application of the biostimulant either once, twice or three times, one month apart, using 12 g of a commercially formulated spore mixture of four strains of T. atroviride per 5 m2 of experimental plots. Treatment with T. atroviride significantly increased tea yield by between 17% and 28% compared to the control over the harvesting season, but there were no statistically significant yield differences among the number of applications. The foliar applied T. atroviride was not detected in the soil or root samples six months after application, in either a soil metabarcoding analysis or on re-isolation media. This was likely due to the dense tea foliage and ground cover under the tea plants which impeded its movement to the soil. While the specific nature of T. atroviride interaction with perennial crops like tea is not known, in this trial it appeared to have remained on the phyllosphere and provided biostimulation without reaching the soil.

Applied Microbiology doi: 10.3390/applmicrobiol6010008

Authors: Michel Leiva-Mora Pamela Elizabeth Mera Guzmán Rafael Isaías Mera-Andrade Alicia Monserrath Zabala Haro Luis Rodrigo Saa Paúl Loján Catherine Lizzeth Silva Agurto Luis Fabián Salazar-Garcés Betty Beatriz González Osorio Dariel Cabrera Mederos Orelvis Portal

Phosphate-solubilizing rhizobacteria associated with the Solanum tuberosum L. cultivar ‘Superchola’ were isolated and characterized to improve our understanding of plant growth promotion in agricultural systems. Bacteria were isolated by serial dilutions, and the morphology of the colonies was characterized on nutrient agar culture medium. In addition, morphological identification was achieved by Gram staining. The ability to solubilize phosphate was assessed in Pikovskaya agar culture medium, while molecular identification involved the amplification of the partial 16S rRNA gene using the polymerase chain reaction. In the Píllaro canton, the highest number of colony-forming units per gram of soil was recorded at 9.72 × 109. Among the isolated strains, 62% exhibited circular morphology, 92% had a smooth texture, and 85% displayed entire margins. Notably, 83% of the isolates were Gram-negative, with 50% exhibiting a bacillary form. The most effective phosphate solubilizers were from the Mocha canton, particularly the isolate CC-FCAGP-BSF6, which showed superior solubilization capacity. Molecular identification revealed bacterial isolates from four genera, i.e., Bacillus, Pseudomonas, Lysinibacillus, and Paenibacillus. These strains exhibited significant phosphate solubilization in vitro and resulted in increased leaf area (0.21–0.49, p = 0.038), fresh mass (0.46–0.87, p = 0.014), dry mass (0.092–0.096, p = 0.047), and leaf area index (0.14–0.33, p = 0.026) in the S. tuberosum cultivar ‘Superchola’ in vitro plants. This study identifies bacterial species associated with the rhizosphere of S. tuberosum in Ecuador and highlights their potential for promoting plant growth and solubilizing phosphates.

Applied Microbiology doi: 10.3390/applmicrobiol6010007

Authors: Elizaveta Vinogradova Almagul Kushugulova Samat Kozhakhmetov Maxim Baltin

Differential abundance analysis (DAA) is a critical task in microbiome research aimed at identifying microbial signatures that reliably characterize groups. Research suggests that microbiome systems are relatively stable and resilient, yet even small changes under certain conditions can trigger dysbiosis. The high dimensionality of microbiome datasets exacerbates the challenge of detecting such changes by posing a multiple comparison problem that requires hypothesis filtration. Standard filtration using multiple comparison correction procedures is designed for scenarios with a high number of true positives and is often too conservative for microbiome data, where the proportion of true signals can be very low. Therefore, there is a substantial need for hypothesis filtration methods tailored to microbiome data. Confidence intervals (CIs) for between-group differences offer a powerful alternative to p-value filtration, as their range simultaneously conveys information about the significance, potential magnitude, and direction of the effect, as well as the certainty of the estimate itself. Microbial data can be adequately modeled using a negative binomial (NB) distribution, and its location parameter can be robustly estimated with the Hodges–Lehmann estimator (HLE). Using synthetic and experimental data, we demonstrate that hypothesis filtration based on CIs for the two-sample HLE is a robust method for comparing microbial data. Our analysis demonstrates that the HLE-CI approach provides the same level of precision as filtration using multiple-adjustment methods while achieving significantly higher recall in microbiome DAA. The results of this study suggest that HLE-CI-based filtration can be an effective step in the search for microbiome biomarkers.

Applied Microbiology doi: 10.3390/applmicrobiol6010006

Authors: Tran Ngoc Han Nguyen Thanh Toan Nguyen Thi Tuyet Hue Le Thi My Thu Phung Thi Hang Nguyen Duc Trong Tran Trong Khoi Nguyen Le Thanh Quang Ly Ngoc Thanh Xuan Ngo Thanh Phong Nguyen Quoc Khuong

Potassium (K) is a vital macronutrient for plant growth and yield, yet most soil K occurs in insoluble mineral forms, limiting availability to crops. Reliance on chemical K fertilizers is unsustainable due to cost and environmental concerns. Microbial solubilization of mineral K, particularly by purple nonsulfur bacteria (PNSB), offers an eco-friendly alternative. This study focused on isolating mica-potassium-solubilizing purple nonsulfur bacteria (MK-PNSB) from in-dyke alluvial soil and assessing their effects on hybrid maize germination and seedling growth. Among the isolates, the results showed that strain M-Wa-19 released the highest amount of soluble K under microaerobic light conditions (27.4 mg∙L−1). Under aerobic dark conditions, M-Wa-24 and M-Wa-26 released 20.1–21.0 mg∙L−1 of soluble K. Strains M-Wa-21, M-Wa-25, and M-Sl-13 solubilized K in the range of 14.3–25.1 mg∙L−1 and 12.9–24.4 mg∙L−1 under both incubation conditions. The selected strains were identified by 16S rRNA as Rhodopseudomonas palustris strain M-Sl-13 (PX588604), Rhodoplanes pokkaliisoli strain M-Wa-19 (PX588605), Afifella marina strain M-Wa-21 (PX588606), Rhodocista pekingensis strain M-Wa-24 (PX588607), Rhodocista pekingensis strain M-Wa-25 (PX588608), and Rhodocista pekingensis strain M-Wa-26 (PX588609). None exhibited toxicity to maize seeds; instead, all enhanced seed vigor indices by up to 99.7% and improved plant height and root biomass by 19.0–26.2% and 14.4–22.9%, respectively, under static hydroponic conditions. At a 1:1000 (bacteria and distilled water) dilution rate, strains M-Wa-26, M-Wa-25, M-Sl-13, M-Wa-24, M-Wa-19, and M-Wa-21, along with the six-strain mixture, improved seed vigor index by 3.96–7.91%. These findings suggest that MK-PNSB, individually or in mixtures, hold promise as biofertilizer candidates for sustainable K management in crop production.

Applied Microbiology doi: 10.3390/applmicrobiol6010005

Authors: Samuel Charles Olabode Barde Yelwa Luka Oluwafemi Abiodun Adepoju Ibrahim Zubairu Waziri Lockta Joel Rabiu Bukar Mohammed Mohammed Nasir Shuaibu Abdullahi Balarabe Sallau Tahir Turaki Mohammed Sa’adiya Halima Mahmud Abdullahi Abdulmalik Salman Ghulam Jeelani Yakubu Kokori Enevehe Ibrahim Emmanuel Oluwadare Balogun

Bacterial cellulose hydrogels (BCHs) are characterized as exopolysaccharides of glucose polymers consisting of β–1–4–glycosidic linkage with various degrees of polymerization which are synthesized by bacteria. There is a paucity of information on the isolation and characterization of a BCH producer isolate from Nigeria. The study, therefore, aimed to characterize a new Acetobacter species that had previously been confirmed to produce BCH. The BCH-producing isolate was characterized by PCR amplification of the full-length 16S rRNA gene, as well as whole-genome sequencing analysis. The whole-genome sequence of the isolate was determined using the Illumina next-generation sequencing (NGS) platform, with downstream analysis of genomic reads through the metaWRAP pipeline. The BCH producer isolate was identified to be Acetobacter orientalis strain Zaria-B1, based on sequence identity with the reference Acetobacter orientalis strain VVS. Based on its annotated genome, the isolate had an approximate genomic size of 3.1 Mbp, 45 total RNAs, a GC content of 52.5%, 3046 total protein-encoding genes, an N50 of 253,774 bp, and an L50 of 4, as well as 30 contigs. The nucleotide BLAST of the cellulose synthase gene sequence confirmed the bin to be Acetobacter orientalis. The whole-genome characterization alongside the 16S rRNA genotyping confirmed the BCH-producing isolate to be Acetobacter orientalis.

Applied Microbiology doi: 10.3390/applmicrobiol6010004

Authors: Doaa S. Aboelwafa Abdel-Hamied M. Rasmey Akram A. Aboseidah Noura S. Dosoky Samar M. Abdelrahman

In this study, we used a culture-dependent approach to explore the biochemical potential of bacteria associated with two genera of soft corals collected from the Red Sea (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonaceae, and family Alcyoniidae). The soft corals were identified as Cladiella sp. and Paralemnalia sp. The associated bacteria were isolated on marine agar, nutrient agar, starch casein agar, ISP2 Agar, and M1 agar. The highest proportion of strains was recovered using marine agar, followed by nutrient agar and M1. We focused on Gram-positive bacteria and evaluated their cytotoxicity and antimicrobial activity. About 24% of the bacterial samples demonstrated promising cytotoxicity against Ehrlich ascites carcinoma (EAC). Out of 12 bioactive isolated strains, two bacterial isolates showed strong cytotoxicity, with IC50 values of 134.47 and 148.5 µg/mL, respectively. Nine isolates displayed significant antimicrobial activity against two tested pathogens. Based on the 16S rRNA gene sequence, two bioactive bacterial isolates were identified as Bacillus subtilis and Microbacterium sp. These findings indicate that bacteria associated with soft corals could be a valuable source of new bioactive compounds with potential uses in drug development. Furthermore, our data add important insights to the understudied field of host-microbiome relationships in soft corals.

