Microorganisms

12 pages, 2036 KB

Open AccessArticle

A Swiss Retrospective Case Series of Pediatric Primary Subacute Hematogenous Osteomyelitis

by Elio Paris, Giacomo De Marco, Ahmer A. Khan, Anne Tabard-Fougère, Oscar Vazquez, Christina Steiger, Romain Dayer and Dimitri Ceroni

Microorganisms 2026, 14(2), 514; https://doi.org/10.3390/microorganisms14020514 - 23 Feb 2026

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Abstract

This study aimed to characterize the clinical spectrum and the bacteriological and microbiological etiology of pediatric primary subacute hematogenous osteomyelitis (PSAHO) and to evaluate a modern diagnostic approach for these infections. A single-center, 25-year retrospective review (2000–2025) of 107 consecutive cases of PSAHO [...] Read more.

This study aimed to characterize the clinical spectrum and the bacteriological and microbiological etiology of pediatric primary subacute hematogenous osteomyelitis (PSAHO) and to evaluate a modern diagnostic approach for these infections. A single-center, 25-year retrospective review (2000–2025) of 107 consecutive cases of PSAHO was performed. Clinical presentation, traditional inflammatory markers, conventional cultures and nucleic acid amplification tests (NAATs) on blood, and bone and oropharyngeal samples were assessed. Most patients (73.8%) were <4 years. Fever was uncommon (15.9%), and inflammatory markers were frequently normal (white blood cell counts in 81.1%, C-reactive protein levels in 60.4%) and abnormal in 69.2% for erythrocyte sedimentation rates and in 53.8% for platelet count. Low diagnostic sensitivity of conventional blood (4.2%) and bone (25.7%) sample cultures has been reported. In contrast, a comprehensive NAAT-based approach identified or strongly suggested a pathogen in 44.9% of cases. Kingella kingae was the predominant pathogen in children under 4. Oropharyngeal PCR tests potentially identified the pathogen in another 20 cases, and its presence could be reasonably suspected in a further 68 (63.6%). MRI was essential for diagnosis, identifying all lesions, whereas the sensitivity of radiographs was low (<50%). All patients recovered completely, regardless of the management strategy. This study provides three critical advances for clinical practice: (1) PSAHO should be considered in a limping toddler even without fever or elevated inflammatory markers, and MRI is the imaging modality of choice; (2) NAATs are indispensable for etiologic diagnosis, revealing age-dependent pathogens; (3) Oropharyngeal PCR is a useful diagnostic adjunct. Full article

(This article belongs to the Special Issue Pediatric Osteoarticular Infections: Diagnosis, Management, and Emerging Challenges)

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18 pages, 2155 KB

Open AccessArticle

Microbial Succession on Honey Bee Body Surfaces Reflects Behavioral Maturation

by Wenbo Wang, Chonghui Zhao, Yane Zhou, Chunling Yi, Mengfan Zhou, Yi Zhang, Shaokang Huang and Wenfeng Li

Microorganisms 2026, 14(2), 513; https://doi.org/10.3390/microorganisms14020513 - 23 Feb 2026

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Abstract

Behavioral maturation is essential for the proper functioning of honey bee societies and is regulated by multiple factors such as juvenile hormone (JH) and nutritional deficiency. Although recent studies have shown that surface-associated microbiota in insects can modulate host behavior, the relationship between [...] Read more.

Behavioral maturation is essential for the proper functioning of honey bee societies and is regulated by multiple factors such as juvenile hormone (JH) and nutritional deficiency. Although recent studies have shown that surface-associated microbiota in insects can modulate host behavior, the relationship between body surface microbiota and behavioral maturation in honey bees remains largely unexplored. This study aimed to determine whether the surface microbial communities of honey bees shift with behavioral maturation. By using 16S rRNA gene amplicon sequencing, we analyzed the surface microbiota of worker bees at different behavioral stages (newly emerged bees, nurses, and foragers) in both Eastern honey bee Apis cerana and Western honey bee Apis mellifera. The results showed that in both honey bee species, nurse bees exhibited the lowest microbial diversity, while forager bees showed the highest, and newly emerged bees had an intermediate level of microbial diversity. Moreover, beta diversity analyses revealed that the body surface microbiota of worker bees significantly varied across behavioral stages in both bee species and differed between the two bee species at the same behavioral stage. Additionally, in both bee species, at the phylum level, Pseudomonadota, Bacillota, and Actinobacteriota dominated the worker bee body surface microbiota; at the genus level, foragers had more Gilliamella, while nurses harbored more Lactobacillus. Together, our findings reveal the emergence of distinct microbial signatures on honey bee body surfaces during behavioral maturation. Full article

(This article belongs to the Topic Diversity of Insect-Associated Microorganisms)

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12 pages, 2038 KB

Open AccessArticle

Intrinsic Acidity of N-Acetylcysteine Mediates Enhanced Inhibition of Klebsiella pneumoniae and Its Biofilms by Polymyxin B

by Fei Wang, Weijie Wang and Haiying Gu

Microorganisms 2026, 14(2), 512; https://doi.org/10.3390/microorganisms14020512 - 23 Feb 2026

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Abstract

This study investigated the combined antibacterial and anti-biofilm activity of polymyxin B (PB) with intrinsically acidic N-acetylcysteine (NAC) against Klebsiella pneumoniae. The minimum inhibitory concentrations (MICs) of PB, acidic NAC, and neutralized NAC against 34 K. pneumoniae strains were determined using the [...] Read more.

This study investigated the combined antibacterial and anti-biofilm activity of polymyxin B (PB) with intrinsically acidic N-acetylcysteine (NAC) against Klebsiella pneumoniae. The minimum inhibitory concentrations (MICs) of PB, acidic NAC, and neutralized NAC against 34 K. pneumoniae strains were determined using the broth microdilution. Drug interactions were assessed by checkerboard assays and the fractional inhibitory concentration index (FICI), while biofilm inhibition was quantified using crystal violet staining. Polymyxin B resistance was identified in the reference multidrug-resistant strain K. pneumoniae ATCC BAA-1705. The PB–NAC combination showed an additive effect (FICI 0.53–0.63) against PB-resistant and PB-intermediately susceptible strains, whereas indifferent interactions were observed in PB-susceptible strains. Furthermore, sub-inhibitory concentrations of the combination produced significantly stronger biofilm inhibition than either agent alone. Neutralization of NAC markedly reduced its antibacterial and anti-biofilm activities, with substantial inhibition observed only at concentrations ≥ 32 mg/mL. These findings demonstrate that the combination of PB and acidic NAC exerts additive antibacterial effects, particularly against resistant K. pneumoniae strains, and enhances biofilm inhibition. Notably, the intrinsic acidity of NAC is essential for its antimicrobial and anti-biofilm activity. Full article

(This article belongs to the Section Antimicrobial Agents and Resistance)

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16 pages, 294 KB

Open AccessArticle

Interplay of Gastrointestinal Parasites, Micronutrient Deficiencies, and Anemia in Children from the Bolivian Highlands

by Washington R. Cuna, Roberto Passera and Celeste Rodriguez

Microorganisms 2026, 14(2), 511; https://doi.org/10.3390/microorganisms14020511 - 22 Feb 2026

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Abstract

Children living in resource-limited regions with inadequate environmental sanitation, such as the Bolivian highlands, are affected by parasitic infections that may compromise nutritional status. Objective: This study aimed to assess the prevalence of intestinal parasitic infections and their associations with nutritional status, micronutrient [...] Read more.

