Journal of Applied Microbiology

Carbapenem-resistant Acinetobacter (CRA) has been associated with increased morbidity and mortality in clinical settings. In this study, we explored the transfer potential of a mobilizable plasmid-harboring blaOXA-72 gene between Acinetobacter species originating from patient, municipal wastewater, and pig farm wastewater. PCR-based evidence suggested putative transfer of blaOXA-72 from Acinetobacter pittii to Acinetobacter baumannii. In this pair, the apparent frequency of PCR-marker-positive putative transconjugants varied depending on temperature and meropenem supplementation, with higher number observed at 27{degrees}C compared to 17{degrees}C and 37{degrees}C. Likewise, the presence of antibiotic pressure yields to higher apparent conjugation frequency, however this observation was limited to a singled donor-recipient pair. Further, we revealed a phenotypic conversion in terms of meropenem susceptibility and a fitness cost in the putative transconjugants. While whole genome sequencing did not conclusively verify the presence of blaOXA-72 or fully resolved plasmid configuration, Oxford Nanopore read mapping consistently detected the chromosomal strA gene in all isolates. In contrast, only a limited number of reads aligned with blaOXA-72 gene, traC, or the complete plasmid sequences. Comparative analyses further revealed variations in the surface-associated factors and defense systems composition of the recipient strains, which could be considered as barriers in conjugation. Lastly, the persistence of PCR-detectable marker genes in putative transconjugants was variable and generally unstable over a 30-day period. Overall, these findings provide preliminary insights into the factors that may influence horizontal gene transfer and short-term maintenance of blaOXA-72.

Bacterial conjugation is the primary mechanism by which antibiotic resistance genes spread in microbial populations, yet our understanding of this process has been largely based on experiments conducted under aerobic conditions. This creates a fundamental disconnect: environments that are considered hotspots for gene exchange (e.g., the gut, abscesses, chronic wounds, and wastewater systems) are predominantly anaerobic. In this study, we investigate whether oxygen availability influences the transfer rate of a set of common ESBL-IncI1- and qnrS1-IncF plasmids in commensal Escherichia coli strains from chickens. We found that oxygen availability significantly shapes conjugation dynamics in a recipient strain-specific manner, with anaerobic conditions promoting higher ESBL-IncI1- plasmid transfer rates to commensal E. coli recipients. Conjugation rates of the ESBL-IncI1- plasmids to a laboratory strain of E. coli were several orders of magnitude higher and independent of oxygen level, while two qnrS1-IncF plasmids showed higher anaerobic rates. Our study reveals critical "oxygen blind spots" in conventional conjugation assays and suggests that conventional aerobic conjugation assays underestimate plasmid transfer rates in natural environments such as the chicken caeca. These findings highlight the importance of aligning experimental conditions with the physiological and ecological environments in which gene exchange naturally occurs. Tailoring these variables is essential for generating results that accurately reflect, predict, and potentially intervene in the horizontal spread of antimicrobial resistance.

Surface waters in urban watersheds receive episodic inputs of wastewater, runoff, and road-salt residues during spring, yet the contribution of these short hydrological windows to antibiotic resistance gene (ARG) loading remains poorly resolved. Weekly samples were collected from offshore and nearshore sites in the Detroit River, a Great Lakes transboundary connecting channel, from February to December 2025. Five clinically relevant ARGs encoding resistance to carbapenems, methicillin, and colistin, alongside the fecal marker pepper mild mottle virus (PMMoV), were quantified by qPCR and paired with ten conventional water-quality variables. blaNDM, mcr-1, and blaVIM-7 were not detected while blaKPC occurred as discrete pulses. One week (5 May) accounted for 37.2% of annual offshore blaKPC loading, and three weeks in late April to early May accounted for 72.8%, with peak concentrations reaching 5.4 x 10E3 and 7.2 x 10E3 copies/L. mecA was detected in nearly all samples without a dominant pulse. PMMoV normalization showed blaKPC did not vary seasonally (Kruskal-Wallis p = 0.198), consistent with diluted wastewater during spring precipitation events rather than an emergent source. mecA/PMMoV varied seasonally (p = 0.003) and was lowest in spring, implicating non-wastewater inputs in summer and autumn. Seven water-quality variables were significantly elevated during blaKPC pulse weeks. PCA distinguished pulse from background conditions, explaining 81.3% of variance. A random forest classifier achieved leave-one-out AUC of 0.917; ROC AUC reached 0.943 for total phosphorus, 0.924 for chloride, and 0.974 for the multivariate model. These results demonstrate that blaKPC and mecA operate through distinct source pathways and that routine water-quality monitoring can flag elevated blaKPC risk without additional sampling infrastructure.