Applied Microbiology doi: 10.3390/applmicrobiol6010003

Authors: Morena India Mokoena Rosina Nkuna Tonderayi Sylvester Matambo

Surfactants are harmful, persistent pollutants that are often found in contaminated soils, wastewater, and industrial effluents in complex mixes. Due to their chemical diversity and persistence, they present a bioremediation challenge. Using long-read shotgun metagenomics, 16S rRNA amplicon sequencing, PICRUSt2 functional prediction, and physicochemical proxies (total organic carbon, dissolved oxygen, chemical oxygen demand, foaming activity, etc.), this study investigated the aerobic biodegradation of SDS, SLS, rhamnolipids, Triton X-100, and CTAB (individually/mixed, 4% w/v) by microbial consortia enriched from oil-contaminated soil for 14 days. Pseudomonadota was dominant (85–90%), with Pseudomonas (60%) driving SLS and SDS degradation, while Paraburkholderia and Bordetella were dominant in recalcitrant surfactant degradation. Among the surfactants, SLS, rhamnolipids, and the combination of all surfactants demonstrated higher degradation by virtue of total organic carbon reductions of 50%, 56%, and 50%, respectively, and a foaming activity decline of 45–64%. The combination of surfactants with CTAB showed a 21% reduction in TOC, most likely due to CTAB’s known bactericidal effects. PICRUSt2 showed differential enrichment in alkyl oxidation, sulfate ester hydrolysis, aromatic ring cleavage, and fatty acid/sulfur genes and pathways. This study establishes inexpensive, scalable proxy indicators for monitoring surfactant bioremediation when direct metabolite analysis is impractical.

Applied Microbiology doi: 10.3390/applmicrobiol6010002

Authors: Lizbeth Mamani-Rojas Raihil Rengifo Leslie Velarde-Apaza Max Ramírez-Rojas Hector Cántaro-Segura

Potato (Solanum tuberosum L.) is a key staple crop in the Peruvian Andes, but its productivity is threatened by fungal pathogens such as Rhizoctonia solani and Alternaria alternata. In this study, 71 native bacterial strains (39 from phyllosphere and 32 from rhizosphere) were isolated from potato plants across five agroecological zones of Peru and characterized for their plant growth-promoting (PGPR) and antagonistic traits. Actinomycetes demonstrated broader enzymatic profiles, with 2ACPP4 and 2ACPP8 showing high proteolytic (68.4%, 63.4%), lipolytic (59.5%, 60.6%), chitinolytic (32.7%, 35.5%) and amylolytic activity (76.3%, 71.5%). Strain 5ACPP5 (Streptomyces decoyicus) produced 42.8% chitinase and solubilized both dicalcium (120.6%) and tricalcium phosphate (122.3%). The highest IAA production was recorded in Bacillus strain 2BPP8 (95.4 µg/mL), while 5ACPP6 was the highest among Actinomycetes (83.4 µg/mL). Siderophore production was highest in 5ACPP5 (412.4%) and 2ACPP4 (406.8%). In vitro antagonism assays showed that 5ACPP5 inhibited R. solani and A. alternata by 86.4% and 68.9%, respectively, while Bacillus strain BPP4 reached 51.0% inhibition against A. alternata. In greenhouse trials, strain 4BPP8 significantly increased fresh tuber weight (11.91 g), while 5ACPP5 enhanced root biomass and reduced stem canker severity. Molecular identification confirmed BPP4 as Bacillus halotolerans and 5ACPP5 as Streptomyces decoyicus. These strains represent promising candidates for the development of bioinoculants for sustainable potato cultivation in Andean systems.

Applied Microbiology doi: 10.3390/applmicrobiol6010001

Authors: Kangsadan Boonprab Vichien Kitpreechavanich Mingkwan Nipitwattanaphon

Companilactobacillus farciminis KUJ 25-S was isolated from fermented fish and identified using 16S rRNA gene sequencing with 30.0 g/L of L-LA (L-lactic acid), with 97% LA per sum of DL-LA. The characteristics of LA and its stereoisomers were confirmed using TLC, chiral-HPLC, and enzymatic techniques. Based on various conditions using liquid MRS broth (static condition, glucose 10%, NaCl 5%, 37 °C for 48 h), the highest growth and LA formation of the culture were at a low temperature (25 °C) and decreased at 37, 45, and 55 °C, respectively. The broth could grow and produce acid at an initial pH in the range 4–11, with a low initial pH of 4 promoting the highest LA formation. LA formation and growth were inversely proportional to the NaCl concentration in the 0.5–30% range. High glucose concentrations suppressed LA formation. The growth-promotion effect varied with glucose concentration (5–40%), with the optimum concentration for LA production being 20% glucose. On the other hand, if used in microoxic conditions, the absence of NaCl was more favorable to acidification than the addition of NaCl (5% NaCl). C. farciminis KUJ 25-S was proposed as a suitable method to produce L-LA based on using the appropriate line for further industrial use.

Applied Microbiology doi: 10.3390/applmicrobiol5040151

Authors: Margarita Kazak Kristina Valavičiūtė-Pocienė Rasa Bernotienė Jurgita Autukaitė Carolina Romeiro Fernandes Chagas

Zoological gardens represent unique sites for vector and vector-borne disease studies. They offer suitable breeding habitats for vector development and a diverse range of vertebrate hosts for blood feeding of insect vectors. This study aimed to assess the prevalence of avian blood parasites (Haemosporida, Trypanosomatida) in wild-caught mosquitoes (Culicidae) and Culicoides biting midges (Ceratopogonidae) from the largest and oldest zoo in Lithuania. Insects were collected in May–August 2023 using UV-light, CDC and BG-Sentinel traps; collected material was analysed using both microscopy and PCR-based methods for parasite detection. Overall, 504 parous biting midges (10 species) and 59 mosquitoes (three species) were investigated. Haemosporidians (Haemoproteus minutus (hTURDUS2), H. homogeneae (hSYAT16), and H. asymmetricus (hTUPHI01)) were identified in 5.4% of the 174 tested biting midges. Haemoproteus asymmetricus hTUPHI01 sporozoites were seen in only one individual of Culicoides kibunensis. Of 108 Culicoides females, 3.7% carried trypanosomatids—parasites infecting birds (Trypanosoma bennetti group) and mammals (T. theileri group). Among the 59 tested mosquitoes, two (3.4%) Cx. pipiens/torrentium mosquitoes were found to be PCR-positive for trypanosomatids (T. culicavium and Crithidia brevicula). No haemosporidian parasite DNA was detected in the mosquitoes examined. This pilot study indicates that avian blood parasites circulate within the Lithuanian Zoo, highlighting the need for further research on transmission pathways, vector–host interactions, and potential risks.

Applied Microbiology doi: 10.3390/applmicrobiol5040150

Authors: Alaa El-Dein Omara Dina Fathi Ismail Ali Naeem M. E. Doha Sahar El-Nahrawy

Drought significantly reduces global crop yields and agricultural productivity. This study aims to isolate drought-tolerant PGPR strains and evaluate their effects, both individually and in combination with salicylic acid (SA), on cowpea plants growth, physiological traits, antioxidant enzymes, and mineral content under both drought stress and non-stress conditions. Among fifteen bacterial isolates, AO7, identified as Streptomyces diastaticus subsp. ardesiacus PX459854 through 16S rRNA sequencing, demonstrated significant plant growth promotion in cowpea under gnotobiotic conditions. On the other hand, varying salicylic acid concentrations (0.5, 1.0, and 2.0 mM) was exposed to assess the plant growth of cowpea plants in a gnotobiotic system. A pot experiment in 2023 used a split-plot design with treatments for irrigation (unstressed and stressed) and different soaking treatments (control, S. diastaticus, salicylic acid (2 mM), and a combination). After 60 days, the combination treatment enhanced growth metrics, outpacing the control under stress. The microbial community in the T4 treatment exhibited the highest counts, while T8 (combination, stressed) showed lower counts but the highest chlorophyll content at 6.32 mg g−1 FW. Notable increases in proline and significant changes in enzyme activities (PO, PPO, CAT, and APX) were observed, particularly in treatment T8 under stress, indicating a positive response to both treatments. Mineral content of cowpea leaves varied with soaking treatments of S. diastaticus and SA (2.0%) especially under drought stress which the highest values were 1.72% N, 0.16% P, and 2.66% K with treatment T8. Therefore, T8 (combination, stressed) > T6 (S. diastaticus, stressed) > T7 (salicylic acid, stressed) > T5 (control, stressed) for different applications under stressed conditions and T4 (combination, unstressed) > T2 (S. diastaticus, unstressed) > T3 (salicylic acid, unstressed) > T1 (control, unstressed) for the other applications under normal conditions. Thus, using S. diastaticus and SA (2.0%) in combination greatly enhanced the growth dynamics of cowpea plants under drought stress conditions.