Children living in resource-limited regions with inadequate environmental sanitation, such as the Bolivian highlands, are affected by parasitic infections that may compromise nutritional status. Objective: This study aimed to assess the prevalence of intestinal parasitic infections and their associations with nutritional status, micronutrient deficiencies, and anemia in school-aged children from La Paz, Bolivia. Methods: A cross-sectional study was conducted among 212 schoolchildren aged 5–13 years in the municipality of La Paz, in highland areas characterized by high poverty levels. Parasitological examination, anthropometric measurements, and biochemical assessment of micronutrients (vitamins A and D, zinc, iron) were performed to evaluate children’s health status. Results: Mild malnutrition was more prevalent than moderate-to-severe forms. Micronutrient analysis revealed substantial deficiencies in vitamin A (39%), zinc (25%), and vitamin D (18%). Zinc deficiency was significantly more common in children aged 11–13 years compared to younger age groups (p = 0.034). Intestinal protozoan infections showed significant associations with micronutrient deficiencies. Giardia lamblia infection was associated with both vitamin A (30.9%, p = 0.042) and vitamin D (78.9%, p = 0.001) deficiencies. Blastocystis spp. infection was similarly linked to higher prevalence of vitamin A (35.8%, p = 0.025) and vitamin D (69.7%, p = 0.004) deficiencies. Entamoeba coli infection was significantly associated with vitamin D deficiency (p = 0.021), while Iodamoeba bütschlii infection showed a significant association with zinc status (p = 0.027), with notably lower zinc deficiency prevalence in infected children (7.7%) compared to non-infected children. Among helminth infections, Ascaris lumbricoides was significantly associated with vitamin D deficiency (37%, p = 0.018). Moderate-to-severe anemia was highly prevalent, affecting over half of the children regardless of sex. Wasting (BAZ) was significantly associated with age (p = 0.030), with moderate-to-severe cases most prevalent in children aged 5–7 years and absent in older groups, while mild wasting increased with age. In univariate logistic regression analysis, zinc deficiency emerged as a significant risk factor for anemia (OR = 2.51, 95% CI: 1.19–5.29, p = 0.016). No significant associations were observed between anemia and sex, age group, vitamin A or D status, or anthropometric indicators including underweight, stunting, or wasting. Conclusions: These findings highlight the substantial burden of micronutrient deficiencies, parasitic infections, and anemia among children in this impoverished region, underscoring the urgent need for targeted public health interventions addressing nutritional supplementation, parasite control, and improved sanitation. Full article

(This article belongs to the Section Gut Microbiota)

14 pages, 3241 KB

Open AccessArticle

Recombinant Cytosolic Truncations of Histidine Kinases Retain Function for Targeted In Vitro Investigations

by Jude Kinkead, Alexander D. Hondros, Aimee M. Figg, Milah M. Young, Richele J. Thompson, Christian Melander and John Cavanagh

Microorganisms 2026, 14(2), 510; https://doi.org/10.3390/microorganisms14020510 - 22 Feb 2026

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Abstract

Histidine kinases are an integral component of bacterial two-component systems (TCSs), playing a pivotal role in signal transduction pathways, resulting in both resistance and virulence. However, their inherent membrane-bound nature often results in poor solubility, making them difficult to isolate and rendering them [...] Read more.

Histidine kinases are an integral component of bacterial two-component systems (TCSs), playing a pivotal role in signal transduction pathways, resulting in both resistance and virulence. However, their inherent membrane-bound nature often results in poor solubility, making them difficult to isolate and rendering them incompatible with most in vitro biochemical techniques. Consequently, much of the research on two-component systems has centered on response regulators, limiting both drug discovery efforts and our broader understanding of key signal transduction mechanisms. To address these challenges, we sought to straightforwardly generate cytosolic truncation mutants of histidine kinases that retain their autophosphorylation and phosphotransfer capabilities. Previously, we successfully developed a cytosolic truncation mutant of PmrB (PmrBc) that maintained these critical functions, demonstrating its suitability as a viable surrogate for in vitro investigations, including inhibitor compound screening. Building upon this foundation, we have refined our methods and here demonstrate these improvements by producing functional histidine kinase truncation mutants from the following diverse bacterial species: Escherichia coli; PhoQ, BasS and Klebsiella pneumoniae; and PmrB and PhoQ. Full article

(This article belongs to the Special Issue The Molecular Mechanisms Regulating Stress-Adaptive Responses in Bacteria)

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14 pages, 3214 KB

Open AccessArticle

Lipidome Disturbances of Vibrio alginolyticus Associated with Citral Exposure

by Yanni Zhao, Zi Wang, Jie Han, Yi Wang, Jiamin Ren, Ting Shao, Hua Li and Huan Liu

Microorganisms 2026, 14(2), 509; https://doi.org/10.3390/microorganisms14020509 - 22 Feb 2026

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Abstract

Vibrio alginolyticus is an important antibiotic-resistant pathogen in aquaculture that can cause mortality in a wide range of aquatic animals and infect humans. It is urgently necessary to discover and develop effective antibiotic alternatives. Citral, a key antibacterial component of lemongrass oil, can [...] Read more.

Vibrio alginolyticus is an important antibiotic-resistant pathogen in aquaculture that can cause mortality in a wide range of aquatic animals and infect humans. It is urgently necessary to discover and develop effective antibiotic alternatives. Citral, a key antibacterial component of lemongrass oil, can be used as a food flavoring and additive. Although the antimicrobial activity and antibiofilm effect of citral against V. alginolyticus have been noted in our previous study, the potential lipidome influence of citral remains unclear. Accordingly, a non-targeted lipidomics approach was employed to investigate citral-induced lipidome disturbances and reveal potential regulated targets of citral against V. alginolyticus. We found that the citral exposure triggered substantial lipidome alterations (i.e., composition, contents, and structure) in V. alginolyticus. Specifically, the content of most phospholipids (e.g., phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), phosphatidylinositols (PIs), and phosphatidylglycerols (PGs)) decreased with the increase in citral concentration, while ceramides (Cers) and lysophospholipids (LPLs) (e.g., lyso-PAs, lyso-PCs, lyso-PEs, and lyso-PGs) showed concentration-dependent accumulation under citral treatment. Notably, the critical lipid remodeling in response to citral exposure mainly involved the phospholipid and sphingolipid metabolic pathways. Collectively, our study reveals the bacterial lipidome response to citral exposure and highlights pivotal metabolic pathways, potentially offering a novel perspective for future investigations into lipid-centric antibacterial targets. Full article

(This article belongs to the Section Biofilm)

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18 pages, 2334 KB

Open AccessArticle

Occurrence and Genomic Characterization of ESBL-, AmpC-, and Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Surface Water in Southern Italy, 2023–2024

by Gaia Nobili, Annachiara Cocomazzi, Maria Grazia Basanisi, Annita Maria Damato, Rosa Coppola, Maria Grazia Cariglia, Ilenia Franconieri, Antonella Stallone, Michelina Notarangelo, Tommaso Scirocco, Valeria Bortolaia and Giovanna La Salandra

Microorganisms 2026, 14(2), 508; https://doi.org/10.3390/microorganisms14020508 - 22 Feb 2026

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Abstract

Antimicrobial resistance (AMR) is recognised as a major global public health threat, with the environment increasingly acknowledged as a key reservoir and dissemination pathway for resistant bacteria and resistance genes. In this study, 148 surface water samples were collected between 2023 and 2024 [...] Read more.

Antimicrobial resistance (AMR) is recognised as a major global public health threat, with the environment increasingly acknowledged as a key reservoir and dissemination pathway for resistant bacteria and resistance genes. In this study, 148 surface water samples were collected between 2023 and 2024 from six rivers and three canals discharging wastewater into two lake waters in Southern Italy to assess the occurrence and genomic features of extended-spectrum β-lactamase (ESBL)-, AmpC-, and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae. Relevant isolates were obtained using selective culturing, and tested for antimicrobial susceptibility by broth microdilution. Major β-lactam resistance genes were detected by real-time PCR. Whole-genome sequencing (WGS) was performed on presumptive carbapenemase-producing isolates. ESBL- and/or carbapenemase-producing Enterobacterales were detected in 67.6% of samples, yielding a total of 176 non-duplicate isolates. The most prevalent gene was bla_CTX-M, detected in 79.3% of positive isolates (96/121), while carbapenemase genes were detected in 20.6% (25/121) of isolates, mainly bla_OXA-48 and bla_VIM. WGS analysis of carbapenemase PCR-positive isolates revealed occurrence of clinically relevant high-risk clones, such as K. pneumoniae ST512/ST307 carrying bla_KPC-3 and E. coli ST10 harbouring bla_OXA-244. These findings highlight a potential risk to public health and underscore the importance of integrating environmental compartments into One Health surveillance frameworks for AMR. Full article

(This article belongs to the Special Issue Antibiotic Resistance in Pathogenic Bacteria)

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17 pages, 810 KB

Open AccessArticle

Phenotypic and Molecular Characterization of ESBL/pAmpC-Associated Resistance in Poultry- and Hatchery-Derived Escherichia coli in Bosnia and Herzegovina

by Amira Koro-Spahić, Adis Softić, Emina Rešidbegović, Šejla Goletić Imamović, Naida Kapo, Aida Kavazović, Ilma Terzić, Dinaida Tahirović, Aida Kustura and Teufik Goletić

Microorganisms 2026, 14(2), 507; https://doi.org/10.3390/microorganisms14020507 - 21 Feb 2026

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Abstract

Antimicrobial resistance (AMR) in poultry-associated Escherichia coli (E. coli) is a persistent One Health concern, particularly when ESBL/pAmpC determinants co-occur with resistance to multiple antimicrobial classes. Between March and October 2024, we investigated commensal E. coli from three interconnected compartments of [...] Read more.

