Microorganisms

13 pages, 859 KB

Open AccessArticle

Molecular Characterization of ‘Candidatus Liberibacter Asiaticus’ Strains from Commercial Citrus-Growing Regions in Cuba Using Polymorphic Regions

by Camilo Paredes-Tomás, Maritza Luis-Pantoja, Miguel Ramos-Leal, Marialea Melle and Assunta Bertaccini

Microorganisms 2025, 13(10), 2381; https://doi.org/10.3390/microorganisms13102381 (registering DOI) - 15 Oct 2025

Abstract

Studies of the genetic diversity of ‘Candidatus Liberibacter asiaticus’ strains based on housekeeping genes have been unsuccessful. The increasing availability of complete genome sequences of several strains from different countries has allowed the identification of regions having greater variability, which have been [...] Read more.

Studies of the genetic diversity of ‘Candidatus Liberibacter asiaticus’ strains based on housekeeping genes have been unsuccessful. The increasing availability of complete genome sequences of several strains from different countries has allowed the identification of regions having greater variability, which have been successfully implemented for the bacterium characterization, including microsatellites, genes of prophage origin, and miniature transposable elements with inverted-repeats (MITEs). In the present work, the genetic structure of 147 ‘Ca. L. asiaticus’ strains from nine provinces of Cuba were investigated using two polymorphic regions, consisting of typing for prophages and MITEs. The results showed an important level of coexistence of type 1 and 2 prophages in the Cuban strains, while the type 3 prophage was not detected. Likewise, a high rate of co-occurrence of both types of MITEs (MCLas-A and -B) was also observed. However, the MITE MCLas-A was detected only in its empty form. The double-locus analysis allowed the identification of eight genotypes. Out of these, seven genotypes were present in the Western region, which constitutes the region with the highest genetic variability. This is the first report of a genetic characterization of Cuban strains of ‘Ca. L. asiaticus’ with polymorphic markers in orchards growing in commercial citrus regions. Full article

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

17 pages, 1718 KB

Open AccessArticle

Neurotoxicity and Intestinal Microbiota Dysbiosis in the Chinese Mitten Crab (Eriocheir sinensis) Induced by Anatoxin-a: A Microbiota–Intestine–Brain Axis Perspective

by Huixia Feng, Shengyu Hu, Zihao Song, Ziqi Lin, Kai Zhang, Xianhui Ning, Cong Zhang and Shaowu Yin

Microorganisms 2025, 13(10), 2380; https://doi.org/10.3390/microorganisms13102380 (registering DOI) - 15 Oct 2025

Abstract

Anatoxin-a (ANTX-a), a potent neurotoxin produced by various cyanobacterial species, poses a serious threat to aquatic organisms. This study investigated the neurotoxicity of ANTX-a on juvenile Chinese mitten crab (Eriocheir sinensis). Different from previous studies on vertebrate models or fish liver [...] Read more.

Anatoxin-a (ANTX-a), a potent neurotoxin produced by various cyanobacterial species, poses a serious threat to aquatic organisms. This study investigated the neurotoxicity of ANTX-a on juvenile Chinese mitten crab (Eriocheir sinensis). Different from previous studies on vertebrate models or fish liver toxicity, we focused on the microbiota–intestine–brain axis. Results demonstrated that ANTX-a exposure induced significant neurotoxicity, marked by the upregulation of apoptosis-related genes and disruption of neurotransmitter homeostasis. Transcriptomic analysis of thoracic ganglia revealed significant dysregulation of metabolic pathways, characterized by upregulated histidine metabolism (elevated histidine decarboxylase-like) and downregulated lipid metabolism (suppressed sphingomyelin phosphodiesterase-like). Additionally, increased intestinal histamine levels and elevated serum diamine oxidase activity indicated intestinal barrier damage. Intestinal microbiota analysis revealed that the abundance of nerve-related bacteria Tyzzerella and Clostridium sensu stricto 1 changed significantly. In summary, these findings indicate that ANTX-a induced neurotoxicity by affecting neurotransmitter systems and gut health, implicating the microbiota–intestine–brain axis. The results underscore the role of microbiota–intestine–brain communication in cyanotoxin toxicity within aquatic invertebrates and provide new insights into the ecotoxicological risks of cyanobacterial blooms in aquatic invertebrates. Full article

(This article belongs to the Section Microbiomes)

16 pages, 2167 KB

Open AccessArticle

Continuous Circulation of Hepatitis E and A Viruses During COVID-19 Pandemic Lockdowns in Munich, Germany—Experience from Three Years of Wastewater Surveillance

by Jasmin Javanmardi, Mathias Schemmerer, Karina Wallrafen-Sam, Jessica Neusser, Raquel Rubio-Acero, Michael Hoelscher, Thomas Kletke, Bernhard Boehm, Michael Schneider, Elisabeth Waldeck, Martin Hoch, Merle M. Böhmer, Christof Geldmacher, Jan Hasenauer, Jürgen J. Wenzel and Andreas Wieser

Microorganisms 2025, 13(10), 2379; https://doi.org/10.3390/microorganisms13102379 - 15 Oct 2025

Abstract

The COVID-19 pandemic has increased interest in wastewater-based epidemiology (WBE) as a reliable and cost-effective framework for monitoring the spread of microbes. However, WBE frameworks have rarely been applied to the study of fecal–oral transmissible diseases, except for poliomyelitis. Here, we investigated the [...] Read more.