Applied Microbiology doi: 10.3390/applmicrobiol5040149

Authors: Peter Ayodeji Idowu Lwando Mbambalala Oluwakamisi Festus Akinmoladun Adeola Patience Idowu

Probiotics have emerged as gut modulators, capable of restructuring microbial communities to enhance animal health and performance. This review synthesizes peer-reviewed studies published between 2015 and 2025, retrieved from Scopus, Web of Science, and Google Scholar. It encompasses both ruminant and monogastric species to evaluate the effects of probiotic supplementation under diverse production environments. Evidence indicates that diet, age, host genetics, and management practices strongly influence gut microbiome composition and function, explaining the context-dependent nature of probiotic efficacy. These interventions improve growth performance, feed efficiency, gut morphology, pathogen resistance, and systemic immune parameters, supporting their potential as sustainable alternatives to antibiotic growth promoters. However, responses vary and are context-dependent, based on differences in strain specificity, dosage, host physiology, and environmental stress. By explaining how probiotic-mediated modulation translates into improved productivity, reduced antimicrobial dependence, and greater resilience in real-world farming systems, this review highlights their practical value for modern livestock production. Future research should focus on field-based validation, multi-omics approaches to resolve host–microbiota–probiotic interactions, and long-term assessments of animal health, productivity, and environmental impacts. Strategic deployment of probiotics, combined with scalable delivery technologies and regulatory alignment, can enhance resilience, sustainability, and efficiency in livestock production systems.

Applied Microbiology doi: 10.3390/applmicrobiol5040148

Authors: Mariah Graham Valbuena Michelle Marie Esposito

The microbiota, comprising microorganisms and their genes, plays a crucial role in health and disease susceptibility. Recent advances have enhanced our understanding of these microbial communities in dogs, which vary by body site—skin, ears, eyes, lungs, etc.—and are influenced by factors such as age, breed, sex, environment, and diet. Dysbiosis, or microbial imbalance, is increasingly linked to various health conditions. Investigating these microbial communities can lead to improved strategies for maintaining pet health. This review explores the impact of microbiota across multiple canine systems, including dental, gut, cardiac, skin, renal, as well as dietary influences. Clinically, microbiome analyses can provide valuable insight into the bacterial changes in healthy versus diseased states and can be used as potential biomarkers for veterinary diagnostics. The information gained from these analyses can also allow for more effective management strategies to be developed, increasing treatment efficacy. This review seeks to demonstrate the crucial role that microbiome analyses could play in future veterinary preventative medicine, diagnostics, and treatment plans. Here we connect microbial dysbiosis, bacterial and fungal, to local and systemic health issues, and then examine the implications towards treatment, especially in cases of high resistance. As microbiota communities on and in dogs are a potential reservoir for human spread, the information elucidated within this review also helps future assessments of pathogenicity and transmission mechanisms that could lead to dangerous zoonotic spread of disease.

Applied Microbiology doi: 10.3390/applmicrobiol5040147

Authors: Ingrid Alarcon-Ancajima Fernando Merino Susana Gutierrez-Moreno

Rhamnolipids (RL) are biosurfactants produced mainly by Pseudomonas aeruginosa strains that have environmental and industrial applications. However, their industrial-scale production still faces the challenge of improving the efficiency and cost-effectiveness of the process. The aim of this work was to optimize the cultivation conditions to increase the RL production by using Response Surface Methodology with key parameters of the process, such as oxygen level, agitation, temperature, nutrients, and residual frying oil as a low-cost carbon source. The optimized parameters were 3.04 g/L of nitrogen, 0.5 vvm of aeration and 180 rpm of agitation, with which 52.2 g/L was obtained in 168 h. The critical micellar concentration (CMC) of this RL was 3.14 g/L, and the Oil Spreading assay confirmed the presence of surface-active compounds in the purified RL that generated an average halo area of 2746.7 ± 72.0 mm2, which represents an increase of 2063.41% ± 28.36% compared to the negative control. These advances could contribute to more sustainable, cost-effective RL production, promoting its application in bioremediation processes and other industries.

Applied Microbiology doi: 10.3390/applmicrobiol5040146

Authors: Enrique José Salazar Llorente Fernando Javier Cobos Mora Aurelio Esteban Amaiquema Carrillo Matteo Radice Luis Humberto Vásquez Cortez Brayan F. Torres Salvatierra

Agro-industrial by-products are rich in polyphenols with potential applications as natural antimicrobials in food systems. This study evaluated the total polyphenol content (TPC) and antimicrobial activity of orange (Citrus sinensis), onion (Allium cepa), cacao (Theobroma cacao), and tamarillo (Solanum betaceum) peel extracts against Listeria monocytogenes, individually and in combination with Saccharomyces cerevisiae. TPC was quantified using the Folin–Ciocalteu method, and minimum inhibitory concentrations (MICs) were determined using broth microdilution. Statistical analysis (two-way ANOVA, p < 0.05) assessed the effect of extract type and yeast addition on MIC values. The highest TPC was recorded in Theobroma cacao peel extract (85.3 ± 2.1 mg GAE/g DW). All extracts inhibited L. monocytogenes, with MICs ranging from 2.5 to 10 mg/mL. This was reduced to 1.25–5 mg/mL when combined with S. cerevisiae, indicating synergism (F = 11.42, p = 0.003). These results suggest that polyphenol-rich peel extracts enhanced by S. cerevisiae can be incorporated into beverage preservation systems, aligning with clean-label trends. This study integrates quantitative and mechanistic analyses to link extraction methods, polyphenol content, and synergistic inhibition with Saccharomyces cerevisiae, providing a coherent analytical framework for sustainable antimicrobial strategies.

Applied Microbiology doi: 10.3390/applmicrobiol5040145

Authors: Pratikchhya Adhikari Peter M. Muriana

The concept of using ‘acid-adapted’ challenge cultures in the microbial validation of food processes that incorporate an acidic treatment is that they would be more resistant to acid and require a robust process to obtain targeted log reductions. The recent confirmation that acid-adapted Salmonella challenge cultures for droëwors and biltong processes are more sensitive to those processes than non-adapted cultures changes that preference for the use of non-adapted cultures for validation studies with these specific processes. However, it is difficult to achieve > 5-log reductions with non-adapted cultures, one of two USDA-FSIS parameters available for validation of processes that are not aligned with traditional process conditions for dried beef products in the USA (i.e., beef jerky). A natural multipurpose (flavor, antimicrobial) commercial product, described as a refined liquid smoke flavorant, provided >7-log reductions with droëwors when challenged with non-adapted cultures of Salmonella (5-serovar mixture), Shiga toxigenic Escherichia coli (STEC, four-strain mixture), and Listeria monocytogenes (four-strain mixture) as well as a >7-log reduction with biltong processing (vs Salmonella). Comparisons between standard droëwors and biltong processes (all <5-log reductions) using non-adapted challenge cultures vs. the same formulation plus 0.75% pyrolyzed liquid smoke extracts (Flavoset) showed greater and significant (p < 0.05) reductions in duplicate trials with triplicate samples at each sampling point in each trial (total n = 6) when analyzed by repeated measures analysis of variance (RM-ANOVA). Although sold as a flavorant, this study examines the antimicrobial properties of Flavoset 5400L to improve the safety of droëwors and biltong by achieving a >5-log reduction with non-adapted pathogenic challenge cultures. Validation processes for droëwors and biltong established with these parameters should result in greater safety of marinaded, non-thermally processed meats from traditional foodborne pathogens commonly associated with meats or meat processing environments.

Applied Microbiology doi: 10.3390/applmicrobiol5040144

Authors: Adeel Farooq Asma Rafique

High-quality bacterial genomes are essential for robust comparative genomics, reliable taxonomic assignment, and accurate pathogen and antimicrobial resistance (AMR) surveillance. Yet, public repositories still contain highly heterogeneous assemblies, and genome quality is often judged using single metrics in isolation. Here we develop an integrative Genome Quality Index (GQI) that combines four complementary metrics—including BUSCO single-copy completeness, contig number, N50, and unmapped read percentage—into a composite, interpretable score. We re-assembled and evaluated 474 pathogenic bacterial genomes submitted from South Korea using a standardized Illumina-based pipeline and validated the framework on an independent Enterobacteriaceae dataset (n = 5781). Species-level analyses and unsupervised clustering revealed pronounced variation in genome quality (one-way ANOVA, p < 1.05 × 10−33), with Cronobacter sakazakii and Listeria monocytogenes showing consistently high GQI scores, whereas Mycobacterium tuberculosis exhibited broad variability, including clear low-quality outliers. After log-transforming skewed variables, contig count and N50 remained strongly negatively correlated (r = −0.83), while BUSCO completeness showed moderate positive association with N50 and negative association with unmapped reads. GQI scores spanned 0.23–0.96, with most genomes clustering between 0.70 and 0.85. A Random Forest classifier trained on the four raw metrics predicted GQI-based quality tiers (low, medium, high) with 97% accuracy. From the top-decile genomes, we derived empirical thresholds like BUSCO ≥ 98.6%, contigs ≤ 30, N50 ≥ 1 Mb, and unmapped reads ≤ 0.82% that refine existing recommendations and provide actionable curation criteria. Our framework complements tools such as CheckM, gVolante, and Hybracter by offering a platform-agnostic composite scoring system that can be integrated into submission workflows and surveillance pipelines to systematically flag low-quality genomes and improve the reliability of microbial genomics.