Antimicrobial resistance (AMR) in poultry-associated Escherichia coli (E. coli) is a persistent One Health concern, particularly when ESBL/pAmpC determinants co-occur with resistance to multiple antimicrobial classes. Between March and October 2024, we investigated commensal E. coli from three interconnected compartments of the poultry production chain in Bosnia and Herzegovina (parent-breeder flocks, commercial broiler farms, hatchery-associated material). A total of 333 samples were examined, and 99 E. coli isolates were recovered (29.7%). Phenotypic characterization included ESBL confirmation, disk diffusion susceptibility testing, and EUVSEC broth microdilution. Targeted real-time PCR assays were used to screen key ESBL/pAmpC-associated genes and selected carbapenemase and plasmid-mediated colistin resistance targets within the targeted panel. ESBL phenotypes were detected in 52/99 isolates (52.5%), and multidrug resistance was highly prevalent across compartments (93/99; 93.9%). ESBL/pAmpC-associated genes were detected in 91/99 isolates (91.9%), with bla_TEM predominating. Gene pattern analysis indicated that bla_TEM occurred most frequently as a single determinant and as part of the predominant multi-gene combinations, most notably bla_TEM + bla_CMY and bla_TEM + bla_CTX-M, while bla_SHV was sporadic. Carbapenemase genes (bla_KPC, bla_NDM, bla_GES, bla_OXA-₄₈) and mcr-1 to mcr-9 were not detected. Overall, our findings indicate a substantial ESBL/MDR burden throughout the poultry production chain, supporting the need for strengthening antimicrobial stewardship and biosecurity measures across both farms and hatcheries. Full article

(This article belongs to the Special Issue Avian Pathogens: Importance in Animal Health and Zoonotic Risks)

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14 pages, 941 KB

Open AccessArticle

Genomic Characterization of Salmonella Isolates Causing Infections in Children with Sickle Cell Disease in Dakar, Senegal

by Amadou Diop, Arfang Diamanka, Adja Bousso Guèye, Baïdy Dièye, El Hadji Aly Niang, Ousmane Sadio, Mouhamadou Abdoulaye Sonko, Aïssatou Ahmet Niang, Momar Ndao, Ken Dewar, Cheikh Fall, François Paillier and Yakhya Dièye

Microorganisms 2026, 14(2), 506; https://doi.org/10.3390/microorganisms14020506 - 21 Feb 2026

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Abstract

Salmonella is a major bacterial pathogen in low-income countries, where it circulates among humans, animals, and the environment. Children with sickle cell disease (SCD) are particularly vulnerable to severe Salmonella infections. This study aimed to characterize Salmonella isolates causing infections in Senegalese children [...] Read more.

Salmonella is a major bacterial pathogen in low-income countries, where it circulates among humans, animals, and the environment. Children with sickle cell disease (SCD) are particularly vulnerable to severe Salmonella infections. This study aimed to characterize Salmonella isolates causing infections in Senegalese children with SCD. Using antimicrobial susceptibility testing, whole-genome sequencing, and bioinformatic analysis, we investigated antibiotic resistance, serovar diversity, and virulence factors on 23 isolates from SCD patients with diverse clinical infections. The isolates belonged to 12 serovars, with Enteritidis predominating (n = 7). Twenty-two isolates were fully susceptible to antibiotics, while one was multidrug-resistant. Eight isolates (Enteritidis and Typhimurium) carried a virulence plasmid harboring the spvRABCD gene cluster. Core Salmonella pathogenicity islands (SPIs-1 to -5, -11, and -13), as well as SPI-10 and SPI-23, were detected in all isolates, whereas other SPIs were variably present. These results show high serovar diversity and low antimicrobial resistance among Salmonella isolates in children with SCD in Dakar, Senegal. Our findings suggest that strains causing diarrhea in healthy individuals may also cause invasive disease in SCD patients, highlighting the need for dedicated surveillance in this vulnerable population. Full article

(This article belongs to the Special Issue Accelerated Topics in Public Health Microbiology and Microbial Food Safety)

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22 pages, 4733 KB

Open AccessArticle

Decoding Virulence Mechanisms of Bacillus anthracis Using a Galleria mellonella Infection Model: Differential Host Response Profiles Elicited by AtxA and PlcR

by Pengyao Wang, Dongshu Wang, Xiaojing Wang, Yufei Lyu, Sicheng Shen, Ruilin Hu, Li Zhu, Xiankai Liu and Hengliang Wang

Microorganisms 2026, 14(2), 505; https://doi.org/10.3390/microorganisms14020505 - 20 Feb 2026

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Abstract

A thorough understanding of the functions of virulence regulators in Bacillus anthracis evolution and host adaptation, particularly the systematic host responses they trigger, requires an efficient infection model capable of resolving subtle mechanisms. This study constructed a high-resolution host immune response decoder based [...] Read more.

A thorough understanding of the functions of virulence regulators in Bacillus anthracis evolution and host adaptation, particularly the systematic host responses they trigger, requires an efficient infection model capable of resolving subtle mechanisms. This study constructed a high-resolution host immune response decoder based on Galleria mellonella to analyze the specific response profiles elicited by different virulence regulators in a capsule-deficient background. By integrating transcriptomic, histopathological, and bacterial colonization analyses, the research delineated distinct host immune stress profiles regulated by AtxA and PlcR. The results showed that the AtxA-deficient strain failed to elicit significant host responses; wild-type infection activated broad systemic immune recognition pathways, while the PlcR-activated strain induced a unique response profile characterized by perturbations in oxidative stress pathways. Its enhanced virulence was associated with the expression of downstream hydrolases and occurred without strong systemic immune activation. This work successfully advanced the G. mellonella model from a phenotypic screener to a mechanistic resolver, providing a new methodological framework for distinguishing B. anthracis virulence regulatory mechanisms at the host response level. This approach not only deciphers pathogen-specific immune signatures but also offers a practical platform for rapid anti-virulence compound screening and guides the design of targeted validation in mammalian systems, thereby accelerating therapeutic strategy development against anthrax. Full article

(This article belongs to the Section Molecular Microbiology and Immunology)

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16 pages, 3889 KB

Open AccessArticle

Diet-Induced Modulation of Gut Microbiota Affects Meat Quality in Grass Carp (Ctenopharyngodon idellus)

by Weiting Chen, Yuqin Fan, Yazhi Huang, Junbao He and Xiongjun Liu

Microorganisms 2026, 14(2), 504; https://doi.org/10.3390/microorganisms14020504 - 20 Feb 2026

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Abstract

Understanding meat quality and gut microbiota hold great potential for promoting healthy and sustainable fish production, while also contributing to fisheries management and conservation. However, differences in meat quality and the diversity, structure, and function of gut microbiota among fish across different feeding [...] Read more.

Understanding meat quality and gut microbiota hold great potential for promoting healthy and sustainable fish production, while also contributing to fisheries management and conservation. However, differences in meat quality and the diversity, structure, and function of gut microbiota among fish across different feeding regimes remain poorly understood. This study compared meat quality and gut microbiota between grass carp (gc) and crisped grass carp (cgc) to support sustainable aquaculture and provide more tasted fish meat. Cgc exhibited higher levels of free amino acids, fatty acids, and collagen, whereas gc had greater concentrations of hydrolyzed amino acids, nucleotides, and antioxidant indices. Fatty acid composition differed significantly between the two. Gut microbiota diversity was higher in cgc, with Proteobacteria, Fusobacteriota, and Firmicutes being dominant, while gc was dominated by Proteobacteria, Bacteroidota, and Firmicutes. The microbial community structures differed significantly. Functional predictions showed 1612 COG and 2032 KEGG pathways varied between groups. Significant correlations were found between microbial abundance and meat quality traits, including fatty acids, hydrolyzed amino acids, and collagen. These findings offered valuable insights for enhancing fish nutrition, optimizing feed formulations, and improving aquaculture practices. Full article

(This article belongs to the Special Issue Microbiome in Fish and Their Living Environment, Second Edition)

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19 pages, 2280 KB

Open AccessArticle

Biosurfactant-Producing Bacteria Isolated from a Microbial Consortium Previously Subjected to Adaptive Laboratory Evolution in Oily Sludge

by Maria Clara Bessa Souza, Rachel Passos Rezende, Natielle Cachoeira Dotivo, Angelina Moreira de Freitas, Elizama Aguiar-Oliveira, Luiz Carlos Salay, Eric de Lima Silva Marques, Suzana Rodrigues de Moura, Erivelton Santana Ferreira, Luana Silva Ferreira, Henrique Andrade Rabelo Bonfim, Fabiano Lopes Thompson, Bianca Mendes Maciel and João Carlos Teixeira Dias

Microorganisms 2026, 14(2), 503; https://doi.org/10.3390/microorganisms14020503 - 20 Feb 2026

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Abstract

Microbial bioprospecting in contaminated environments is a promising strategy for identifying biosurfactant-producing bacteria; however, translating environmentally adapted strains into predictable cultivation processes remains challenging. In this study, a microbial consortium subjected to long-term evolutionary laboratory adaptation in oily sludge was investigated to evaluate [...] Read more.