The COVID-19 pandemic has increased interest in wastewater-based epidemiology (WBE) as a reliable and cost-effective framework for monitoring the spread of microbes. However, WBE frameworks have rarely been applied to the study of fecal–oral transmissible diseases, except for poliomyelitis. Here, we investigated the presence of hepatitis A virus (HAV) and hepatitis E virus (HEV) in wastewater in Munich. We collected wastewater samples between July 2020 and November 2023. A total of 186 samples were processed using centrifugation and analyzed for HAV- and HEV-RNA using RT-qPCR. As a reference, we used notification data from clinically or laboratory-diagnosed hepatitis A and E cases. Lockdown stringency levels were derived from official documentation. Our results show that 87.6% of wastewater samples were positive for HEV at concentrations of 9.0 × 10¹ to 2.5 × 10⁵ copies/L, while HAV was only detectable in 7.5% of the samples at viral loads of 4.6 × 10¹ to 2.4 × 10³ copies/L. We also detected differences in HEV concentrations but not in case numbers when comparing lockdown and no-lockdown periods. This study covers all but the first lockdowns in Bavaria. We present a unique real-world dataset evaluating the impact of lockdown interventions on hepatitis A and E case numbers, as well as on the concentrations of HAV and HEV in wastewater. Person-to-person spread and eating out appear to have contributed to the transmission of HEV. In addition, the consistently high HEV concentrations in sewage support the findings of serological studies, indicating a substantial burden of undetected subclinical infections. Full article

(This article belongs to the Special Issue Surveillance of Health-Relevant Pathogens Employing Wastewater)

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37 pages, 2442 KB

Open AccessReview

Preharvest Control of Campylobacter Colonization in Chickens, with a Special Emphasis on Vaccination Strategies

by Chaitanya Gottapu, Lekshmi K. Edison, Gary D. Butcher and Subhashinie Kariyawasam

Microorganisms 2025, 13(10), 2378; https://doi.org/10.3390/microorganisms13102378 - 15 Oct 2025

Abstract

Campylobacter is a leading cause of human gastroenteritis, with poultry serving as the primary reservoir host. Effective preharvest control strategies are crucial for preventing or reducing Campylobacter contamination on meat surfaces. As concerns grow regarding the use of antimicrobials in animal agriculture, the [...] Read more.

Campylobacter is a leading cause of human gastroenteritis, with poultry serving as the primary reservoir host. Effective preharvest control strategies are crucial for preventing or reducing Campylobacter contamination on meat surfaces. As concerns grow regarding the use of antimicrobials in animal agriculture, the importance of non-antimicrobial preharvest strategies in poultry production has become increasingly significant. This comprehensive review focuses on the biology of Campylobacter, its impact on public health, and current and emerging preharvest strategies, with a special emphasis on vaccination. Preharvest strategies are broadly classified into biosecurity measures, gut microbiota modifications using prebiotics, probiotics, postbiotics, feed additives, and vaccination. While some vaccines have proven to be effective in research settings, no commercial vaccines are currently available. Because no single strategy can effectively combat Campylobacter, integrating multiple approaches, such as improved biosecurity measures, immunization, and dietary modifications, may provide a solution for reducing Campylobacter loads in poultry. Embracing a “One Health” approach, gaining a deeper understanding of Campylobacter pathophysiology, advancing vaccine technology, and implementing holistic farm management practices will be essential for the sustainable control of Campylobacter and for reducing the risk of human campylobacteriosis. Full article

(This article belongs to the Special Issue Poultry Pathogens and Poultry Diseases, 2nd Edition)

15 pages, 1743 KB

Open AccessArticle

Rumen-Protected Leucine Improved Growth Performance of Fattening Sheep by Changing Rumen Fermentation Patterns

by Shu Li, Jiantao Zhao, Peng Zhang, Shaoyang Pang, Mingyu Ma, Yifan Nie, Zhenzi Xu, Sijin Li, Yuanyuan Li and Wenju Zhang

Microorganisms 2025, 13(10), 2377; https://doi.org/10.3390/microorganisms13102377 - 15 Oct 2025

Abstract

This experiment investigated the effects of rumen-protected leucine (RP-Leu) supplementation on growth performance, rumen fermentation parameters, and rumen microbiota of fattening sheep. Forty fattening sheep (19.03 ± 0.38 kg) were randomly divided into four groups. The four groups were fed the basal diet [...] Read more.