Applied Microbiology doi: 10.3390/applmicrobiol5040143

Authors: Matteo Calcagnile Andrea Giuliano Fabio Paladini Stefania Villani Salvatore Maurizio Tredici Francesco Dondero Vincenzo Nassisi Pietro Alifano

Widespread environmental contamination by perfluoroalkyl and polyfluoroalkyl substances (PFAS) is raising particular concerns. PFAS are remarkably resistant to microbial degradation and have a profound impact on the structure and function of microbial communities. In this study, we analyzed the effect of perfluorooctanoic acid (PFOA) on bacterial quorum sensing, a communication process that in marine Vibrio species regulates biofilm formation and dissolution, virulence factors, swimming/swarming motility and bioluminescence. A system to continuously monitor bioluminescence during the growth on agar medium of Vibrio campbellii BB120 and isogenic luxS-, cpsA- and luxM-defective mutants, unable to synthesize, respectively, the autoinducers AI-2, CAI-1, and HAI-1, was utilized. By this system, we found that PFOA has dramatic effects on bacterial growth on agar and light emission kinetics, with specific effects in the different strains depending on the set of the autoinducers produced. Furthermore, we found that PFOA inhibited swarming motility in cqsA- and luxM-defective mutants which exhibited a very robust swarming phenotype in the absence of PFOA due to the lack of CAI-1 or HAI-1 that inhibit motility. The inhibitory effect on motility could be due to increased adherence of bacterial colonies to the agar substrate caused by the presence of PFOA. These results, although obtained in an in vitro system, suggest that PFOA may strongly interfere with bacterial growth kinetics and quorum sensing-regulated responses.

Applied Microbiology doi: 10.3390/applmicrobiol5040142

Authors: Lyudmila Asyakina Pavel Barsukov Yuliya Serazetdinova Olga Baturina Natalya Fotina Alexander Prosekov Marsel Kabilov

Chemical fungicides play a key role in protecting crops, but their use can result in environmental problems. We tested a novel fungicide, composed of endophytic microorganisms, for its effect on wheat yield, grain quality, plant development, and the rhizosphere microbiome, assessed by 16S and ITS metabarcoding. The fungicide increased the grain yield, the effect being similar to a well-known commercial bacterial fungicide, without affecting its quality. Ascomycota, Zygomycota and Mucoromycota together comprised 80% of the mycobiome. Mucoromycota/Mucoromycetes/Rhizopodaceae/Rhizopus arrhizus were significantly decreased. The dominant (≥10%) bacterial phyla were Pseudomonadota, Acidobacteriota, Bacteroidota and Actinomycetota, but their fungicide-related differences were small or random. Different modes of fungicide application (seeds only, seeds plus one or two foliar applications) had no effect on wheat characteristics. Neither of the fungicide’s agents (Raoultella ornithinolytica and Pantoea allii) were found in the rhizosphere. The changes in the mycobiome seemed more pronounced than in the bacteriobiome. The proposed preparation is concluded to have good prospects as a fungicide. However, the low species/strain resolution of the DNA metabarcoding did not allow us to fully interpret shifts in the microbiome diversity, both agronomically and environmentally. These aspects need more comprehensive investigation, using methodology with higher species resolution.

Applied Microbiology doi: 10.3390/applmicrobiol5040141

Authors: Waleed Almomani Deniz Al-Tawalbeh Khaled Alwaqfi Ali BaniHani Lujain Abuirsheid Raghad Ayasreh Mohammad BaniHani Andre Barreiros Mohammad Albataineh

The gut microbiome plays a vital role in metabolism and can be significantly influenced by body mass index (BMI). This study investigated the variations in gut microbial composition and function across different BMI categories by analyzing 16S rRNA sequencing data of 126 stool samples. While our analysis of microbial diversity did not reveal significant differences among BMI groups, a differential abundance analysis identified specific bacterial genera associated with BMI status. Notably, Lachnospira, Lactobacillus, and Roseburia were enriched in non-obese individuals, while Phascolarctobacterium showed greater abundance in obese subjects. Functional profiling utilizing PICRUSt2 and DESeq2 revealed fifteen KEGG pathways that exhibited significant alterations across varying BMI groups. Notably, several of these pathways were associated with short-chain fatty acid (SCFA)-producing taxa, including Lactobacillales and Tannerellaceae. Additionally, covariance network analysis identified the microbial genera Alistipes and Bilophila as central participants in multiple metabolic pathways, particularly those associated with steroid biosynthesis and pathogenic Escherichia coli, which showed a notable enrichment in individuals with obesity. These findings suggest that BMI influences the composition and metabolic potential of the gut microbiome, highlighting the importance of functional profiling to better understand the mechanisms underlying obesity.

Applied Microbiology doi: 10.3390/applmicrobiol5040140

Authors: Ryan Page Kayanush Aryana

Lactobacillus acidophilus is a widely researched probiotic bacterium with broad applications in health and biotechnology; however, its protoplast formation has not been extensively investigated. This study aimed to optimize conditions for L. acidophilus protoplast formation. Freeze-dried cells were suspended in 20 mM HEPES buffer (pH 7) supplemented with sucrose (1.0 M, 1.5 M, and 2.0 M) to induce hyperosmotic conditions, yielding a final cell density of 108 cells/mL. The suspensions were treated with 125 µg/mL lysozyme and incubated at 37 °C for 30 min, 1 h, or 2 h. Prior to enzymatic treatment, the buffer, lysozyme, and cell suspensions were equilibrated at either 22 °C (room temperature) or 37 °C. Phase contrast microscopy was used to evaluate protoplast formation across all treatment combinations, and a three-way ANOVA was conducted to assess the effects of buffer molarity, incubation time, and temperature. Protoplasts are valuable tools for genetic manipulation, cell fusion, and cell wall studies, yet optimized protocols for their generation in L. acidophilus are lacking. The highest protoplast yield with minimal lysis was observed under 2.0 M sucrose conditions after 2 h of incubation, particularly when all components were equilibrated at 37 °C. Prolonged lysozyme exposure increased lysis, especially at lower buffer molarities. Elevated buffer molarity conferred a protective effect by maintaining cell integrity during enzymatic digestion. These findings highlight the importance of osmotic strength and thermal equilibration in optimizing protoplast formation and provide a reproducible framework for controlled enzymatic treatments in L. acidophilus.

Applied Microbiology doi: 10.3390/applmicrobiol5040139

Authors: Arman Mussakhmetov Dmitriy Silayev

Esterases are ubiquitous enzymes found in all living organisms, including animals, plants, and microorganisms. They are involved in several biological processes, including the synthesis and breakdown of biomolecules, such as nucleic acids, lipids, and esters; phosphorus metabolism; detoxification of natural and artificial toxicants; polymer breakdown and synthesis; remodeling; and cell signaling. The present review focuses on the most industrially important esterases, namely lipases, phospholipases, cutinases, and polyethylene terephthalate hydrolases (PETases). Esterases are widely used in industrial and biotechnological applications. Notably, the biotechnological production of esters, including methyl acetate, ethyl acetate, vinyl acetate, polyvinyl acetate, and ethyl lactate, as an alternative to chemical production, represents a multi-billion-dollar industry. Currently, most enzymes (>75%) used in industrial processes are hydrolytic. Among them, lipases and phospholipases are primarily used for lipid modification. Lipases are the third most commercialized enzymes after proteases and carboxyhydrases, and their production is steadily increasing, currently representing over one-fifth of the global enzyme market. Esterases, particularly lipases, phospholipases, and cutinases, are employed in cosmetics, food, lubricants, pharmaceuticals, paints, detergents, paper, and biodiesel, among other industries. Overall, biotechnological production using enzymes is gaining global traction owing to its environmental benefits, high yields, and efficiency, aligning with green economy principles.

Applied Microbiology doi: 10.3390/applmicrobiol5040138

Authors: Janet Leandro Marmolejo María Magdalena Crosby Galván Elsa Margarita Crosby Galván Laura Haydée Vallejo Hernández María Teresa Sánchez-Torres Esqueda César Cortez Romero Ricardo Daniel Améndola Massiotti

Streptomyces avermitilis is a soil actinobacterium and has a complex metabolism in its natural habitat. Because of this, the environmental fluctuations present in the seasons can activate or silence the biosynthetic pathways involved in its metabolism. The objective of this research was to analyze the morphological characteristics of the metabolism of Streptomyces avermitilis, isolated during the four seasons of the year and from four types of soil. Isolation was performed on oat agar ISP-3 and nystatin as an antifungal agent. The planting methods were rod drag and cross striations. The Petri dishes were incubated for 10 days at 30 °C in complete darkness. For 10 days, a colony count was performed to analyze the growth curves, as was an evaluation of the diffusible pigments in each Petri dish. The isolates presented the diffusible pigments white, yellow, orange, red and pink with a higher proportion in spring and summer compared to in autumn and winter. Under laboratory conditions, the isolates in summer presented the three phases of bacterial growth: lag (24 h), exponential (48–96 h) and stationary (120–168 h). A doubling time of 35.30–62.92 h was obtained. The morphological characteristics of the metabolism of Streptomyces avermitilis show differences according to the climatic conditions of each season of the year.