Microbial bioprospecting in contaminated environments is a promising strategy for identifying biosurfactant-producing bacteria; however, translating environmentally adapted strains into predictable cultivation processes remains challenging. In this study, a microbial consortium subjected to long-term evolutionary laboratory adaptation in oily sludge was investigated to evaluate strain-specific phenotypic responses related to biosurfactant production. Phylogenetic analysis based on 16S rDNA sequencing identified three taxonomically distant isolates: Faucicola sp. strain BS5C, Pseudomonas sp. strain BS16B, and Enterobacter sp. BS14MR. Biosurfactant production was evaluated using a sequential Design of Experiments (DOE) approach, including fractional factorial and central composite rotatable designs, with the emulsification index (E24) used as a semi-quantitative response variable. Initial screening revealed a statistically significant negative effect (p < 0.10) of high dextrose concentrations for all isolates. Strain-specific differences in model adequacy were observed, with a statistically adequate quadratic model obtained for Pseudomonas sp. BS16B (R² = 0.8658, p = 0.0225), whereas the other isolates showed significant lack of fit (p < 0.05). ATR-FTIR analysis revealed spectral profiles consistent with lipopeptide-like compounds. Overall, these results indicate that isolates derived from the same long-term adapted system may differ substantially in process predictability, suggesting that productivity-based screening alone may be insufficient for selecting robust strains. Full article

(This article belongs to the Section Microbial Biotechnology)

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25 pages, 3194 KB

Open AccessArticle

Biodiversity and Biotechnological Potential of Dunaliella sp. Isolates from Kalloni Solar Saltworks (Lesvos, Greece)

by Athina Petridi, Aikaterini Koletti, Sofia Marka, Maria-Eleftheria Zografaki, Ioanna Fouskari, Ioannis Karavidas, Alexandros Ntzouvaras, Ioannis Tzovenis, Rodica C. Efrose, Emmanouil Flemetakis, George Tsirtsis and Chrysanthi Kalloniati

Microorganisms 2026, 14(2), 502; https://doi.org/10.3390/microorganisms14020502 - 20 Feb 2026

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Abstract

Hypersaline solar saltworks represent unique ecological niches that harbor extremophilic microalgae with considerable biotechnological potential. Within these environments, members of the genus Dunaliella are particularly noteworthy due to their remarkable metabolic plasticity and ability to accumulate high-value biomolecules. In the present study, we [...] Read more.

Hypersaline solar saltworks represent unique ecological niches that harbor extremophilic microalgae with considerable biotechnological potential. Within these environments, members of the genus Dunaliella are particularly noteworthy due to their remarkable metabolic plasticity and ability to accumulate high-value biomolecules. In the present study, we investigated the biodiversity of Dunaliella in hypersaline saltworks by isolating and identifying autochthonous strains and assessing their growth kinetics and biomass biochemical composition in the context of potential biotechnological applications. Specifically, sixteen strains of Dunaliella were isolated from evaporation and crystallizer ponds of the Kalloni saltworks in Lesvos, Greece, and subjected to an integrative characterization combining morphological observations, molecular phylogenetics, growth kinetics, and biochemical profiling. Phylogenetic analyses based on four genetic markers (18S, ITS, rbcL, tufA) consistently resolved the isolates into three distinct clades: one corresponding to Dunaliella salina/D. minutissima, one to D. parva, and a third representing a clearly divergent lineage. Growth assays revealed marked variability in cell density, biomass productivity and specific growth rate, with certain strains exhibiting enhanced proliferation under controlled conditions. Biochemical analyses demonstrated distinct allocation patterns, with evaporation pond isolates comparatively enriched in proteins (up to 60.8% DW), whereas crystallizer pond isolates accumulated higher levels of carbohydrates (up to 19.0% DW), carotenoids (up to 7.34% mg g⁻¹ DW) and phenolic compounds (up to 8.68% mg GAE g⁻¹ DW). Antioxidant assays (FRAP, TEAC) further indicated significantly elevated reducing and radical scavenging activities among crystallizer isolates. These findings expand current knowledge on the biodiversity of autochthonous Dunaliella strains and support their potential as sustainable sources of bioactive compounds for applications in the agri-food, nutraceutical, pharmaceutical, and cosmeutical sectors. Full article

(This article belongs to the Special Issue Advances in Genomics and Ecology of Environmental Microorganisms)

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13 pages, 1086 KB

Open AccessArticle

Effectiveness of COVID-19 Vaccines Against Hospitalization and Severe Disease in Children with Diabetes Mellitus During Pandemic and Post-Pandemic Eras

by Laura G. Coelho, Lilian M. Diniz, Stella C. Galante, Cristiane S. Dias, Maria Christina L. Oliveira, Enrico A. Colosimo, Ana Cristina Simões e Silva, Fernanda N. Duelis, Maria Eduarda T. Bernardes, Daniela R. Martelli, Fabrício Emanuel S. Oliveira, Hercílio Martelli-Junior, Robert H. Mak and Eduardo A. Oliveira

Microorganisms 2026, 14(2), 501; https://doi.org/10.3390/microorganisms14020501 - 20 Feb 2026

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Abstract

Pediatric patients with SARS-CoV-2 infection are at an increased risk of severe disease and adverse outcomes. Nevertheless, comprehensive data on COVID-19 vaccine effectiveness (VE) in children with diabetes during the post-pandemic period remain limited. This study assessed the VE against severe COVID-19 outcomes [...] Read more.

Pediatric patients with SARS-CoV-2 infection are at an increased risk of severe disease and adverse outcomes. Nevertheless, comprehensive data on COVID-19 vaccine effectiveness (VE) in children with diabetes during the post-pandemic period remain limited. This study assessed the VE against severe COVID-19 outcomes during both the pandemic and post-pandemic phases in children with and without diabetes mellitus (DM). A cohort study based on population data was carried out, including all patients under 18 years of age with symptomatic SARS-CoV-2 infection as registered in the Brazilian national surveillance systems from February 2020 to June 2025. The main outcomes were hospitalization due to COVID-19 and severe illness, which included admission to the intensive care unit (ICU), need for invasive ventilation, and death. Utilizing a propensity score-matched cohort, we estimated the VE and the number needed to vaccinate (NNV) for a booster dose against these outcomes by comparing vaccinated and unvaccinated individuals, employing conditional logistic regression adjusted for confounding variables. The cohort comprised 3,730,007 pediatric patients with COVID-19, of whom 7675 (0.2%) had DM. At baseline, children with DM exhibited a significantly higher prevalence of hospitalization (11.2% vs. 2.0%), severe COVID-19 (6.4% vs. 0.6%), and mortality (1.9% vs. 0.1%) than those without DM (all p < 0.001). During the pandemic period, the adjusted VE was consistently higher in children with DM. Against severe disease, the VE was 72.8% (95% CI: 12.3–93.2) in the DM cohort compared with 45.7% (28.1–59.0) in the non-DM cohort. This increased effectiveness corresponded to a more favorable NNV; the NNV to prevent one severe case was 24 (95% CI: 12–232) for children with DM versus 243 (168–440) for those without DM. In the post-pandemic period, the VE remained significantly higher in the DM cohort. Against severe disease, the VE was 76.2% (11.5–93.5) for children with DM and 52.9% (32.7–67.1) for those without. The NNV to prevent one severe case was consistently lower in the DM cohort (8 vs. 591). In conclusion, a complete vaccination regimen, including a booster dose, substantially mitigated severe COVID-19 outcomes in children with DM in the pandemic and post-pandemic periods. Full article

(This article belongs to the Special Issue SARS-CoV-2: Infection, Transmission, and Prevention)

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12 pages, 7161 KB

Open AccessArticle

Sand Fly Fauna (Diptera: Psychodidae): Association Between Climatic Variables and Natural Leishmania Infection in Araçatuba, Brazil

by Graziella Borges Alves, Debora Regina Romualdo da Silva, Elis Domingos Ferrari, Lilian Aparecida Colebrusco Rodas, Alex Akira Nakamura, Carolina Beatriz Baptista, Camila Pedrozo Rodrigues Furlan, Keuryn Alessandra Mira Luz Requena, Gabriele Zaine Teixeira Debortoli, Thais Rabelo Santos-Doni and Katia Denise Saraiva Bresciani

Microorganisms 2026, 14(2), 500; https://doi.org/10.3390/microorganisms14020500 - 20 Feb 2026

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Abstract

Visceral leishmaniasis (VL) is a zoonosis of major public health importance. In urban areas, Lutzomyia longipalpis is the primary vector of Leishmania (L.) infantum. This study assessed the seasonality, spatiotemporal distribution, and climatic factors associated with L. longipalpis abundance in Araçatuba, São [...] Read more.