This experiment investigated the effects of rumen-protected leucine (RP-Leu) supplementation on growth performance, rumen fermentation parameters, and rumen microbiota of fattening sheep. Forty fattening sheep (19.03 ± 0.38 kg) were randomly divided into four groups. The four groups were fed the basal diet supplemented with RP-Leu at 0 (L-0), 0.5 (L-0.5), 1.0 (L-1.0), or 1.5 (L-1.5) g/d. The experimental period lasted 67 d. The results demonstrated that compared with the L-0 group, the L-1.0 and L-1.5 groups significantly increased the average daily gain (ADG) by 22.2% and 18.0%, respectively (p < 0.05), and reduced the feed-to-gain ratio (F:G ratio) by 20.0% and 13.4% (p < 0.05); the L-1.5 group significantly increased the concentrations of propionate and isovalerate (p < 0.05) and significantly decreased the acetate to propionate ratio (A:P) by 25.7% (p < 0.05); and the L-1.0 group significantly decreased the concentrations of free branched-chain amino acids (BCAAs) in rumen (p < 0.05). A microbiota analysis showed that RP-Leu enriched the abundance of fiber-degrading bacteria. A correlation analysis showed that acetic acid and A:P were positively correlated with norank_f_F082 and norank_f_UCG-011 (p < 0.05). Phenylalanine, isoleucine, valine, and leucine were negatively correlated with norank_o_Bacteroidales (p < 0.05). In conclusion, RP-Leu improved the growth performance of fattening sheep by changing the rumen fermentation parameters and patterns; the optimal supplementation level is 1.0 g/d. Full article

(This article belongs to the Section Gut Microbiota)

24 pages, 1969 KB

Open AccessReview

History and Evolution of the Hypervirulent Clostridioides difficile Ribotype 027 Lineage

by Isabella A. Tickler, Richard V. Goering and Fred C. Tenover

Microorganisms 2025, 13(10), 2376; https://doi.org/10.3390/microorganisms13102376 - 15 Oct 2025

Abstract

Clostridioides difficile was first identified in 1935 and subsequently emerged over the next several decades as the predominant bacterial cause of healthcare-associated gastrointestinal infections, placing a significant burden on healthcare systems worldwide. A major driver of the rapid rise in the incidence of [...] Read more.

Clostridioides difficile was first identified in 1935 and subsequently emerged over the next several decades as the predominant bacterial cause of healthcare-associated gastrointestinal infections, placing a significant burden on healthcare systems worldwide. A major driver of the rapid rise in the incidence of C. difficile infection (CDI) was the emergence and spread of a hypervirulent strain, which became known as PCR ribotype 027 (RT027). The C. difficile RT027 strain produced not just the typical toxin A and toxin B virulence factors but also expressed a third toxin called binary toxin that enhanced pathogenicity. Interestingly, the C. difficile RT027 strain apparently emerged at least twice in geographically distinct areas. The two lineages can be differentiated by their resistance or susceptibility to fluoroquinolones. Other ribotypes of C. difficile that also express binary toxin have emerged recently, some of which are highly related genetically to RT027. The aim of this study is to integrate genomic data and published literature to clarify the emergence, divergence, and apparent decline of C. difficile RT027. Full article

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

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17 pages, 1554 KB

Open AccessArticle

Sodium Butyrate Supplementation in Whole Milk Modulates the Gastrointestinal Microbiota Without Altering the Resistome and Virulome in Preweaned Calves

by Liwen Xing, Song Niu, Donglin Wu, Zhanghe Zhang and Ming Xu

Microorganisms 2025, 13(10), 2375; https://doi.org/10.3390/microorganisms13102375 - 15 Oct 2025

Abstract

This study investigated the effects of supplementing whole milk (WM) with sodium butyrate (SB) on the gastrointestinal microbiota of preweaned calves. Twelve newborn Holstein calves (4 days old, 39.21 ± 1.45 kg) were randomly assigned into one of two dietary treatments: (1) WM [...] Read more.

This study investigated the effects of supplementing whole milk (WM) with sodium butyrate (SB) on the gastrointestinal microbiota of preweaned calves. Twelve newborn Holstein calves (4 days old, 39.21 ± 1.45 kg) were randomly assigned into one of two dietary treatments: (1) WM without SB (CON) and (2) WM supplemented with SB (8.8 g/d; SB). At 74 days of age, all calves were slaughtered to collect ruminal and cecal digesta. Metagenomic analysis was used to characterize the microbial composition, virulence factor genes (VFGs), and antibiotic resistance genes (ARGs). SB supplementation altered the ruminal microbial composition and increased the abundance of beneficial bacteria, including Actinobacteria, Bifidobacterium, and Olsenella (p < 0.05). Although SB did not significantly affect the overall microbial composition or diversity in the cecum (p > 0.05), it promoted the growth of beneficial genera such as Flavonifractor and Subdoligranulum (p < 0.05). Furthermore, SB supplementation did not significantly alter the composition of VFGs or ARGs in either the rumen or the cecum (p > 0.05). However, significant differences in VFGs and ARGs were observed between the rumen and the cecum, with a greater diversity of both VFGs and ARGs detected in the cecum compared to that in the rumen (p < 0.05). In summary, supplementing whole milk with sodium butyrate modulates gastrointestinal health in preweaned calves by favorably shaping the microbial community without significantly altering the antibiotic resistome or virulome. Full article

(This article belongs to the Section Gut Microbiota)

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21 pages, 1168 KB

Open AccessReview

Role of IFN-γ from Different Immune Cells in Chlamydia Infection

by Xuan Chen, Wenjing Yang, Yuchen Hu, Yang Zhou and Zhou Zhou

Microorganisms 2025, 13(10), 2374; https://doi.org/10.3390/microorganisms13102374 - 15 Oct 2025

Abstract

Chlamydia invades multiple mucosal tissues in humans and animals. The body’s first line of defense against chlamydial infection is provided by innate immunity, whereas adaptive immunity plays a crucial role in managing the infection’s progression and preparing the immune system to combat reinfection. [...] Read more.