Applied Microbiology doi: 10.3390/applmicrobiol5040137

Authors: Nguyen Quoc Khuong Ly Kim Quyen Tran Trong Khoi Nguyen Nguyen Duc Trong Le Thi My Thu Vo Yen Ngoc Le Thanh Quang La Cao Thang Pham Thi Phuong Thao

Microorganisms are considered a potential source of biofertilizers for mobilizing nutrients from insoluble mineral potassium (K). This study was conducted to evaluate the effects of liquid potassium-solubilizing bacteria, Cereibacter sphaeroides M-Sl-09, Rhodopseudomonas thermotolerans M-So-11, and Rhodopseudomonas palustris M-So-14 (LPS-PNSB), on soil K content, plant K uptake, growth, and yield of hybrid maize cultivated on alluvial soil in the dyke-protected area of An Phu, An Giang, Vietnam. Results showed that the application of LPS-PNSB significantly improved exchangeable soil K from 0.428 to 0.460–0.470 meq 100 g−1, total plant K uptake from 181.5 to 205.8–259.4 kg ha−1, and yield from 11.1 to 12.2–12.6 ton ha−1, compared with the recommended 100% NPK fertilization. The addition of LPS-PNSB allowed a 100% reduction in K fertilizer compared with the recommended rate while still maintaining yield. Hybrid maize grain yield further increased when 100% recommended K was applied in combination with LPS-PNSB, surpassing the yield obtained with 100% K alone.

Applied Microbiology doi: 10.3390/applmicrobiol5040136

Authors: Asmae Aboulkacem Hanane Zaki Amina Aboulkacem Tarik Ainane Rafail Isemin Fatouma Mohamed Abdoul-Latif Ayoub Ainane

Groundwater represents an essential resource for domestic and agricultural use, and its physicochemical and microbiological quality directly affects public health. This study assessed the bacteriological quality of untreated well water in the province of Fez-Meknes, specifically in the Aïn Tawjdate area, and evaluated seasonal variations in bacterial contamination. During the spring and summer of 2023, groundwater samples were collected from several wells. A total of 139 bacterial strains were isolated and identified using API biochemical galleries. The most frequently detected species were Aeromonas hydrophila gr.1 (6.47%), Aeromonas hydrophila gr.2 (9.35%), Enterobacter cloacae (7.19%), Pseudomonas aeruginosa (10.07%), and Flavimonas oryzihabitans (6.47%), among others. Genetic variability among ten E. cloacae isolates was further explored using ERIC-PCR profiling; the strains differed by more than three fragments and showed less than 80% similarity; therefore, they were considered as distinct ERIC types. Statistical analyses (Chi-square, Fisher’s exact, Tukey HSD, one-way ANOVA, and two-sided Dunnett tests) revealed no significant differences in bacterial load between wells within the same season, with p-values > 0.05 according to ANOVA. However, a significant increase in contamination levels was observed in summer compared with spring. These findings highlight the potential health risks associated with the consumption of untreated groundwater and underline the need for regular microbiological monitoring and improved water treatment practices in rural communities.

Applied Microbiology doi: 10.3390/applmicrobiol5040135

Authors: David Bulnes Irene Albertos José-María Jiménez María José Castro-Alija Alexandra Díez-Méndez

Donkey milk is an underexplored biological niche with distinctive nutritional and microbiological properties, potentially harboring lactic acid bacteria (LAB) with technological or probiotic value. In this study, raw milk from the endangered Zamorano-Leonesa donkey breed was stored at 4 °C for 24 h to simulate realistic cold-chain conditions and favor the recovery of cold-tolerant microorganisms. Fourteen isolates were obtained, eight of which belonged to LAB or species with potential technological interest and were selected for functional evaluation. Phenotypic screening showed that most isolates tolerated acidic conditions (pH 2.5) and that four also resisted 0.3% bile salts. Acidification assays in pasteurized donkey milk revealed variable fermentation performance, with L. mesenteroides subsp. mesenteroides B8 and Lacticaseibacillus paracasei subsp. tolerans B19 displaying the most favorable profiles. These two strains were selected for genome sequencing. Genomic analysis revealed genes associated with acid and bile resistance, adhesion, cold and environmental stress responses, and carbohydrate metabolism. Both genomes also encoded biosynthetic gene clusters linked to secondary metabolites, including β-lactones, lincosamides, and RiPP-like compounds. No acquired antimicrobial resistance genes were detected. To our knowledge, this is the first study combining isolation, phenotypic screening, and genome-based characterization of cold-tolerant LAB from Zamorano-Leonesa donkey milk. Our findings highlight this milk as a valuable reservoir of safe, cold-adapted microorganisms with promising applications in functional dairy products and food biotechnology.

Applied Microbiology doi: 10.3390/applmicrobiol5040134

Authors: Priyanka Mishra Sidharth P. Mishra Aryamav Pattnaik Swoyam Singh Ahmad R. Shakri Jhasketan Badhai Soumyajit Ganguly Ashirbad Guria Satyanarayana S. Cheekatla

The gut microbiota, a dynamic and metabolically active microbial ecosystem, plays a pivotal role in regulating host digestion, immune homeostasis, metabolism, and hormone signaling. Among its specialized functions, the estrobolome (a collection of bacterial genes involved in estrogen metabolism) has emerged as a key regulator of systemic estrogen levels. Through microbial β-glucuronidase activity, estrogens undergo deconjugation and reabsorption, influencing the pathogenesis of hormone-receptor-positive breast cancers. Disruption of the gut microbial balance, termed dysbiosis, can result from dietary changes, antibiotic use, environmental toxins, and psychosocial stress. Dysbiosis alters intestinal permeability, immune responses, and microbial metabolite profiles, contributing to chronic inflammation and endocrine disruption. Mechanistic links between gut microbiota and breast cancer include altered estrogen recirculation, immunomodulation, shifts in microbial metabolites (e.g., SCFAs, bile acids, tryptophan derivatives), and stress-mediated signaling through the microbiota–gut–brain axis. Accumulating preclinical and clinical evidence reveals distinct microbial signatures in breast cancer patients, supporting a causal or contributory role of gut dysbiosis in tumorigenesis. In parallel, biotics (including probiotics, prebiotics, synbiotics, and postbiotics) offer promising avenues for modulating the microbiota. Certain strains of Lactobacillus (L.) and Bifidobacterium (B.) exhibit anti-inflammatory and estrogen-modulating effects, while dietary fibers and microbial metabolites may enhance epithelial integrity and immunocompetence. This review critically examines the interplay between gut microbiota and breast cancer, elucidates the mechanistic pathways involved, and evaluates the current evidence on microbiota-targeted interventions. We also highlight research gaps, safety considerations, and the potential for integrating microbiome modulation into personalized oncologic care. This review uniquely integrates mechanistic pathways with those supported by preclinical and clinical evidence on biotics, highlighting microbiome-based precision strategies for breast cancer prevention and management.

Applied Microbiology doi: 10.3390/applmicrobiol5040133

Authors: Akale Assamere Habtemariam Péter Cseh Mihály Csizmár Ferenc Fodor Zoltán Bratek

We hypothesized that Tuber melanosporum colonization enhances growth and photosynthetic performance in Corylus avellana seedlings. Forty-eight seedlings were assessed for root colonization (stereomicroscopy, ITS sequencing) and photosynthetic traits (Li-6800F) under short-term disturbed and undisturbed rhizosphere conditions. Mycorrhizal colonization was found in 97.9% of seedlings (47/48). The mean colonization was 33.1% (SD = 16.1), 16.7% of seedlings showed more than 50% colonization per seedling, and 65.0% showed more than 30% colonization per seedling. Colonization declined with root depth and correlated with seedling length (r = 0.371, p = 0.01). In disturbed roots, longer root length predicted higher Gsw (r = 0.60), PhiCO2 (r = 0.77), and PhiPSII (r = 0.70), while collar diameter negatively affected transpiration (r = −0.60). In undisturbed roots, collar-proximal colonization improved PhiPSII (r = 0.69, p = 0.02). Undisturbed seedlings showed ~2× higher CO2 assimilation, stomatal conductance, quantum yield, and transpiration. These findings confirm that T. melanosporum enhances seedling physiology, especially under undisturbed conditions.

Applied Microbiology doi: 10.3390/applmicrobiol5040132

Authors: Rakesh Kaushik Sanjeev Anand

Cheddar cheese ripening creates favorable conditions for desired microbial changes but also allows survival and outgrowth of spores like Clostridium tyrobutyricum and Bacillus licheniformis, leading to late-blowing defects. In the first phase of the study, NSLAB dynamics were evaluated in the presence of spores, where pilot-scale cheeses (110 L) were produced in four treatments: control, T1 (BL), T2 (CT), and T3 (BL+CT), each inoculated at 2.0 Log10 CFU/mL with spores. Results showed that SLAB declined from 8.0 to 0.2 Log10 CFU/g, while NSLAB increased from 2.0 to 8.5 Log10 CFU/g by the third month and remained stable thereafter. Spore counts reached 2.94 ± 0.02 (T2) and 2.48 ± 0.03 (T3) Log10 CFU/g, with visible spoilage signs appearing after five months, indicating that native NSLAB populations were inadequate to control late-blowing defects. In this study, the effect of soluble fiber (inulin) in stimulating NSLAB was evaluated by incorporating 1% inulin into Cheddar cheese across four treatments: T1 (C SF), T2 (BL SF), T3 (CT SF), and T4 (BL+CT SF). Inulin addition resulted in significantly higher NSLAB counts (>10.5 Log10 CFU/g) and suppressed spore levels (<0.91 ± 0.03 Log10 CFU/g), with no spoilage observed. Inulin addition selectively enhanced beneficial NSLAB, suppressing spore-forming bacteria and preventing late-blowing defects without affecting cheese quality. This provides a natural, sustainable strategy to enhance microbial safety during Cheddar cheese ripening.