Visceral leishmaniasis (VL) is a zoonosis of major public health importance. In urban areas, Lutzomyia longipalpis is the primary vector of Leishmania (L.) infantum. This study assessed the seasonality, spatiotemporal distribution, and climatic factors associated with L. longipalpis abundance in Araçatuba, São Paulo State, and detected Leishmania spp. DNA in captured females. Monthly collections were conducted from March 2023 to February 2024 in 72 households across eight urban areas using CDC-type light traps placed indoors and in peridomestic environments. A total of 1641 specimens (1516 males and 125 females) were captured, with 92.4% originating from peridomestic areas. Area 3 had the highest density (n = 671) and was the only area with PCR-positive females (n = 3). Vector activity peaked in December 2023 (n = 335). Male abundance differed significantly among peridomestic areas, particularly between Areas 3, 5, 6, and 7. In peridomestic areas, higher final temperatures increased vector abundance, whereas higher initial temperatures and humidity reduced it. Indoors, final temperature, humidity, and month were significant predictors. L. longipalpis exhibited a defined seasonal and spatial pattern influenced by climatic conditions. The detection of PCR-positive females (Area 3) highlights the epidemiological role of the vector and underscores the need for targeted interventions to control VL. Full article

(This article belongs to the Special Issue Research on Leishmania and Leishmaniasis: Second Edition)

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16 pages, 2017 KB

Open AccessArticle

In Vitro Evaluation of Ethanolic Medicinal Plant Extracts from the Aseer Region Against Selected Cultured Oral Bacterial Isolates from Healthy Volunteers

by Samah Noor, Aisha Shathan and Azhar Najjar

Microorganisms 2026, 14(2), 499; https://doi.org/10.3390/microorganisms14020499 - 19 Feb 2026

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Abstract

This work provides an in vitro assessment of the antibacterial efficacy of ethanolic extracts derived from four medicinal plants historically utilized in the Aseer region (Foeniculum vulgare, Solanum incanum, Forsskaolea tenacissima, and Abutilon pannosum) against cultured oral bacterial [...] Read more.

This work provides an in vitro assessment of the antibacterial efficacy of ethanolic extracts derived from four medicinal plants historically utilized in the Aseer region (Foeniculum vulgare, Solanum incanum, Forsskaolea tenacissima, and Abutilon pannosum) against cultured oral bacterial isolates obtained from healthy volunteers. Oral samples from a subset of 50 healthy female participants were included in this analysis, yielding independent cultured bacterial isolates. Isolates were identified using morphological and biochemical characterization combined with partial 16S rRNA gene sequencing and included representatives of common oral-associated genera. Antibacterial activity was assessed using agar disk diffusion and broth microdilution assays. Abutilon pannosum and Solanum incanum had lower values of MIC (range of 16–128 µg/mL), whereas Forsskaolea tenacissima had higher values of MIC (maximum to >512 µg/mL) with the tested isolates. Qualitative microscopic observations and crystal violet biofilm staining showed extract-associated varying cellular morphology, aggregation patterns and surface coverage under sub-inhibitory conditions. Representative isolate scanning electron microscopy (SEM) qualitatively validated descriptive cell surface morphology and organization changes. The research presents preliminary in vitro results of inconsistent antibacterial and antibiofilm effects of crude ethanolic extracts of four plants in the Aseer region on a small (n = 13) group of cultured oral bacteria isolates in healthy volunteers, which requires fractionation and further testing. Full article

(This article belongs to the Section Antimicrobial Agents and Resistance)

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27 pages, 1538 KB

Open AccessReview

From Basics to Breakthroughs: A Review on the Evolution of Campylobacter spp. Culture Media

by Ana Rita Barata, Maria José Saavedra and Gonçalo Almeida

Microorganisms 2026, 14(2), 498; https://doi.org/10.3390/microorganisms14020498 - 19 Feb 2026

Viewed by 579

Abstract

Since their recognition as human pathogens in the 1970s, Campylobacter spp. have posed persistent challenges to microbiologists due to their fastidious growth requirements and environmental sensitivity. The continuous refinement of selective and differential culture media has been crucial for improving their detection, isolation, [...] Read more.

Since their recognition as human pathogens in the 1970s, Campylobacter spp. have posed persistent challenges to microbiologists due to their fastidious growth requirements and environmental sensitivity. The continuous refinement of selective and differential culture media has been crucial for improving their detection, isolation, and characterization in both clinical and food microbiology. This comprehensive review provides a chronological overview of the evolution of Campylobacter culture media, highlighting the scientific milestones that shaped current cultivation practices—from early blood- and charcoal-based formulations to modern selective, chromogenic, and systems permitting incubation under less stringent atmospheric conditions. Emphasis is placed on the rationale behind medium composition, the transition from empirical experimentation to standardized formulations, and the integration of molecular and metabolic insights into media design. The evolution of Campylobacter growth media mirrors the broader trajectory of microbiology itself, moving from artisanal experimentation toward precision-driven innovation. Ongoing advancements in culture technology, including sustainable and data-guided formulations, will continue to enhance global surveillance, food safety, and pathogen ecology research. Full article

(This article belongs to the Special Issue Advances in Food Microbial Biotechnology)

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13 pages, 2203 KB

Open AccessArticle

Microbial Community Composition and Major Environmental Factors Influencing Changes in Different Vegetation Soils of Coastal Wetlands

by Dongmei He, Weixiang Liu, Lei Wang, Wenwen Xu, Jiaojiao Zhang, Qihang Lu and Ting Luo

Microorganisms 2026, 14(2), 497; https://doi.org/10.3390/microorganisms14020497 - 19 Feb 2026

Viewed by 278

Abstract

The soil microbial community in coastal wetlands plays a crucial role in biogeochemical cycling. In this study, Spartina alterniflora soil (HB), found near the sea; Spartina alterniflora soil (ZY), found near land; and Phragmites australis soil (KA), found in coastal wetlands, were selected [...] Read more.

The soil microbial community in coastal wetlands plays a crucial role in biogeochemical cycling. In this study, Spartina alterniflora soil (HB), found near the sea; Spartina alterniflora soil (ZY), found near land; and Phragmites australis soil (KA), found in coastal wetlands, were selected to study the microbial community structure and major environmental influencing factors. The results showed that environmental factors had a significant difference in the three soils. Compared with the ZY and KA sites, the soil at the HB site had the highest value of salinity (14.1 g/kg) and the lowest value of total organic carbon (TOC) (2.9 g/kg) in summer. At the KA site, the values of soil temperature, soil humidity (SH), TOC, and NH₄⁺ were higher than those at HB and ZY sites, while the values of EC (159.8 μS/cm in summer) and salinity (4.4 g/kg) were the lowest. Furthermore, the microbial community structure had significant differences at the three sites. Pseudomonas and Bacteroidota dominated at the HB site, while Chloroflexota and Gemmatimonades were more abundant at the ZY and KA sites. Microbial alpha diversity analysis indicated that the microbial community diversity of Phragmites australis soil was the most uniform, and the microbial species richness in the soil of Spartina alterniflora near the sea was the highest. Salinity, TOC, and SH might be the key environmental factors that affect the structure and diversity of microbial communities in soils. High-salt environments may promote the enrichment of salt-tolerant microbial communities, while high TOC and suitable soil humidity may enhance the uniformity of microbial communities. Full article

(This article belongs to the Section Environmental Microbiology)

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13 pages, 9863 KB

Open AccessArticle

Rapid Detection of Vibrio vulnificus with Recombinase Polymerase Amplification Assays Based on Specific Sequence Tags of Core Genome

by Bing Yuan, Jianhao Xu, Jiaxin Zhang, Jinglin Wang and Yuan Yuan

Microorganisms 2026, 14(2), 496; https://doi.org/10.3390/microorganisms14020496 - 19 Feb 2026

Viewed by 320

Abstract

Vibrio vulnificus (V. vulnificus) is a motile, Gram-negative, opportunistic human pathogen capable of causing severe to life-threatening infections in individuals with predisposing conditions. It has the highest mortality rate among foodborne pathogens. Rapid and accurate detection of V. vulnificus is crucial [...] Read more.