Introduction: Rapid pathogen identification, resistance detection, and susceptibility profiling improve antimicrobial prescribing and associated outcomes, but fragmented workflows lead to inefficiencies and are costly. We evaluated a research-use-only (RUO) approach using ASTar(R) remnant bacterial suspension from routine AST for MALDI-TOF MS pathogen identification and Lateral Flow Assay (LFA)-based detection of targeted resistance mechanisms. Methods: Gram-negative (GN) bacterial strains from reference and curated resistance collections (CDC1, ARLG2, ATCC3) [n=119] were contrived into blood culture bottles and processed in the ASTar 16 System using the ASTar BC G- Kit (Q-linea AB, Sweden). Under RUO conditions, remnant bacterial suspensions were collected ~1-2 h after ASTar run initiation and analyzed using NG Test CTX-M Multi, NG Test CARBA-5, NG Test Acineto-5 RUO, and MALDI-TOF MS. Results: Mean ({+/-} SD) remnant suspension volume was 2722 L ({+/-} 300 L). All samples yielded high confidence MALDI-TOF MS scores (>2.0), with five initially scoring <2.0 and resolving on repeat testing. LFA results showed full agreement with reference isolates for blaCTX-M positive/negative (30/30) and with 60 or 61 target carbapenemase-positive/negative isolates. Testing of a subset of samples to mimic reflex workflows with ASTar phenotypic results did not affect LFA performance 24 (n=26; 23 Enterobacterales, 3 P. aeruginosa and 9 A. baumannii). Cost savings can be realised versus commercial multiplex PCR. Conclusion: This integrated approach of ~6 h rapid phenotypic AST with same-run identification and resistance detection (1-2 h from instrument start) or reflex testing upon availability of ASTar results may support earlier susceptibility results and offer cost savings to current workflows.

Background: Urinary tract infections (UTIs) rank among the most common infections globally, with many linked to indwelling urinary catheters. Our prior culture-based longitudinal evaluation of long-term catheterized nursing home residents revealed persistent asymptomatic colonization by pathogens and demonstrated that CAUTI onset was not necessarily due to new pathogen acquisition. In this study, we optimized metagenomics methods to examine the ecological structure underlying persistent colonization and the transition to infection. Results: We present a comprehensive longitudinal metagenomic analysis of catheterized urine specimens, revealing colonization dynamics of 69 microbial species across 198 samples from 9 individuals. Descriptive ecological metrics were combined with Bayesian mixed-effects models that accounted for repeated within-participant sampling to identify clusters of co-occurring species, determine the impact of perturbations such as antibiotic exposure and catheter changes on community structure, and identify taxa predictive of infection sign and symptom onset. Longitudinal specimens clustered into three main ecological phenotypes: 1) moderate diversity, unstable communities (3 participants); 2) high diversity, stable communities that resisted disruption even after multiple catheter changes (3 participants); and 3) low diversity, pathogen-dominated communities (3 participants). Catheter changes alone did not significantly disrupt community composition, while antibiotic exposures induced major shifts often followed by re-colonization with the same genera within subsequent weeks. Six clusters of species were identified for which relative abundances correlated across perturbations to the microbial community, including a mutually exclusive Enterobacterales cluster and fastidious-anaerobe group cluster. 24 species were found to correlate with onset of signs and symptoms of infection, 11 of which were missed by standard urine culture. Conclusions. The catheterized urinary tract represents a novel ecosystem that is resilient to disruption by catheter changes but susceptible to antibiotic perturbation. Antibiotic exposure did deplete all species associated with signs and symptoms but also depleted potentially benign microbes. Our findings have direct implications for catheter management protocols and antibiotic stewardship in long-term catheterized patients. Prospective evaluation using this framework in a larger cohort can help translate these ecological insights into clinical decision-making tools.