Applied Microbiology doi: 10.3390/applmicrobiol5040131

Authors: Ales Eichmeier Milan Spetik Lucie Frejlichova Jakub Pecenka Jana Cechova Lukas Stefl Pavel Simek

The genus Tilia (Malvaceae) comprises long-lived broadleaf trees of considerable ecological, cultural, and historical importance in temperate Europe and Asia. Among these, Tilia × europaea L. (common European linden) is a key native species in Central and Northern Europe, with individuals documented to live for several centuries. While the phyllosphere and soil-associated microbiomes of linden have been studied, the internal fungal communities inhabiting ancient trees remain poorly understood. In this study, the complete mycobiome of linden tree wood was analyzed. Wood-inhabiting fungi (the wood mycobiome) include endophytes, saprotrophs, and potential pathogens that can strongly influence host vitality and ecosystem processes. Advances in high-throughput amplicon sequencing (HTAS) now provide unprecedented opportunities to characterize these hidden communities. In this study, we investigated the trunk wood mycobiome of an ancient T. × europaea L. individual using a culture-independent HTAS approach. The results reveal a diverse fungal assemblage, including taxa like Arthinium or Phialemonium not previously reported from living linden wood, and highlight potential implications for tree health and longevity. This work provides a first baseline characterization of the internal mycobiome of the ancient Tilia tree and contributes to broader efforts to conserve its biological and cultural value.

Applied Microbiology doi: 10.3390/applmicrobiol5040130

Authors: Iskra Vitanova Ivanova Yavor Rabadjiev Maria Ananieva Ilia Iliev Svetoslav Dimitrov Todorov

Fructophilic lactic acid bacteria (FLAB), Apilactobacillus kunkeei strains AG8 and AG9 were selected in the current study for in-depth analysis. Cultivation on fructose yeast peptone (FYP) medium with varying fructose concentrations (1%, 10%, and 30%) revealed that higher fructose levels promoted acetate production over lactate, confirming a heterofermentative metabolic profile. Ethanol production was negligible, consistent with the absence of alcohol dehydrogenase (ADH) activity. Enzyme assays showed fructokinase activity doubled at 30% fructose, while acetate kinase activity increased and L-lactate dehydrogenase activity decreased. This shift in enzyme ratios from 1:1 at 1% fructose to 10:1 or 15:1 at higher concentrations explains the metabolic preference for acetate. Apb. kunkeei is an obligate FLAB, growing poorly on glucose unless supplemented with external electron acceptors like pyruvate or oxygen. It lacks ADH, but retains acetaldehyde dehydrogenase (ALDH), enabling acetate production and additional ATP generation, enhancing biomass yield. The absence of the adhE gene contributes to NAD+/NADH imbalance and favors acetate production. Gene expression studies targeting fructose transport enzymes showed elevated expression of ABC transporters and carbohydrate metabolism genes in response to fructose. ADH expression remained low across sugar concentrations. Fructokinase gene expression was shown to be strain specific. Neither strain expressed the ABC transporter ATP-binding protein gene on glucose, nor the bacteriocin ABC transporter gene, correlating with the absence of antibacterial activity. These findings underscore the metabolic specialization of Apb. kunkeei, its reliance on fructose, and the role of ABC transporters in optimizing fermentation. The strain-specific gene expression and metabolic flexibility highlight its potential as a probiotic and feed additive in apiculture and biotechnology.

Applied Microbiology doi: 10.3390/applmicrobiol5040129

Authors: Anyelo Florez Angie Patiño-Montoya Hernan Florez-Ríos Madelaine Piedrahita Juan Pablo Arias Marmolejo Néstor Roncancio-Duque Diana López-Alvarez Andrés Castillo

The microbiome of howler monkeys is being studied as a potential indicator of forest health. This explorative research aimed to analyze the microbiome, antibiotic resistance genes, and virulence factors of the howler monkey (Alouatta seniculus) in two Colombian Andean forests. A total of six samples were collected from three monkeys in two different forests. The samples were processed and sequenced using 16S rRNA V3-V4 metabarcoding and shotgun metagenomics. No significant differences in microbial diversity were observed between locations. A total of 43 possible resistance genes were identified, 11 of which were associated with plasmids, while 66 virulence genes were detected. The bacterial genera with the highest number of resistance genes were Escherichia and Enterococcus, whereas Escherichia and Citrobacter exhibited the highest number of virulence factors. The bacteria were predominantly resistant to fluoroquinolones, macrolides and beta-lactams, while adherence was the dominant virulence mechanism. This exploratory study suggests that the locations provide similar habitats for howler monkeys and that the presence of resistance genes is primarily due to intrinsic bacterial resistance mechanisms and natural resistance in wild populations despite the environmental presence of bacterial genera with resistance genes and virulence factors. However, acquisition through interaction with domestic animals was not evaluated.

Applied Microbiology doi: 10.3390/applmicrobiol5040128

Authors: David Talavera-Cortés Laureano E. Carpio Patricia Serrano-Candelas Carlos Lafita María José Tàrrega Marti Ángela Baeza-Serrano Pau Granell Rafael Gozalbes Eva Serrano-Candelas

Microorganisms such as methanogenic archaea play a key role in wastewater treatment plants (WWTPs) by breaking down organic matter and pollutants and producing methane, a potential renewable energy source. However, sulphate-reducing bacteria (SRB) compete with archaea for the same substrates under anaerobic conditions, lowering methane production and generating harmful hydrogen sulphide (H2S). Inhibiting SRB is therefore crucial to enhance methane yield and reduce toxic by-products. By means of manual screening of public databases (KEGG, BRENDA, PDB, PubChem) 12 potential inhibitors of SRB were found. After computational ecotoxicological assessment, four candidates were selected, and one of them experimentally increased methane production, demonstrating that SRB inhibition favours the anaerobic digestion of sludges. In order to further explore new candidates, Quantitative Structure–Activity Relationship (QSAR) models were developed showing reliable predictive performance. These models enabled the virtual screening of COCONUT, a natural product database, identifying 73 potential SRB inhibitors. After an ecotoxicological assessment, five commercially available compounds remained. The identified candidates may reduce competition between SRB and methanogenic archaea, leading to higher methane production and supporting WWTPs in generating their own biogas. This would contribute to a circular economy and help mitigate greenhouse gas emissions.

Applied Microbiology doi: 10.3390/applmicrobiol5040127

Authors: Francisco Zea-Gamboa Claudia Clavijo-Koc Jose Fernando Sandoval-Niebles Virginia Liliana Chipana-Laura Jhonny Paredes-Escobar Dayana Araceli Condori-Pacoricona Daladier Castillo-Cotrina

Bioleaching represents a sustainable and economically viable method for recovering metals from sulfide ores. This study evaluates the bioleaching potential of eight native mesophilic strains isolated from chalcopyrite ore sourced from the Toquepala mine in Peru. The strains were molecularly and phylogenetically identified as Leptospirillum ferriphilum based on 16S rRNA gene sequencing. Bioleaching process was evaluated over 648 h in triplicate flask assays at 30 °C and 120 rpm. All strains demonstrated leaching activity, achieving an average copper recovery above 30%. Notably, strains M1D and M3E demonstrated superior performance, with iron oxidation reaching approximately 50%, copper recoveries of 34.78% and 32.61%, respectively, and peak cell counts of up to 2.45 × 109 cells/mL. Specific growth rates were determined as 0.03469 h−1 for M1D and 0.03651 h−1 for M3E. A positive correlation was observed among microbial growth, iron oxidation, and copper recovery. These results confirm the potential of native mesophilic L. ferriphilum strains as efficient agents for chalcopyrite bioleaching at moderate temperatures, supporting their application in biotechnological metal recovery processes.

Applied Microbiology doi: 10.3390/applmicrobiol5040126

Authors: Gloria Anaí Valencia-Luna Damián Lozada-Campos Liliana Pardo-López Karla Sofía Millán-López Octavio Loera Armando Tapia-Hernández Beatriz Pérez-Armendáriz

This study explored the potential of bacterial consortia to remediate real diesel-contaminated agricultural soils. Two consortia were tested: a native consortium isolated from contaminated soil and an exogenous consortium derived from vermicompost. Bacterial communities (consortia and soils) were characterized through high-throughput sequencing. Within 30 days, total petroleum hydrocarbons (TPH) were removed most efficiently by bioaugmentation with the native consortium (53.32%), followed by the exogenous vermicompost consortium (47.14%) and the indigenous microbiota (42.52%). Gas chromatography confirmed the reduction of polycyclic aromatic hydrocarbons (PAHs) with 2–5 rings; however, terphenyl, chrysene, and pyrene persisted. The highest TPH biodegradation rate was observed in the treatment inoculated with the native consortium (208.5 mg/kg per day), followed by the treatment with indigenous microbiota (181.8 mg/kg per day) and the exogenous consortium (161.9 mg/kg per day). Furthermore, hydrocarbon-degrading bacterial populations increased significantly during the first week but declined after day 21, showing a negative correlation with TPH concentrations across all treatments, indicating that the highest bacterial activity and degradation occurred during the first 14 days. Taxonomic analysis identified Actinobacteria as the most abundant phylum in the initial soil, whereas Proteobacteria dominated both the consortia and the bioremediated soils. Significant differences in community structure and composition were observed between the consortia according to their origin, influencing removal efficiency. Dominant genera shifted from Nocardioides and Streptomyces in untreated soil to Pseudomonas, Sphingobium, and Pseudoxanthomonas following biological treatments, while Nocardia, Rhodococcus, and Bacillus remained nearly constant. These findings underscore the effectiveness of adapted bacterial consortia in restoring real diesel-contaminated agricultural soils and highlight potential microbial succession patterns associated with biodegradation and soil ecological recovery.