Vibrio vulnificus (V. vulnificus) is a motile, Gram-negative, opportunistic human pathogen capable of causing severe to life-threatening infections in individuals with predisposing conditions. It has the highest mortality rate among foodborne pathogens. Rapid and accurate detection of V. vulnificus is crucial for preventing and controlling acute deaths caused by infection with this bacterium. However, identifying V. vulnificus is challenging due to its high genomic plasticity. We analyzed 518 V. vulnificus genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish V. vulnificus from its closely related species. Specifically, one specific sequence tag with the minimal mutations was selected for V. vulnificus detection, combined with a recombinase polymerase amplification (RPA) method. The results showed that the developed RPA detection method displayed high specificity and enabled the identification of a specific 462 bp band from V. vulnificus. The reaction involved isothermal incubation at 39 °C for 20 min with a compact portable instrument. The sensitivity of the method was determined to be as low as 0.5 aM (1.65 fg/μL) of genomic DNA, 0.96 copies/μL of pUC57-Vv plasmid and 1 CFU/mL of V. vulnificus cells. The RPA method accurately detected target DNA within 5–10 min. Additionally, specificity testing was performed using 33 different strains of V. vulnificus. In conclusion, the established RPA method exhibits excellent high sensitivity and rapid discriminative capability, making it suitable for clinical applications and rapid detection in the field. Full article

(This article belongs to the Section Microbial Biotechnology)

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13 pages, 3265 KB

Open AccessArticle

Seasonal Variations in the Occurrence of SARS-CoV-2 RNA Recovered from Wastewater Treatment Facilities (WWTFs) Within South Africa

by Kingsley Ehi Ebomah, Luyanda Msolo, Velisa Vuyolwethu Qongwe, Okuhle Mayoyo, Piwe Athi Ntlati, Balisa Ngqwala, Nolonwabo Nontongana, Renee Street, Rabia Johnson and Anthony Ifeanyi Okoh

Microorganisms 2026, 14(2), 495; https://doi.org/10.3390/microorganisms14020495 - 18 Feb 2026

Viewed by 354

Abstract

Several researchers have documented the occurrence of the unfamiliar severe acute respiratory syndrome coronavirus 2 ribonucleic acid (also known as SARS-CoV-2 RNA) in various raw wastewater (WW) samples analyzed globally. The efficiency of strategic WW-based epidemiology (WBE) approach as a timely cautioning tool [...] Read more.

Several researchers have documented the occurrence of the unfamiliar severe acute respiratory syndrome coronavirus 2 ribonucleic acid (also known as SARS-CoV-2 RNA) in various raw wastewater (WW) samples analyzed globally. The efficiency of strategic WW-based epidemiology (WBE) approach as a timely cautioning tool for human coronavirus disease-2019 (COVID) and other similar outbreaks is highly promising. This strategy offers a cost-effective, population-wide surveillance tool that can detect rising case trends, from days to weeks before clinical reports, thus enabling proactive public health interventions. This study aimed to detect the occurrence of the viral genome in WW over four seasons, which contributes to the database for multi-plant surveillance research in South Africa. About 480 WW influent samples were amassed from ten sampling points situated in nine wastewater treatment facilities (WWTFs) in Amathole District Municipality (ADM) located in the Province of Eastern Cape (EC), South Africa (SA). The study was carried out for a period of one year. Quantitative real-time polymerase chain reaction (i.e., RT-PCR) was operated to identify the viral genomes in the respective total RNA samples. Of the 480 extracted RNA samples, 210 (44%) were positive with viral genome copies (gc) that ranged from 700 to 40,000 GC/mL. Our results were contrasted with existing COVID-19-positive cases throughout the COVID omicron wave in the ECP. Variations in gc were observed across different seasons, with the highest GC observed in winter. In contrast, there were significant inconsistencies in the existing data of COVID-19 clinical cases, thus indicating no connection between both data. However, with more similar studies, advanced innovative WBE strategies could possibly act as prompt warning tools to signal public health officials about potential future outbreaks. Full article

(This article belongs to the Special Issue Pathogen Surveillance in Wastewater)

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15 pages, 3366 KB

Open AccessArticle

Polycyclic Aromatic Hydrocarbon Pollution Stress Impairs Soil Enzyme Activity and Microbial Community

by Yuancheng Wang, Donglei Wu, Junxiang Liu and Haolong Xu

Microorganisms 2026, 14(2), 494; https://doi.org/10.3390/microorganisms14020494 - 18 Feb 2026

Viewed by 289

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are widely prevalent harmful organic pollutants. Enzymatic activities (such as those of dehydrogenases, catalase, protease and urease), as well as the microbial community structure and assembly (through 16S and ITS amplicon sequencing), were evaluated 90 days after PAH contamination [...] Read more.

Polycyclic aromatic hydrocarbons (PAHs) are widely prevalent harmful organic pollutants. Enzymatic activities (such as those of dehydrogenases, catalase, protease and urease), as well as the microbial community structure and assembly (through 16S and ITS amplicon sequencing), were evaluated 90 days after PAH contamination and compared to those in normal soils. Microbial activity, as indicated by soil urease, catalase, and protease activities, was inhibited under PAH stress. Furthermore, PAH stress exerted significant impacts on the soil microbial community structure. Notably, PAH stress reduced soil bacterial and fungal biomass and inhibited the abundance of microbial taxa involved in soil carbon and nitrogen cycling (e.g., Marmoricola, Pedobacter, and Streptomyces), along with the majority of predicted responsive metabolic functions, particularly those related to amino acid and carbohydrate metabolism. PAH stress enriched PAH-degrading microorganisms, including Pseudomonas, Mycobacterium, Bacillus, Cycloclasticus, and Flavobacterium. The niche breadth of bacterial and fungal communities decreased significantly under PAH stress (51.5 and 14.1, respectively) compared to that in normal soil (63.7 and 22.3), which was further supported by Beta Nearest Taxon Index and co-occurrence network analysis. PAH stress increased the contribution of heterogeneous selection to soil microbial assembly (100%) compared to that in normal soil (80%). Thus, the majority of microbial community responses to PAH stress were adversely affected. These results suggest that PAH contamination may profoundly affect the soil quality by restricting the survival space of bacteria and fungi. Full article

(This article belongs to the Section Environmental Microbiology)

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18 pages, 1741 KB

Open AccessArticle

Unraveling the Coevolutionary Dynamics of Phage and Bacterial Protein Warfare Occurring in the Drains of Beef-Processing Plants

by Vignesh Palanisamy, Joseph M. Bosilevac, Darryll A. Barkhouse, Sarah E. Velez and Sapna Chitlapilly Dass

Microorganisms 2026, 14(2), 493; https://doi.org/10.3390/microorganisms14020493 - 18 Feb 2026

Viewed by 318

Abstract

Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic [...] Read more.

Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic analysis. Metagenomic data collected from 75 drain samples from beef-processing plants were analyzed to investigate phage–bacterial interactions. First, assembled contigs were screened to identify viral sequences, which were then taxonomically annotated to determine the viral composition, including phages. Functional annotation of these viral sequences provided information about the viral genes and their roles in bacterial interactions specifically associated with attack and counterattack of bacteria. In parallel, bacterial contigs were examined to identify genes associated with antiphage defense systems, providing insights into the strategies adapted by bacteria to resist phage infection. Taxonomic annotation of viral sequences from the bulk metagenomic data revealed the presence of phages targeting Pseudomonas, Klebsiella, and Enterococcus. The higher abundance of Pseudomonas phages aligns with our previous study, where Pseudomonas was identified as the dominant bacterial genus, suggesting potential copersistence of phages and their hosts. Functional annotation of phage contigs revealed infective and lysis-related genes, highlighting their potential role in bacterial attack. Conversely, bacterial contigs encoded antiphage defense systems, including CRISPR-Cas, restriction–modification, and other defense-related genes. The study also uncovered the presence of anti-CRISPR proteins in phages, suggesting a counterattack on the bacterial defense. These findings provide evidence for phage attack, bacterial defense, and phage counterattack and may showcase the ongoing coevolutionary arms race between phages and bacteria. While this evidence looks promising, these results remain preliminary and further studies are needed to validate these findings. Still, this study provides a foundational understanding of bacteria–phage coexistence in beef-processing plant drains and paves the way for further explorations of these intricate interactions and their possible applications in controlling pathogenic microorganisms within biofilms. Full article

(This article belongs to the Section Environmental Microbiology)

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19 pages, 1157 KB

Open AccessReview

Current Applications and Immunological Considerations of Salmonella enterica Serovar Typhimurium as a Vaccine Vector

by Adam S. Hassan, Kaitlin Winter, Charles M. Dozois, Brian J. Ward and Momar Ndao

Microorganisms 2026, 14(2), 492; https://doi.org/10.3390/microorganisms14020492 - 18 Feb 2026

Viewed by 362

Abstract

Live attenuated Salmonella enterica serovar Typhimurium has been investigated for decades as an orally delivered vaccine vector due to its ability to target the intestinal mucosa and engage both innate and adaptive immune responses. In humans, S. Typhimurium infection is largely restricted to [...] Read more.