ObjectivesFosfomycin (FOS) remains an important therapeutic option for urinary tract infections caused by uropathogenic Escherichia coli (UPEC), but specific virulence traits as biofilm formation, metabolic adaptation, and antimicrobial resistance may limit its efficacy. This study investigated whether the natural compound, trans-cinnamaldehyde (t-CA) potentiates FOS activity against UPEC and explored the underlying mechanisms of its effect MethodsThe interaction between t-CA and FOS was assessed using checkerboard assays, time-kill analysis. We evaluated biofilm viability and structure using confocal and scanning microscopy as well as catheter-associated biofilm models. Next, effects on membrane integrity, cell-surface properties, membrane potential, intracellular pyruvate levels, and resistance evolution during serial passage were evaluated. Molecular docking was used to explore potential interactions of t-CA with enzymes involved in pyruvate metabolism. Galleria mellonella infection model was employed to evaluate in vivo therapeutical efficiency. Resultst-CA potentiated FOS activity against laboratory, reference, and clinical UPEC strains, with synergistic or additive interactions observed across the tested collection. The combination enhanced bacterial killing, reduced biofilm viability and biomass, and disrupted biofilm architecture. In catheter-associated biofilms, combined treatment markedly impaired surface-associated UPEC communities. t-CA reduced extracellular matrix abundance and altered cell-surface hydrophobicity and membrane potential without inducing detectable oxidative stress. Mechanistically, t-CA affected pyruvate homeostasis, reduced intracellular pyruvate levels, and phenotypically intersected with the BtsSR pyruvate-sensing pathway. Serial exposure to FOS alone rapidly increased MIC, whereas t-CA limited this phenomenon and did not itself promote reduced susceptibility. The compounds combination also improved survival of UTI89-infected G. mellonella larvae. Conclusionst-CA enhances FOS activity against UPEC through complementing the antibiofilm and metabolic effects. By weakening biofilm matrix integrity, perturbing pyruvate homeostasis, and limiting FOS-associated MIC elevation, t-CA represents a promising adjuvant candidate for improving FOS efficacy against biofilm-associated UPEC infections.

Climate-driven increases in sea surface temperature have been associated with the expansion of Vibrio species and a corresponding rise in vibriosis cases in both human populations and aquaculture systems. Coastal waters across the south of England are increasingly becoming suitable for the growth and establishment of both human- and aquaculture-associated Vibrio species, potentially increasing vulnerability to the types of infections and disease outbreaks already reported elsewhere in the world. In this study, we report the presence of a diverse and well-established Vibrio community within the Teign Estuary, (Southwest, UK), including the human-pathogenic species V. parahaemolyticus, V. cholerae (non-O1/non-O139), V. alginolyticus, and V. diabolicus, as well as the important aquaculture pathogens V. jasicida, V. aestuarianus, and V. anguillarum. We identified V. diabolicus, a species that was indistinguishable from V. alginolyticus using conventional biochemical identification methods and could only be accurately resolved by whole-genome sequencing and developed novel PCR targets to differentiate these species in the lab. Using the insect infection model Galleria mellonella, we demonstrate that environmental isolates of V. cholerae (non-O1/non-O139), V. parahaemolyticus, and V. alginolyticus possess virulence potential. We also investigated the effects of sewage effluent on the growth of Vibrio isolates from the Teign Estuary and found that sewage can preferentially promote the growth of Vibrio species. Furthermore, several Vibrio isolates were multidrug resistant and carried antimicrobial resistance genes, highlighting the potential role of environmental Vibrio populations in the Teign Estuary as reservoirs of antimicrobial resistance. Together, these findings demonstrate how rising sea surface temperatures and sewage pollution may influence the emergence, persistence, and public health and aquaculture significance of Vibrio species in UK coastal waters.