Applied Microbiology doi: 10.3390/applmicrobiol5040125

Authors: John Samelis Athanasia Kakouri

Autochthonous enterococci surviving mild thermization of raw milk (RM) before traditional Greek cheese processing may simultaneously comprise safe and virulent thermoduric strains with multiple antibiotic resistances (ARs). Therefore, this study biotyped and then compared the ARs of 60 Enterococcus isolates from two antilisterial sheep milks of native Epirus breeds before (RM) and after (TM) thermization at 65 °C for 30 s; the RM isolates were previously genotyped and evaluated for primary safety traits, namely, hemolytic activity, vanA/vanB, cytolysin, and virulence genes, by molecular methods. Biochemically typical and atypical strains of Enterococcus faecium (six biotypes), E. durans (five biotypes), E. faecalis (two biotypes), and E. hirae (one biotype), which were subdominant to other LAB species in RM (19 isolates), prevailed in TM (41 isolates). E. faecium biotypes 1A, 1D, and 1H included multiple-Ent+ (entA/entB/entP or entA/entB) strains with strong antilisterial CFS activity, whereas E. faecium 1X (entA), E. durans 2A, 2B, 2C, and 2X (entA/entP or entP), E. faecalis 3B, and E. hirae 4A (entA) biotypes displayed direct in vitro antilisterial activity only. Biotypes 1D, 1X, and 2A were selected in TM. All E. faecium/durans isolates were susceptible to vancomycin, but the m-Ent + E. faecium biotype 1A and 1D strains were resistant to penicillin, erythromycin, ciprofloxacin, and ampicillin. In contrast, all biotype 1X isolates were susceptible to all antibiotics tested. All E. faecalis and most E. durans isolates were resistant to penicillin but susceptible to erythromycin and ciprofloxacin. Biotype 2X isolates and one virulent (ace; gelE) E. faecalis isolate from RM were tetracycline-resistant. A sporadic RM isolate of E. hirae that was resistant to penicillin and vancomycin was not retrieved from the counterpart TM, but the inclusion of three vancomycin-resistant isolates from TM in the primary biotype 3B of E. faecalis was a cause for concern. In conclusion, based on the results, antibiotic-susceptible representatives of all strain biotypes of the E. faecium/durans group, as well as antagonistic m-Ent+ E. faecium strains from sheep milk that were susceptible to vancomycin and ampicillin and lacking virulence genes, can be included in safe complex natural starters to be developed for onsite use in traditional Greek hard cheese technologies.

Applied Microbiology doi: 10.3390/applmicrobiol5040124

Authors: Sergejs Kolesovs Inese Strazdina Linards Klavins Armands Vigants

The use of lactose-utilizing microalgae offers a sustainable and cost-effective approach for the bioconversion of dairy industry side-streams and the reduction in microalgae production costs. This work aims to improve the biomass productivity of the lactose-utilizing microalgal strain Graesiella emersonii MSCL 1718 in concentrated cheese whey permeate. It was demonstrated that the mixotrophic growth of the axenic G. emersonii culture resulted in a significantly higher biomass productivity in 20% permeate medium compared to the heterotrophic cultivation. Furthermore, supplementation of the permeate medium with iron, zinc, cobalt, and molybdenum resulted in 12.8%, 12.9%, 9.3%, and 28.9% significant increases (p < 0.05) in biomass synthesis, respectively, compared to the control permeate group. In the subsequent experiment, G. emersonii cultivated in molybdenum-supplemented permeate resulted in 0.34 ± 0.02 g/(L·d) biomass productivity and twofold higher lipid content (30.21 ± 1.29%) compared to the photoautotrophic control in defined synthetic medium. Analysis of the fatty acid composition revealed a twofold increase in saturated fatty acids, reaching 62.16% under mixotrophic cultivation in permeate, compared with the photoautotrophic control. Overall, concentrated cheese permeate proved to be a suitable medium for G. emersonii biomass production, supporting both enhanced growth and increased lipid accumulation.

Applied Microbiology doi: 10.3390/applmicrobiol5040123

Authors: Md. Motaher Hossain Farjana Sultana Mahabuba Mostafa Md. Tanbir Rubayet Nusrat Jahan Mishu Imran Khan Mohammad Golam Mostofa

Soil-borne plant pathogens pose a serious threat to global food security by causing extensive yield losses and compromising crop quality. Conventional chemical-based control methods often prove inadequate, environmentally harmful, and disruptive to beneficial soil microbiota, highlighting the urgent need for sustainable alternatives. Plant growth-promoting fungi (PGPF) have emerged as effective biocontrol agents capable of suppressing diverse soil-borne pathogens while simultaneously enhancing plant growth and resilience. This review synthesizes current knowledge on the tripartite interactions among plants, pathogens, and PGPF within the rhizosphere, with emphasis on their roles in disease suppression, rhizosphere competence, and plant health promotion. The findings highlight that PGPF such as Trichoderma, Penicillium, Aspergillus, non-pathogenic Fusarium, hypovirulent binucleate Rhizoctonia and sterile fungi can significantly reduce diseases caused by fungi, oomycetes, bacteria, nematodes, and protists through mechanisms including antibiosis, hyperparasitism, competition, and induction of systemic resistance. Evidence also indicates that consortium approaches and bioformulations enhance field efficacy compared to single-strain applications. Despite this progress, challenges such as variability in field performance, limited shelf life of inoculants, and gaps in understanding ecological interactions constrain large-scale use. Overall, the review underscores that PGPF-based strategies represent a promising and sustainable alternative to chemical pesticides, with strong potential for integration into holistic crop disease management under changing climatic conditions.

Applied Microbiology doi: 10.3390/applmicrobiol5040122

Authors: Manal Al-Kattan Afra Baghdadi Afnan Sahloli

Diabetic foot ulcers (DFUs) and other chronic wounds are major global health challenges, often complicated by infections and delayed healing due to excessive collagen accumulation. Microbial collagenases offer an enzymatic alternative to surgical debridement by selectively degrading collagen and potentially limiting microbial colonization. In this study, an isolated and characterized thermostable collagenase from Streptomyces scabies from rhizospheric soil in Al-Lith thermal springs, Saudi Arabia, is investigated. Identification was confirmed via 16S rRNA sequencing, and enzyme production was optimized on gelatin agar. Partial purification was achieved through ammonium sulfate precipitation and dialysis, and molecular weight (~25 kDa) was determined by Sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Activity was assessed under varying temperatures, pH, substrates, and metal ions, while antibacterial potential was tested against Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The collagenase exhibited optimal activity at 80 °C and pH 9, stability under thermophilic and alkaline conditions, activation by Fe2+, and notable antibacterial effects at higher concentrations. These results demonstrate that S. scabies collagenase exhibits selective antibacterial activity in vitro, suggesting its potential as an enzymatic tool for further evaluation in diabetic foot debridement and infection control.

Applied Microbiology doi: 10.3390/applmicrobiol5040121

Authors: Nahla Fadel Mohammed Abu-Elghait Hassan Gebreel Takeshi Zendo HebatAllah Youssef

This study reports the biosynthesis of selenium nanoparticles (Se-NPs) using four newly isolated strains of lactic acid bacteria, molecularly identified as Lactiplantibacillus pentosus, Lactiplantibacillus plantarum, Lactiplantibacillus plantarum, and Lactobacillus acidophilus. The synthesized Se-NPs were characterized using Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Spectroscopy (EDX), Fourier Transform Infrared Spectroscopy (FTIR), and UV-Vis Spectroscopy, and zeta potential analysis. The result revealed that their size ranged from 16 nm to 90 nm with favorable stability and purity. The Se-NPs exhibited significant antimicrobial and antibiofilm activities against certain Gram-positive, Gram-negative bacteria, and Candida albicans, particularly those produced by isolate S4, which showed the lowest MIC values and highest biofilm inhibition. Furthermore, MTT assays revealed selective cytotoxicity against the A549 cancerous lung cell line, with minimal toxicity toward normal Wi38 cells. These findings suggest that biosynthesized Se-NPs are a promising, biocompatible candidate for combating antibiotic-resistant pathogens and biofilm-associated infections.

Applied Microbiology doi: 10.3390/applmicrobiol5040120

Authors: Nikita Vasilchenko Maksim Kulikov Yaroslav Brislavsky Ludmila Khmelevtsova Anna Aleshukina Iraida Berezinskaya Andrey Gorovtsov Evgeniya Prazdnova Vladimir Chistyakov

This study investigates the range of secondary metabolites with antifungal activity produced by bacterial strains that have been previously characterised as antagonistic strains to phytopathogenic fungi. The properties of ten strains were analyzed in silico through functional genomic analysis and in vitro by mass spectrometry. The research has demonstrated that Bacillus and Paenibacillus strains possess diverse gene clusters for biosynthesis of secondary metabolites, including peptides and polyketides. Mass spectrometry analysis confirmed the synthesis of key antimicrobial peptides, such as surfactin, fengycin, macrolactin, and fusaricidin, which are associated with antifungal activity. These strains have been observed to exhibit robust antagonism against phytopathogenic fungi—the diameter of the inhibition zones for the presented strains was at least 20 mm. This feature highlights their potential as biocontrol agents. The presence of numerous uncharacterized gene clusters suggests the possibility of discovering previously undescribed bioactive compounds.