Chlamydia invades multiple mucosal tissues in humans and animals. The body’s first line of defense against chlamydial infection is provided by innate immunity, whereas adaptive immunity plays a crucial role in managing the infection’s progression and preparing the immune system to combat reinfection. Host resistance to chlamydial infection necessitates a coordinated effort between innate and adaptive immune cells. Numerous cell types are capable of producing interferon gamma (IFN-γ) as a defense mechanism against chlamydial infection, thereby effectively mediating the clearance of infection. However, the distinct roles of various immune cell populations in responding to chlamydial infection, as well as their functions during infection progression, remain poorly understood. Therefore, we will discuss the various roles of IFN-γ released by different immune cells in chlamydial infection, focusing primarily on experimental animal models and a review of available data from in vivo cellular immunological studies in humans. Full article

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

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24 pages, 3029 KB

Open AccessArticle

Spatiotemporal Dynamics of Microbial and Fish Communities in the Thracian Sea Revealed by eDNA Metabarcoding

by Maria Tokamani, Panagiotis Liakopoulos, Konstantinos Tegopoulos, Aristea-Marina Zigkou, George Triantaphyllidis, Nikolaos Kamidis, Maria E. Grigoriou, Raphael Sandaltzopoulos and Petros Kolovos

Microorganisms 2025, 13(10), 2373; https://doi.org/10.3390/microorganisms13102373 - 15 Oct 2025

Abstract

The Thracian Sea, a semi-enclosed coastal basin in the northeastern Aegean Sea, represents a dynamic marine environment influenced by freshwater inputs, stratification, and seasonal variability. Here, we investigated the spatiotemporal dynamics of microbial and ichthyofaunal communities using environmental DNA (eDNA) and high-throughput sequencing [...] Read more.

The Thracian Sea, a semi-enclosed coastal basin in the northeastern Aegean Sea, represents a dynamic marine environment influenced by freshwater inputs, stratification, and seasonal variability. Here, we investigated the spatiotemporal dynamics of microbial and ichthyofaunal communities using environmental DNA (eDNA) and high-throughput sequencing across various stations in the vicinity of the Thracian Sea, in consecutive months (through spring and summer). Seawater samples were collected from the surface and thermocline layers, and environmental parameters were recorded to examine their influence on biodiversity patterns. Microbial communities exhibited strong seasonal and depth-related structuring. Alpha diversity was highest in spring and declined during summer, while beta diversity analyses revealed clear clustering by month and depth. Dominant taxa included Alphaproteobacteria (SAR11), Cyanobacteria (Synechococcus, Prochlorococcus), with distinct core microbiomes. Fish communities, identified via CytB metabarcoding, displayed marked temporal turnover but limited spatial segregation. While alpha diversity metrics did not differ significantly, beta diversity analyses showed seasonal shifts with dominant taxa such as Raja spp., Engraulis spp., and Diplodus sargus. Multivariate and co-structure analyses (Mantel, Procrustes) revealed moderate but significant concordance between microbial and fish communities and support the existence of similar biodiversity responses to environmental parameters across temporal and spatial variability. Co-occurrence networks further present depth-specific associations, with surface communities being more cooperative and phototrophic, while thermocline networks showed modularity and potential ecological specialization. This study highlights the value of integrated eDNA-based monitoring in revealing seasonal biodiversity dynamics and ecological interactions in coastal marine ecosystems, supporting future spatial planning and conservation strategies in the Thracian Sea. Full article

(This article belongs to the Special Issue Microbiome Research for Animal, Plant and Environmental Health: Second Edition)

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17 pages, 2535 KB

Open AccessArticle

Sustainable Recycling of Mushroom Residue as an Effective Substitute for Cotton Hull Waste in Volvariella volvacea Cultivation: Evidence from Physicochemical and Microbiome Analyses

by Pattana Kakumyan, Lin Yang, Shunjie Liu, Kritsakorn Saninjuk, Qin Dong, Xueyu Pan, Changxia Yu and Yan Zhao

Microorganisms 2025, 13(10), 2372; https://doi.org/10.3390/microorganisms13102372 - 15 Oct 2025

Abstract

Mushroom residue (MR) is extensively produced during the industrialized cultivation of mushrooms, and its utilization is environmentally sustainable. Cotton hull waste (CW) serves as a common raw material for the cultivation of Volvariella volvacea in China. This study compared MR- and CW-based cultivation [...] Read more.