The use of nitrogen (N) fertilizers to meet global food demands is expected to continue rising. However, up to 70% of N applied to agricultural soils is lost through microbially mediated processes such as nitrification. Inhibiting nitrification is thus a key strategy to reduce N losses and improve fertilizer N use efficiency. Various plant-derived compounds, termed biological nitrification inhibitors (BNIs), have been shown to reduce accumulation of nitrification products, intermediates, and byproducts (nitrite, nitrate, nitric and nitrous oxides). However, the mechanisms by which BNIs affect nitrifiers, along with their specificity and persistence in soil are not well understood. Here, we evaluated the effects of three BNIs: methyl 3-(4-hydroxyphenyl) acrylate (MHPA), 6-methoxy-2(3H)-benzoxazolone (MBOA), and limonene, on ammonia-oxidizing, total microbial, and fungal communities in two soils with contrasting pH. Their persistence in each soil was also evaluated. Although ammonia-oxidizing archaea initially dominated nitrifier communities in both soils, their bacterial counterparts significantly increased after mineral N addition but also were more sensitive to BNI application. Limonene and the synthetic inhibitor DMPP stimulated ammonium immobilization, as total soil mineral N was significantly reduced. Limonene and MHPA had the strongest off-target effects, increasing the relative abundance of hydrocarbon-degrading bacteria and potential fungal pathogens, respectively. In contrast, MBOA inhibited nitrification with minimal off-target effects. Among the tested BNIs, MBOA was also the most persistent in the high-pH, high-nitrification-rate soil. Our results show that MBOA is a promising biological inhibitor and highlight the importance of understanding BNIs ecological effects to develop targeted and sustainable N management strategies.

Background: Antimicrobial resistance (AMR) is a global health crisis, necessitating alternative antibacterial strategies. Bacteriophages (phages) offer a promising solution, and their use as a therapeutic agent relies on stringent bioinformatic characterisation using whole genome sequencing (WGS) technologies. However, phages are highly diverse, with no clear consensus on best practices concerning phage DNA extraction or sequencing platform. Efficient and repeatable DNA extraction, sequencing, and bioinformatics processes are critical for safety assessments but remain poorly defined. Additionally, the impact of sequencing platform choice and DNA extraction methods on downstream genomic analyses is not well understood. Methods: We evaluated multiple DNA extraction, library preparation, and sequencing approaches using a diverse collection of Pseudomonas phages from the PhageWA biobank. Column-based and precipitation-based DNA extraction methods were compared for DNA yield and recovery efficiency. Genome sequencing was performed using short-read (Illumina) and long-read (Oxford Nanopore Technologies) platforms, incorporating multiple library preparation kits and Nanopore basecalling models. Assemblies were assessed for completeness, quality, and sequence concordance using standardised bioinformatics pipelines, with hybrid Illumina-Nanopore assemblies used as references for comparison. Results: DNA extraction efficiency varied substantially between protocols, with the Puregene precipitation-based method yielding significantly higher DNA recovery than column-based approaches when normalised to phage titre. Illumina sequencing consistently generated complete genome assemblies, although assembly fragmentation was observed for several jumbo phages when using the SeqWell ExpressPlex 2.0 library preparation method. For Nanopore sequencing, ligation-based native barcoding libraries produced longer reads than rapid barcoding libraries, while selection of the Dorado v5.0.0 basecalling model significantly improved read quality. Genome assembly success was dependent on phage genus; native Nanopore sequencing failed to assemble several Pbunavirus genomes, likely due to modified DNA bases, but an amplification-based library preparation successfully resolved these genomes. Across successfully assembled samples, Illumina and Nanopore platforms produced highly concordant genomes with comparable completeness scores, and hybrid polishing identified only minor sequence differences. Conclusions: DNA extraction methodology, sequencing chemistry, and basecalling model selection significantly influence phage WGS outcomes. Precipitation-based DNA extraction improved DNA recovery, while both Illumina and Nanopore sequencing generated high-quality phage genomes suitable for therapeutic characterisation. Nanopore sequencing provided assemblies comparable to Illumina with minimal benefit from hybrid polishing, supporting its routine use for phage genomics. These findings provide practical guidance for phage genome characterisation workflows and contribute to the development of standardised, regulatory-grade approaches for therapeutic phage assessment.