Live attenuated Salmonella enterica serovar Typhimurium has been investigated for decades as an orally delivered vaccine vector due to its ability to target the intestinal mucosa and engage both innate and adaptive immune responses. In humans, S. Typhimurium infection is largely restricted to the gastrointestinal tract, distinguishing it from Salmonella Typhi and providing a rationale for its use in mucosal vaccine strategies. In this review, we discuss the biological features of S. Typhimurium that support its use as a vaccine vector and summarize current understanding of the immune responses generated during wild-type infection, including innate activation and downstream T cell and B cell responses. We compare key biological differences between Salmonella Typhi and S. Typhimurium and outline emerging vector design strategies, including delayed attenuation and chromosomal integration of heterologous antigens. We then review applications of attenuated S. Typhimurium vectors targeting viral, bacterial, and parasitic pathogens, highlighting shared immunological outcomes and design principles across platforms. Finally, we discuss recent advances in vector engineering, including chromosomal integration of heterologous antigens, as well as remaining gaps in knowledge related to the durability of immune responses and translational considerations. Full article

(This article belongs to the Section Molecular Microbiology and Immunology)

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17 pages, 2040 KB

Open AccessArticle

Use of Thermal and Emerging Non-Thermal Treatments Reveal Biomolecular and Morphological Changes in Pathogenic E. coli

by Maxsueli Machado, Jelmir Craveiro Andrade, Eduardo Eustáquio de Souza Figueiredo and Carlos Adam Conte-Junior

Microorganisms 2026, 14(2), 491; https://doi.org/10.3390/microorganisms14020491 - 18 Feb 2026

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Abstract

(1) Background: We sought to explore the changes in the biomolecular profile and morphology of Pathogenic heat-resistant E. coli isolated from animal-based food. (2) Methods: Six strains underwent heat (60 °C for 6 min), ultrasound (US; 299 W), UVC (4950 mJ/cm²), [...] Read more.

(1) Background: We sought to explore the changes in the biomolecular profile and morphology of Pathogenic heat-resistant E. coli isolated from animal-based food. (2) Methods: Six strains underwent heat (60 °C for 6 min), ultrasound (US; 299 W), UVC (4950 mJ/cm²), and combined treatments (UVC+US and heat+UVC). Afterwards, biomolecular characterization across four spectral regions was evaluated by Fourier transform infrared (FT-IR) spectroscopy and analyzed by principal component analysis (PCA) for treated and non-treated strains (control group). These regions are fatty acids (3010–2800 cm⁻¹), proteins and peptides (1700–1200 cm⁻¹), carbohydrates (1200–900 cm⁻¹), and amide A (3280–3120 cm⁻¹). Additionally, treated and untreated strains were assessed for surface damage using scanning electron microscopy (SEM). (3) Results: Among all the regions studied, the amide A and fatty acids regions exhibited the most significant variations in absorbance for treated strains compared to the control. Treatments such as US, heat, and UVC+US tended to increase Principal Components (PCs) and, consequently, absorbance. On the other hand, UVC and heat+UVC showed the opposite trend in these regions. SEM images showed filamentous cells for strains treated with UVC and UVC+US, indicating that cells continued to replicate under these conditions. These results highlight how thermal and non-thermal treatments influence specific biomolecular and morphological regions of E. coli. The methodologies used provide reliable data for understanding stress responses, which can guide the development of more effective technologies for eliminating multi-resistant pathogens. Full article

(This article belongs to the Special Issue The Evolution of Foodborne Pathogens in the 4.0 Era: A Food Safety Perspective)

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17 pages, 2680 KB

Open AccessArticle

Characteristics, Whole Genome Analysis of a Virulent Phage from Avian-Derived Enterococcus faecalis and Its Application in Poultry Product Processing Safety

by Xiaoming Li, Mengli Zhao, Lei Zhang, Guobo Sun, Xiujun Duan and Guoshun Chen

Microorganisms 2026, 14(2), 490; https://doi.org/10.3390/microorganisms14020490 - 18 Feb 2026

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Abstract

To explore high-quality phage resources for controlling Enterococcus faecalis (E. faecalis) contamination, a virulent phage vB-Efa1 was isolated and purified from poultry slaughterhouse sewage in this study. Its biological characteristics, whole-genome features, and potential in ensuring poultry product processing safety were [...] Read more.

To explore high-quality phage resources for controlling Enterococcus faecalis (E. faecalis) contamination, a virulent phage vB-Efa1 was isolated and purified from poultry slaughterhouse sewage in this study. Its biological characteristics, whole-genome features, and potential in ensuring poultry product processing safety were systematically investigated. The phage belongs to the Siphoviridae family, with an optimal multiplicity of infection (MOI) of 0.1 and a titer of 8.87 lg PFU/mL; it has a 30 min latent period and stable lytic activity, retaining good stability at 25–37 °C, pH 6–8, and 4 °C. Its circular whole genome is 166,586 bp in length with a GC content of 35.46%, encoding 276 genes; no antibiotic resistance genes were detected, and only one low-pathogenic-risk virulence-related sequence was identified. Application tests in poultry products revealed that temperature is the key factor regulating phage titer: the titer stably maintained 5.5–6.6 lg PFU/mL at 4 °C, while proliferating significantly at 25 °C, reaching 7.55–8.38 lg PFU/mL at 12 h. Collectively, vB-Efa1 exhibits superior biological traits, environmental adaptability, and biosafety, making it a promising biocontrol candidate for mitigating E. faecalis contamination in poultry products. Full article

(This article belongs to the Section Microbial Biotechnology)

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26 pages, 4050 KB

Open AccessArticle

Uncovering the Prokaryotic Diversity of Hypersaline Soils of Odiel Saltmarshes Natural Area Through Metagenome-Assembled Genomes

by Cristina Galisteo, Fernando Puente-Sánchez, Rafael R. de la Haba, Stefan Bertilsson, Antonio Ventosa and Cristina Sánchez-Porro

Microorganisms 2026, 14(2), 489; https://doi.org/10.3390/microorganisms14020489 - 18 Feb 2026

Viewed by 434

Abstract

The hypersaline soils of the Odiel Saltmarshes Natural Area in Southwest Spain harbor highly diverse microbial communities adapted to extreme conditions. However, their genomic diversity remains largely unexplored. In addition to high salinity, these soils are contaminated with heavy metals, creating a hostile [...] Read more.

The hypersaline soils of the Odiel Saltmarshes Natural Area in Southwest Spain harbor highly diverse microbial communities adapted to extreme conditions. However, their genomic diversity remains largely unexplored. In addition to high salinity, these soils are contaminated with heavy metals, creating a hostile environment of great interest for studying extremophilic microorganisms and their metabolic adaptations. This study aims to characterize the uncovered prokaryotic taxa as Candidatus species inhabiting the hypersaline soils of the Odiel Saltmarshes, based on their metagenomic assembled genomic sequences. The reconstructed genomes were assessed for quality based on completeness and contamination thresholds and subsequently taxonomically classified. Comparative genomic analysis of six high-quality MAGs revealed key metabolic traits related to survival under extreme salinity and heavy metal conditions. The findings provide new insights about microbial diversity of hypersaline environments and expand the catalog of known prokaryotic genomes. Detailed characterization of six novel Candidatus taxa highlights the unique adaptations of these microorganisms, enhancing our understanding of life in extreme habitats. Full article

(This article belongs to the Special Issue Halophiles)

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14 pages, 1141 KB

Open AccessArticle

Paenibacillus–Pseudomonas Consortium Improves Barley Performance with Minimal Impact on Native Rhizobacterial Community

by Jakub Dobrzyński, Aleksandra Naziębło, Iryna Kulkova, Magdalena Szpytma, Adrianna Antosik, Monika Sitarek-Andrzejczyk and Barbara Wróbel

Microorganisms 2026, 14(2), 488; https://doi.org/10.3390/microorganisms14020488 - 18 Feb 2026

Viewed by 504

Abstract

The intensive use of mineral nitrogen fertilizers in cereal production contributes to environmental degradation, highlighting the need for more sustainable crop management strategies. Plant growth-promoting bacteria (PGPB) offer a promising alternative; however, their effects on native rhizosphere communities remain underexplored, particularly in barley. [...] Read more.