Mushroom residue (MR) is extensively produced during the industrialized cultivation of mushrooms, and its utilization is environmentally sustainable. Cotton hull waste (CW) serves as a common raw material for the cultivation of Volvariella volvacea in China. This study compared MR- and CW-based cultivation formulas with respect to their physicochemical characteristics, bacterial communities, and functional dynamics during substrate fermentation (composting). Xylanase production was greater in the MR formula than in the CW formula. Conversely, cellulase (CMCase) was generated at higher levels in the CW formula compared to the MR formula. Interestingly, the biological efficiency of MR was found to be comparable to that of CW, but the cost of MR was much lower. The dynamics of bacterial communities and their associated metabolic functions during substrate fermentation were monitored using 16S rRNA metagenomics techniques. Significant alterations in bacterial community structure were observed within both formulas throughout the preparation phase. Indicator species analysis revealed distinct patterns of bacterial diversity development between MR- and CW-based composts during fermentation. Metabolic function analysis indicated that carbohydrate and amino acid metabolism remained relatively active throughout this process. These results suggest that the MR formula is equally effective as conventional CW compost for supporting V. volvacea cultivation, while also offering a lower raw material cost. Full article

(This article belongs to the Special Issue Microbes for Sustainable Production)

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20 pages, 5516 KB

Open AccessArticle

Multi-Omics Insights into Gingivitis from a Clinical Trial: Understanding the Role of Bacterial and Host Factors

by Niranjan Ramji, Ping Hu, Alejandra Muñoz Bodnar, Camila Pereira Braga, John Snowball, Dionne Swift, Hao Ye, Sancai Xie, Rachel Trenner, Malgorzata Klukowska, Eva Schneiderman and Aaron R. Biesbrock

Microorganisms 2025, 13(10), 2371; https://doi.org/10.3390/microorganisms13102371 - 15 Oct 2025

Abstract

Poor oral health is a neglected epidemic, potentially contributing to systemic health issues. We employed a multi-omics approach to investigate the biological changes associated with gingivitis and the effects of stannous fluoride (SnF₂) dentifrice on microbial composition and salivary proteomics in [...] Read more.

Poor oral health is a neglected epidemic, potentially contributing to systemic health issues. We employed a multi-omics approach to investigate the biological changes associated with gingivitis and the effects of stannous fluoride (SnF₂) dentifrice on microbial composition and salivary proteomics in an eight-week clinical trial involving 39 participants categorized as high (n = 20) and low bleeders (n = 19). Baseline assessments revealed significant microbial dysbiosis in high bleeders, characterized by a higher abundance of Porphyromonas and Fusobacterium, alongside compromised epithelial barriers and increased inflammation. Following SnF₂ treatment, a substantial reduction in these bacteria, and an increase in Rothia and Haemophulis, were observed, correlating with improved clinical measures, including reduced bleeding and inflammation indices. In total, 80 proteins (including pro-inflammatory cytokines, alarmin keratins, and matrix metalloproteinases) showed a significant reduction in high bleeders after treatment, with 29 overlapping the disease biomarkers in the plasma atlas, supporting the role of SnF₂ in mitigating oxidative stress and enhancing epithelial integrity. Furthermore, SnF₂ treatment significantly reduced collagen degradation, suggesting the preservation of tissue integrity. These findings highlight that SnF₂ not only improves local oral health but may also benefit systemic health, showcasing the value of a multi-omics approach in understanding the interconnections among oral microbiota, inflammatory responses, and systemic health outcomes. Full article

(This article belongs to the Special Issue Crosstalk Between Host and Pathogen: Innate Immunity, Infection and Metabolic Reprogramming)

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3 pages, 141 KB

Open AccessEditorial

Antimicrobial Testing: Advancing Methodologies and Addressing Global Challenges

by Suresh G. Joshi

Microorganisms 2025, 13(10), 2370; https://doi.org/10.3390/microorganisms13102370 - 15 Oct 2025

Abstract

An increasing prevalence of antimicrobial resistance (AMR) represents a formidable threat to global health, demanding the urgent evolution of diagnostic methods and therapeutic strategies [...] Full article

(This article belongs to the Special Issue Antimicrobial Testing (AMT) 2.0)

25 pages, 3767 KB

Open AccessArticle

Seasonal Variations of the Nebraska Salt Marsh Microbiome: Environmental Impact, Antibiotic Resistance, and Unique Species

by Emma K. Stock, Ketlyn Rota, Brandi Dunn, Madelynn Vasquez, Daniela Hernandez-Velazquez, Alyssia Lespes, Solenn Bosmans, Jace C. Smith and John A. Kyndt

Microorganisms 2025, 13(10), 2369; https://doi.org/10.3390/microorganisms13102369 - 15 Oct 2025

Abstract

The Nebraska Salt Marshes are unique inland saltwater ecosystems, and this exploratory study is aimed at understanding the microbial composition and diversity that is providing the underlying support for these ecosystems. The microbiome shows both temporal and spatial variations that are concurrent with [...] Read more.