Applied Microbiology doi: 10.3390/applmicrobiol5040119

Authors: Catherine W. Y. Wong Xinyi Zhou Laura M. Carroll Megan L. Fay Joelle K. Salazar Wei Zhang

Listeria monocytogenes and Salmonella enterica Typhimurium are leading causes of foodborne illness in the United States and frequently implicated in produce outbreaks. Conventional decontamination methods, such as cold-water washes with chlorine, have limited antibacterial efficacy and environmental sustainability. Power ultrasound has emerged as a promising non-thermal alternative, but the molecular mechanisms remain insufficiently elucidated. This study evaluated transcriptomic responses of L. monocytogenes and S. enterica Typhimurium to (i) ultrasound (20 kHz), (ii) chlorine (50 ppm), and (iii) combined ultrasound + chlorine treatments. RNA-seq analysis identified differentially expressed genes, as well as enriched Gene Ontology and KEGG terms. Results showed that ultrasound and chlorine triggered distinct transcriptomic responses. L. monocytogenes exhibited broad transcriptional shifts under ultrasound, including significant upregulation of phosphotransferase system components and central metabolism. Chlorine alone induced a narrower response, with fewer differentially expressed genes clustering into limited functional categories. In contrast, the combined ultrasound + chlorine treatment elicited the strongest response in S. enterica Typhimurium, with enrichment of multiple energy- and metabolism-related pathways, including the citrate cycle, carbon metabolism, and microbial metabolism in diverse environments. These findings provide new insights into ultrasound-triggered responses in foodborne pathogens and may inform development of optimized ultrasound-based hurdle sanitization strategies for produce safety.

Applied Microbiology doi: 10.3390/applmicrobiol5040118

Authors: Chinedu Eucharia Joseph Aashika Jain Muneer Oladipupo Yaqub Lekshmi K. Edison

CRISPR-Cas systems are best known as adaptive immune defenses in prokaryotes, but they also function as versatile regulators bridging bacterial immunity with host-related processes. Beyond neutralizing invasive phages and plasmids, these systems influence core aspects of bacterial physiology, such as modulating gene expression, stress responses, biofilm formation, quorum sensing, and virulence. Notably, CRISPR-mediated regulation can facilitate immune evasion at the host-pathogen interface, underscoring these systems as central orchestrators of microbial survival and host interactions. In addition, CRISPR-Cas has rapidly become a cornerstone of synthetic biology and microbiome engineering. Recent strategies repurpose native and engineered CRISPR systems to precisely modulate microbiome composition or deliver sequence-specific antimicrobials, underscoring the expanding translational potential of this system. Collectively, emerging insights highlight both the canonical immune function and non-canonical regulatory roles of CRISPR-Cas, as well as their broad biological and biotechnological relevance. This review provides a critical synthesis of these developments, illustrating how CRISPR-Cas bridges adaptive immunity and microbial physiology, and outlines future directions for harnessing this duality to deepen understanding of microbial physiology and inform new translational applications.

Applied Microbiology doi: 10.3390/applmicrobiol5040117

Authors: Naomi Canourgues Emilie Adicéam Benoît Beitz Scott Atwell Maroussia Roelens Abdessalem Rekiki Christophe Vedrine Ilia Belotserkovsky

The intestinal microbiota plays a crucial role in maintaining epithelial barrier integrity, while its impairment and the resulting inflammation contribute to numerous human pathologies. To preserve intestinal homeostasis, various probiotics are being developed; however, their selection and validation require accessible yet physiologically relevant models. We recently established a high-throughput Gut-on-Chip model comprising human epithelial (Caco-2) cells and peripheral blood mononuclear cells (PBMCs), demonstrating epithelial barrier disruption and pro-inflammatory cytokine secretion upon inflammation induction. The present study aimed to evaluate the feasibility of co-culturing anaerobic members of the human intestinal microbiota within this model and to assess their effects on inflammation-induced epithelial damage. We successfully co-cultured five intestinal anaerobic bacterial species in direct contact with the epithelial monolayer for two days. As proof of concept, we demonstrate that live Bacteroides thetaiotaomicron and its supernatant preserve epithelial barrier integrity and attenuate CCL2 secretion by Caco-2 cells. In contrast, Clostridium scindens did not prevent epithelial damage but suppressed CCL20 secretion, revealing a promising target for future studies. By recapitulating some of the key aspects of intestinal inflammation, we suggest that the current Gut-on-Chip model has potential as an easy-to-use platform for screening next-generation probiotics and live biotherapeutics with homeostatic and immunomodulatory properties.

Applied Microbiology doi: 10.3390/applmicrobiol5040116

Authors: Allondra M. Woods Catherine J. Pettinger Catherine Harris Tanya Soule Garth Farley Erica L.-W. Majumder

Fats, oils, and grease (FOG) deposits are hardened, sticky, insoluble solids that accumulate in sewage systems globally. These deposits contribute to pipe blockages and sanitary sewer overflows, releasing pathogens and pollutants into the environment, posing significant environmental and public health risks. Current removal methods are labor-intensive and costly, emphasizing the need for alternatives. While biological strategies offer a viable alternative, the microbial breakdown of FOG is poorly understood. In this study, we evaluated the potential of individual microbial strains and synthetic microbial communities to biodegrade wastewater-derived FOG deposit samples. These biological agents were applied to a range of FOG samples, and biodegradation was assessed through visual observations such as color change or gas bubbles, particle size, cell counts, pH, weight loss, and changes in fatty acid profile. Results demonstrate that microbial augmentation can enhance FOG degradation, offering an alternative or complementary approach for reducing maintenance burdens and preventing sewer blockages.

Applied Microbiology doi: 10.3390/applmicrobiol5040115

Authors: Alice Nishigaki Kurt Arden Siân-Marie Frosini

The role of petting zoo animals in the dissemination of disease has been widely studied, yet understanding the potential reservoir of antimicrobial resistance (AMR) in these centres has not been explored in the United Kingdom (UK). To understand the carriage of AMR pathogens within petting zoos, this study aimed to identify AMR in E. coli and Staphylococcus intermedius group (SIG) isolated from faeces and skin, respectively, including selective cultures for ESBL-E. coli and methicillin-resistant staphylococci. Faecal samples and skin swabs were collected from 166 petted mammals across eight UK centres to recover E. coli and coagulase-positive staphylococci (CoPS), respectively, through enrichment culture methods, plating onto non-selective (tryptone bile-x agar, mannitol salt agar) and selective media (ESBL ChromID, mannitol salt agar with 6 mg/L oxacillin). Antimicrobial susceptibility was assessed using Kirby-Bauer disc diffusion, covering eight classes of antimicrobials. Antimicrobial usage records from the past 12-months were obtained from 7/8 centres. Overall, 145/166 faecal samples yielded 223 E. coli isolates, with an overall AMR prevalence of 42.6%. Thirteen E. coli isolates (from 8.5% of animals) were classified as multidrug-resistant. ESBL-producing E. coli were detected in 5/166 faecal samples. From 166 skin swabs, 84 yielded CoPS isolates, with S. aureus (n = 70), SIG (n = 13) and S. hyicus (n = 1) identified. Overall, 25.3% of SIG isolates exhibited resistance to at least one antimicrobial. Antimicrobial usage correlated positively with AMR prevalence for E. coli (p < 0.001), though was not associated with multidrug-resistance. This study demonstrates for the first time the presence of AMR within bacteria isolated from UK petting zoo animals, highlighting this reservoir of AMR bacteria.

Applied Microbiology doi: 10.3390/applmicrobiol5040114

Authors: Camila Buitrago-Zuluaga Carol Vanessa Osorio-Giraldo Aida Esther Peñuela-Martínez

In efforts to enhance the sensory profile of coffee, fermentation variations have been implemented, including extending the process for prolonged periods. Such practices create imbalances among the microbial groups involved and increase populations of filamentous fungi, compromising product safety. To identify the filamentous fungi present in fermentations for up to 192 h, coffee samples were collected from fermentations conducted under semi-anaerobic (SA) and self-induced anaerobic fermentation (SIAF) conditions. Microscopic, metabolic, and rDNA sequencing techniques were applied to identify the filamentous fungi. Relative abundance and taxonomic classification were obtained through High-Throughput Sequencing of the ITS region. In addition, the presence of Ochratoxin A (OTA) was evaluated through HPLC/FLD. The most abundant genera identified was Aspergillus in SA fermentations, and Fusarium in SIAFs both at 192 h. 3438 OTUs of filamentous fungi were obtained, distributed across 11 orders, 20 families, and 17 genera. The results suggest a greater presence of mycotoxin-producing genera in fermentations with longer processing times, particularly under semi-anaerobic conditions. However, OTA levels remained below 0.8 ppb (µg/kg). These findings provide essential information for microbiological control of fermentation, supporting the maintenance of product safety in subsequent stages and ensuring the safety of the final product.