The intensive use of mineral nitrogen fertilizers in cereal production contributes to environmental degradation, highlighting the need for more sustainable crop management strategies. Plant growth-promoting bacteria (PGPB) offer a promising alternative; however, their effects on native rhizosphere communities remain underexplored, particularly in barley. This study evaluates the impact of a bacterial consortium composed of Paenibacillus sp. Z15 and Pseudomonas sp. KR227 on barley growth, yield, and rhizosphere bacteria under field conditions in temperate climate (2025). Plant biometric traits, photosynthetic pigment content, and soil properties were measured, and rhizobacterial communities were analyzed using 16S rRNA gene (V3–V4) sequencing. The PGPB consortium significantly increased early root biomass (120%), shoot height (7.8%), and grain yield (15.5%), while no significant effects were observed on soil chemistry or photosynthetic pigments. Sequencing revealed no major changes in alpha or beta diversity; however, transient shifts in the relative abundance of specific taxa were detected relatively shortly after inoculation and mostly disappeared by harvest. These findings indicate that the Paenibacillus–Pseudomonas consortium can enhance barley performance without disrupting native rhizobacterial communities. Overall, the results support the potential of PGPB as a sustainable agronomic tool and provide new insights into PGPB–microbiome interactions in barley under field conditions. Full article

(This article belongs to the Special Issue Advances in Agro-Microbiology)

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18 pages, 3423 KB

Open AccessArticle

Responses of Biofilm-Forming Halophilic Calothrix and Coelastrella Strains to Environmental Stressors Associated with Climate Change

by Gabrielle Zammit, Kristina Fenech and Emmanuel Sinagra

Microorganisms 2026, 14(2), 487; https://doi.org/10.3390/microorganisms14020487 - 17 Feb 2026

Viewed by 238

Abstract

Research into the effects of environmental stressors associated with global climate change (GCC) on cyanobacteria and microalgae is scarce, with bloom-forming planktonic cyanobacteria being the exception. This study aimed to address the issue by assessing morphological and biochemical changes in cyanobacterial and microalgal [...] Read more.

Research into the effects of environmental stressors associated with global climate change (GCC) on cyanobacteria and microalgae is scarce, with bloom-forming planktonic cyanobacteria being the exception. This study aimed to address the issue by assessing morphological and biochemical changes in cyanobacterial and microalgal cells exposed to an increased temperature (T), ultraviolet radiation (UVR) and carbon dioxide (CO₂) concentration. The strains selected were Calothrix sp. SLM0211 and Coelastrella sp. SLM0503, which were isolated from a coastal environment in the central Mediterranean island of Malta. Elevated UVR had a pronounced effect on Calothrix sp. filaments, which produced screening compounds and resorted to trichome coiling to enhance self-shading. Enhanced growth was observed in cultures of Calothrix sp. grown at an increased CO₂ concentration, which produced significantly high amounts of biomass, chlorophylls and carotenoids. An increased T resulted in stunted growth and low biomass accumulation in both strains. Each strain exhibited a unique response to T and UVR stressors, which stimulated the production of exopolymeric substances (EPS) and mycosporine-like amino acids (MAAs) in cultures of Calothrix sp. and lipid production in Coelastrella sp. cells. Our findings indicate that the effects of stressors related to GCC on cyanobacterial and microalgal cells are strain-specific, making changes at community and ecosystem levels difficult to predict. Full article

(This article belongs to the Special Issue Microorganisms: Climate Change and Terrestrial Ecosystems)

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11 pages, 787 KB

Open AccessArticle

Role of Next-Generation Sequencing in Excluding the Nosocomial Origin of a Case of Legionnaires’ Disease Integrating Environmental Surveillance and Clinical Diagnosis

by Francesco Paglione, Cataldo Maria Mannavola, Marilena La Sorda, Maria Luisa Ricci, Maria Scaturro, Silvia Laura Bosello, Roberta Masnata, Francesca Romana Monzo, Sara Vincenti, Patrizia Laurenti, Maurizio Sanguinetti and Flavio De Maio

Microorganisms 2026, 14(2), 486; https://doi.org/10.3390/microorganisms14020486 - 17 Feb 2026

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Abstract

Legionella pneumophila (Lp) remains one of the major causes of community- and hospital-acquired pneumonia, yet its diagnosis and source attribution continue to pose significant challenges. Here, we describe the case of an immunocompromised patient who developed Legionnaires’ disease during hospitalization. Following [...] Read more.

Legionella pneumophila (Lp) remains one of the major causes of community- and hospital-acquired pneumonia, yet its diagnosis and source attribution continue to pose significant challenges. Here, we describe the case of an immunocompromised patient who developed Legionnaires’ disease during hospitalization. Following activation of the hospital’s internal surveillance system, Lp and Legionella anisa (L. anisa) were recovered from multiple water distribution points using a simplified culture-based protocol. Whole-genome sequencing (WGS) demonstrated that all environmental isolates belonged to a single clonal strain, whereas the clinical isolate was genetically unrelated, thereby excluding the hospital water system as the source of infection. Although not implicated in the patient’s disease, the detection of both Lp and L. anisa within the plumbing system highlighted underlying structural contamination and the potential masking effect of non-L. pneumophila species during culture-based surveillance. These findings support the integration of conventional microbiological methods with high-resolution genomic tools to enhance surveillance accuracy, support outbreak investigations, and strengthen public health responses. Overall, this case underscores the value of WGS as a decisive tool for source attribution, including the robust exclusion of a suspected nosocomial source, in complex clinical and environmental scenarios. Full article

(This article belongs to the Special Issue Legionella pneumophila: A Microorganism with Thousand Faces, 2nd Edition)

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21 pages, 1856 KB

Open AccessArticle

Draft Genome Sequence of Bacillus sp. Strain 11B20, a Promising Plant-Growth Promoting Bacterium Associated with Maize (Zea mays L.) in the Yaqui Valley, Mexico

by Alina Escalante-Beltrán, Pamela Helué Morales-Sandoval, Amelia Cristina Montoya-Martínez, Edgar A. Cubedo-Ruíz, Rubén Félix-Gastélum, Fannie Isela Parra-Cota and Sergio de los Santos-Villalobos

Microorganisms 2026, 14(2), 485; https://doi.org/10.3390/microorganisms14020485 - 17 Feb 2026

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Abstract

Strain 11B20 was isolated from a commercial field of maize (Zea mays L.) located in the Yaqui Valley, Mexico. The draft genome sequence revealed a genomic size of 3,759,824 bp, 41.6% G + C content, 973,288 bp N50, 2 L50, and 29 [...] Read more.

Strain 11B20 was isolated from a commercial field of maize (Zea mays L.) located in the Yaqui Valley, Mexico. The draft genome sequence revealed a genomic size of 3,759,824 bp, 41.6% G + C content, 973,288 bp N50, 2 L50, and 29 contigs. According to the 16S rRNA gene, strain 11B20 belongs to the genus Bacillus. Genome annotation revealed 3952 coding DNA sequences (CDSs) grouped into 319 subsystems. Among these, several CDSs were associated with traits related to plant growth promotion, including (i) virulence, disease, and defense (33 CDSs); (ii) iron acquisition and metabolism (28 CDSs); and (iii) secondary metabolism (6 CDSs), among others. In vitro, metabolic analysis (IAA, siderophore biosynthesis; phosphorus solubilization; and tolerance to thermal, hydric, and saline stress) confirmed the genomic background of this strain. Finally, in planta assays showed that the inoculation of Bacillus sp. 11B20 significantly (p ≤ 0.05) increased the root length (48.2%) and root dry weight (35.4%) versus non-inoculated maize plants. Thus, this is the first report of Bacillus sp. 11B20 as a promising beneficial strain for sustainable corn production, and further research is needed to ensure the success of the application of this strain in agriculture. Full article

(This article belongs to the Special Issue Advances in Plant–Soil–Microbe Interactions)

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Microorganisms, Volume 14, Issue 2 (February 2026) – 253 articles

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