The Nebraska Salt Marshes are unique inland saltwater ecosystems, and this exploratory study is aimed at understanding the microbial composition and diversity that is providing the underlying support for these ecosystems. The microbiome shows both temporal and spatial variations that are concurrent with seasonal variations in salinity, temperature, and vegetation growth. Whole genome metagenomics analysis showed the predominance of purple non-sulfur bacteria in each season, indicating their importance in the marsh ecosystem. The fall season showed the highest microbial diversity and coincided with the highest levels of antimicrobial resistance markers to a variety of natural and synthetic antibiotics. In addition to the metagenomics approach, we also isolated and sequenced several unique species, most of them belonging to what appear to be new species of purple non-sulfur or purple sulfur bacteria. Both the metagenomics analysis and isolated species indicate that the nitrogen and sulfur cycling is well balanced in these marshes by a high relative abundance of purple bacteria. Noteworthy is the isolation of a new strain of Vibrio cholerae, which is a known human intestinal pathogen, that was predominantly present in the fall samples carrying several antibiotic resistance markers. Overall, the Nebraska salt marsh microbiome showcases both seasonal variations in microbial composition, a concerning prevalence of multiple antibiotic resistance, and the presence of unique bacterial species well-adapted to its distinctive alkaline and saline environment. Full article

(This article belongs to the Special Issue Interaction Between Microorganisms and Environment)

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13 pages, 280 KB

Open AccessArticle

Entomopathogenic Nematodes and Bioactive Compounds of Their Bacterial Endosymbionts Act Synergistically in Combination with Spinosad to Kill Phthorimaea operculella (Zeller, 1873) (Lepidoptera: Gelechiidae), a Serious Threat to Food Security

by Ebubekir Yüksel, Rachid Lahlali, Aydemir Barış, Muhammad Sameeullah, Furkan Ulaş, Abdurrahman Sami Koca, Essaid Ait Barka, Mustafa İmren and Abdelfattah Dababat

Microorganisms 2025, 13(10), 2368; https://doi.org/10.3390/microorganisms13102368 - 15 Oct 2025

Abstract

As a staple food, potato (Solanum tuberosum L.) (Solanaceae) is one of the most produced food crops to ensure food security. The potato tuber moth (PTM), Phthorimaea operculella (Zeller, 1873) (Lepidoptera: Gelechiidae), is a major pest of potato, damaging both the growing [...] Read more.

As a staple food, potato (Solanum tuberosum L.) (Solanaceae) is one of the most produced food crops to ensure food security. The potato tuber moth (PTM), Phthorimaea operculella (Zeller, 1873) (Lepidoptera: Gelechiidae), is a major pest of potato, damaging both the growing and storage processes. In recent years, green pest control strategies have been gaining importance to reduce the adverse effects of chemicals and protect the environment. Entomopathogenic nematodes (EPNs) and their bacterial endosymbionts (Xenorhabdus and Photorhabdus spp.) have been one of the top topics studied in sustainable pest control approaches. In the present study, the two most common EPN species, Steinernema feltiae and Heterorhabditis bacteriophora, and their bacterial associates, Xenorhabdus bovienii and Photorhabdus luminescens subsp. kayaii were evaluated against PTM larvae separately and in combination with spinosad. The survival rates of infective juveniles (IJs) of EPNs were over 92% after 72 h of direct exposure to spinosad. Co-application of EPNs and bioactive compounds (BACs) of endosymbiotic bacteria with spinosad induced synergistic interactions and achieved the maximum mortality (100%) in PTM larvae 48 h post-treatment. Spinosad and BAC combinations were highly efficient in controlling the PTM larvae and provided LT₅₀ values below 23.0 h. Gas chromatography mass spectrometry (GC-MS) analysis identified 29 compounds in total, 20 of which belonged to P. luminescens subsp. kayaii. The results indicate that the integration of EPNs and BACs of endosymbiotic bacteria with spinosad presents a synergistic interaction and enhances pest control efficacy. Full article

(This article belongs to the Special Issue Beneficial Microorganisms Against Phytopathogens: Innovations in Sustainable Plant Protection)

26 pages, 2735 KB

Open AccessArticle

Bioengineering Caulobacter vibrioides for Xylanase Applications in the Bakery Industry

by Bruna Simioni, Paula Maria Carneiro Rocha, Adriano Fávero, José Luis da Conceição Silva, Rinaldo Ferreira Gandra, Alexandre Maller, Marina Kimiko Kadowaki and Rita de Cássia Garcia Simão

Microorganisms 2025, 13(10), 2367; https://doi.org/10.3390/microorganisms13102367 - 15 Oct 2025

Abstract

The present study investigated the impact of genetic engineering strategies to produce a cell-free xylanase for applications in the baking industry. The xynA1 gene from the nonpathogenic bacterium Caulobacter vibrioides was integrated into the pAS22 vector with a xylose-inducible promoter and introduced back [...] Read more.

The present study investigated the impact of genetic engineering strategies to produce a cell-free xylanase for applications in the baking industry. The xynA1 gene from the nonpathogenic bacterium Caulobacter vibrioides was integrated into the pAS22 vector with a xylose-inducible promoter and introduced back into the bacteria, resulting in the creation of the BS-xynA1. This construct exhibited substantial secreted xylanase 1 (XynA1) activity, reaching 17.22 U/mL, and a specific activity of 278.64 U/mg after an 18 h growth period with 0.3% (v/v) xylose plus 0.2% (w/v) corn straw. RT-qPCR analysis confirmed that higher xylanase activity in C. vibrioides cells was correlated with increased transcription of the xynA1 gene in the induction medium. Moreover, BS-xynA1 cells coexpress other enzymes, including xylanase 2 (XynA2), cellulase, pectinase, α-amylase, β-glucosidase, β-xylosidase, and α-L-arabinosidase, at low levels (≤2 U/mL). In vitro comparison of cell-free xylanases from BS-xynA1 with three commercially available xylanase-containing mixtures commonly utilized in baking protocols revealed its superior specific activity (163.4 U/mg) across a broad temperature range (30–100 °C), with optimal performance at 50 °C. In practical baking tests, the addition of cell-free XynA1 led to a reduction in dough kneading time and increase in bread height compared to those of the control. Notably, the incorporation of XynA1 resulted in enhanced alveolar structure formation within the bread crumb. Specifically, the following changes were observed in the mass parameters compared to those of the control: an increase in extensibility, elasticity, and deformation energy, and subsequent improvements in strength. Additionally, XynA1 addition led to a reduction in toughness and toughness/elasticity index, indicating a reduction in the mass stiffness of the enzyme-treated bread. To date, this is the first successful application of recombinant XynA1 from C. vibrioides in biotechnological processes related to baking, underscoring the potential and prospects in the food industry. Full article

(This article belongs to the Special Issue Microbial Enzymes—Tools for Biotechnological Processes)

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15 pages, 1097 KB

Open AccessSystematic Review

Comparative Meta-Analysis of Long-Read and Short-Read Sequencing for Metagenomic Profiling of the Lower Respiratory Tract Infections

by Giovanni Lorenzin and Maddalena Carlin

Microorganisms 2025, 13(10), 2366; https://doi.org/10.3390/microorganisms13102366 - 15 Oct 2025

Abstract

Metagenomic next-generation sequencing (mNGS) is increasingly employed for the diagnosis of lower respiratory tract infections (LRTIs). However, the relative diagnostic performance of long-read versus short-read sequencing platforms remains incompletely defined. For this systematic review, a search was conducted in PubMed, Embase, Scopus, Web [...] Read more.

Metagenomic next-generation sequencing (mNGS) is increasingly employed for the diagnosis of lower respiratory tract infections (LRTIs). However, the relative diagnostic performance of long-read versus short-read sequencing platforms remains incompletely defined. For this systematic review, a search was conducted in PubMed, Embase, Scopus, Web of Science, and Google Scholar to identify studies directly comparing long-read (e.g., Oxford Nanopore, PacBio) and short-read (e.g., Illumina, Ion Torrent, BGISEQ) metagenomic sequencing for the diagnosis of LRTI. Eligible studies reported diagnostic accuracy or comparative performance between platforms. Risk of bias was evaluated using the QUADAS-2 tool. Thirteen studies met inclusion criteria. Reported platforms included Illumina, Oxford Nanopore, PacBio, Ion Torrent, and BGISEQ-500. A total of 13 studies met inclusion criteria. Across studies reporting sensitivity, average sensitivity was similar for Illumina (71.8%) and Nanopore (71.9%). Specificity varied substantially, ranging from 42.9 to 95% for Illumina and 28.6 to 100% for Nanopore. Concordance between platforms ranged from 56 to 100%. Illumina consistently produced superior genome coverage (approaching 100% in most reports) and higher per-base accuracy, whereas Nanopore demonstrated faster turnaround times (<24 h), greater flexibility in pathogen detection, and superior sensitivity for Mycobacterium species. Risk of bias was frequently high or unclear, particularly in patient selection (6 studies), index test interpretation (5), and flow and timing (4), limiting the robustness of pooled estimates. Long-read and short-read mNGS platforms exhibit comparable strengths in the diagnosis of LRTIs. Illumina remains optimal for applications requiring maximal accuracy and genome coverage, whereas Nanopore offers rapid, versatile pathogen detection, particularly for difficult-to-detect organisms such as Mycobacterium. However, there are certain limitations of the review, including a lack of comparable outcomes reported in all studies; therefore, further research is warranted to address this. Full article

(This article belongs to the Section Medical Microbiology)

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14 pages, 2340 KB

Open AccessCommunication

Bacteria That Made History: Detection of Enterobacteriaceae and Carbapenemases in the Waters of Southern Brazil’s Largest Flood

by João Vitor Barboza Cardoso, Dariane Castro Pereira, William Latosinski Matos, Gabriela Simões de Oliveira, Victória Rodrigues de Carvalho, Louidi Lauer Albornoz, Afonso Luis Barth, Salatiel Wohlmuth da Silva and Andreza Francisco Martins

Microorganisms 2025, 13(10), 2365; https://doi.org/10.3390/microorganisms13102365 - 15 Oct 2025

Abstract

Floods seriously threaten public health by promoting the spread of antimicrobial-resistant (AMR) bacteria, particularly in urban areas with poor sanitation. In May 2024, the state of Rio Grande do Sul, Brazil, experienced the most severe flood in its history, affecting over 2.3 million [...] Read more.

Floods seriously threaten public health by promoting the spread of antimicrobial-resistant (AMR) bacteria, particularly in urban areas with poor sanitation. In May 2024, the state of Rio Grande do Sul, Brazil, experienced the most severe flood in its history, affecting over 2.3 million people and resulting in extensive dissemination of sewage, contaminating the environment. This study aimed to investigate the presence of Enterobacteriaceae and clinically relevant carbapenemase genes (bla_KPC and bla_NDM) in floodwaters from Porto Alegre using molecular methods. Seventy-nine water samples were collected during four sampling campaigns conducted between May and June 2024. Samples were obtained from flooded areas and points across Guaíba Lake. DNA was extracted with the DNeasy PowerWater Kit, and qPCR was performed using TaqMan assays targeting Enterobacteriaceae, bla_KPC and bla_NDM. Of the 79 samples, 75 yielded sufficient DNA for analysis. Enterobacteriaceae were detected in 100% of the samples, across all collections. The bla_KPC gene was detected in 100% of the first collection pools, and in 94.7%, 94.7%, and 85.7% of samples from the second, third, and fourth collections, respectively. The bla_NDM gene was present in 81.3% of the first collection pools, and in 78.9%, 89.4%, and 80.9% of samples from the subsequent collections. The high prevalence of Enterobacteriaceae and carbapenemase genes in floodwaters reveals an alarming environmental dissemination of AMR genetic markers. These findings underscore the need for environmental AMR surveillance, especially in disaster settings, and support the implementation of the One Health approach to mitigate the spread of resistance genes across human, animal, and environmental interfaces. Full article

(This article belongs to the Special Issue From Sewers to Solutions: Wastewater Surveillance in the Fight Against Antimicrobial Resistance)

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16 pages, 1199 KB

Open AccessArticle

FucR Functions as a Transcriptional Regulator for L-Fucose Utilization in Campylobacter jejuni

by Wayne T. Muraoka, Nicholas Lizer, Peng Liu, Zhangqi Shen, Qingqing Xia, Muslum Ilgu and Qijing Zhang

Microorganisms 2025, 13(10), 2364; https://doi.org/10.3390/microorganisms13102364 - 14 Oct 2025

Abstract

Campylobacter jejuni is an enteric pathogen and a major cause of foodborne illness worldwide. It has been shown that C. jejuni primarily utilizes amino acids as its preferred energy source, but its ability to utilize L-fucose can grant a competitive advantage during intestinal [...] Read more.

Campylobacter jejuni is an enteric pathogen and a major cause of foodborne illness worldwide. It has been shown that C. jejuni primarily utilizes amino acids as its preferred energy source, but its ability to utilize L-fucose can grant a competitive advantage during intestinal colonization. In C. jejuni, fucose utilization is encoded by a variable region named plasticity region 2 (PR2); however, the regulatory mechanism for the region remains unknown and is investigated in this study. Genomic sequence analysis revealed that immediately upstream of the fucose utilization operon is a putative IclR-type transcriptional regulator, cj0480c (named fucR here). To determine whether fucR regulates the expression of the fucose utilization operon, we generated a knock-out mutant of fucR. RT-PCR and microarray analysis found that all the genes in the operon were polycistronic and significantly upregulated in the fucR mutant compared with their expression in the wild-type strain. In the presence of fucose, expression of the fucose utilization genes was induced in the wild-type strain but no longer inducible in the fucR mutant, suggesting that FucR functions as a repressor for the fucose utilization operon. To determine whether FucR directly or indirectly regulates the fucose utilization operon, a 6xHis-tagged full-length FucR was produced in Escherichia coli, and the purified recombinant FucR was used in electrophoretic mobility shift assay, which demonstrated that FucR bound specifically to the promoter region of the fucose utilization operon. Together, these results indicate that the L-fucose utilization operon in C. jejuni is directly regulated by FucR, which functions as a transcriptional repressor and modulates the expression of the operon in response to fucose. Full article

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

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44 pages, 1083 KB

Open AccessReview

Major Bacterial Foodborne Pathogens in Poultry: Implications for Human Health and the Poultry Industry and Probiotic Mitigation Strategies

by Shreeya Sharma, Sukhman Kaur, Mostafa Naguib, Ari Bragg, Abigail Schneider, Raveendra R. Kulkarni, Ali Nazmi and Khaled Abdelaziz

Microorganisms 2025, 13(10), 2363; https://doi.org/10.3390/microorganisms13102363 - 14 Oct 2025

Abstract

Poultry production has become the fastest-growing sector in global meat supply. However, the intensification of poultry farming has increased the risk of zoonotic transmission of bacterial pathogens such as Salmonella spp., Campylobacter spp., Escherichia coli, Clostridium perfringens, and Listeria monocytogenes. [...] Read more.

Poultry production has become the fastest-growing sector in global meat supply. However, the intensification of poultry farming has increased the risk of zoonotic transmission of bacterial pathogens such as Salmonella spp., Campylobacter spp., Escherichia coli, Clostridium perfringens, and Listeria monocytogenes. These bacterial agents pose major public health concerns, contributing to millions of human infections annually and substantial economic losses. Historically, antibiotic growth promoters (AGPs) were widely used to mitigate disease burden and improve poultry productivity. Yet, the global shift away from AGPs due to concerns over antimicrobial resistance has spurred interest in antimicrobial alternatives. Among these, probiotics have been explored as a promising preharvest intervention. This review investigates major bacterial foodborne pathogens associated with poultry and evaluates the practical implementation of probiotic-based strategies in modern poultry production systems, with the goal of reducing pathogen load and enhancing overall food safety. Full article

(This article belongs to the Special Issue Enhancing Gut Health: Early-Life Microbiome Manipulation in Poultry Through Probiotics)

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