Probiotics produce antimicrobial peptides and small molecules that are secreted into the medium. Antimicrobial activity of cell-free supernatants can be tested using various qualitative and quantitative methods. Many of these techniques employ methods which introduce uncontrolled variables, impacting reproducibility and making comparison of reported results difficult. Here, we present a simple procedure for quantitative estimation of antimicrobial activity of cell-free supernatants which overcomes drawbacks of commonly used methods.

Combined sewer overflows (CSOs) are a major pathway for untreated wastewater into urban streams, yet their role in shaping antimicrobial resistance (AMR) dynamics remains poorly understood. Here, we used high-frequency, time-resolved sampling during two storm-triggered CSO events across two monitoring locations and one stormwater-only control site in an urban stream to quantify how these disturbances affect microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) in an urban stream. CSO events caused rapid, up to two orders of magnitude, increases in bacterial, pathogen, and ARG abundance, with multiple transient peaks occurring within single overflow episodes. However, these increases were largely proportional to the total bacterial load, and most ARGs and MGEs did not change in relative abundance, indicating that CSOs primarily act as mass-transfer events rather than drivers of in situ selection. Downstream attenuation was governed by hydrological dilution despite additional CSO inputs: Both microbial and resistance signals largely returned to baseline within short time frames. This demonstrates that CSOs function as hydrologically driven pulse disturbances that generate acute but transient AMR exposure. Because CSO events lack the sustained pressure associated with continuous wastewater discharges, rapid washout prevents the long-term establishment of sewage-derived resistance. These findings highlight that AMR risk in CSO-impacted systems is driven primarily by short-term exposure rather than by persistent ecological transformation, with important implications for urban water management under increasingly extreme rainfall conditions.

Background: Human activities such as mining generally lead to increased heavy metal concentrations in the environment. While traditional remediation techniques are often costly, the use of fungi as bioremediators, known as mycoremediation, is increasingly gaining attention as a sustainable approach for removal of heavy metals. Here, we evaluated heavy metal levels inside the Kiirunavaara iron ore mine in Northern Sweden and analysed fungal responses to various metal concentrations by comparing growth and metal uptake in mine-derived isolates and closely related control isolates. Results: Sediments inside the mine were enriched in heavy metals compared to those from the outlet of the mine to natural lakes. Six Fusarium isolates were recovered from contaminated mining environments: five isolates from inside the mine were identified as Fusarium oxysporum, and one isolate from the outlet was identified as Fusarium tricinctum. Isolates from the mine and outlet showed overall higher survival and biomass production in presence of copper, iron, and zinc across a range of concentrations (up to 1000 mg/L) compared to control isolates. At the same time, these isolates often exhibited reduced relative metal uptake. As a result, mycoremediation potential, assessed as total uptake in the grown mycelium, was isolate-dependent. Conclusions: Based on these results, we conclude that Fusarium isolates from the Kiirunavaara mine show increased growth in media enriched with heavy metals compared to closely related control isolates. We additionally show that mycoremediation potential is not necessarily associated with environmental origin. Instead, mycoremediation potential should be evaluated on a case-by-case basis for each isolate and based on specific needs for mycoremediation.

BackgroundReliable detection of Salmonella remains a major challenge for public health surveillance and food safety due to the growing diversity of circulating serovars and the limitations of existing molecular targets. This study aimed to identify an optimal molecular target and develop a TaqMan real-time PCR assay for the detection of Salmonella spp. MethodsBased on the results screening for Salmonella genes suitability as molecular markers, a TaqMan real-time PCR assay targeting the hilA gene was developed and validated. Analytical sensitivity, analytical specificity, and performance on bacterial isolates and artificially contaminated food samples were assessed. ResultsAmong all candidate targets, hilA demonstrated the broadest coverage and was detected in all tested Salmonella isolates, including representatives of rare serological groups, whereas invA conventionally used for this pathogen detection, was absent in a subset of strains. The assay exhibited a limit of detection of 100 bacterial cells/mL and 100 fg/L of genomic DNA. No cross-reactivity was observed with DNA from Shigella flexneri, Shigella sonnei, Yersinia pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. kristensenii, Bacillus anthracis, Vibrio cholerae, or Francisella tularensis. The assay successfully detected Salmonella DNA in all artificially contaminated food samples tested. Evaluation using a collection of 25 bacterial isolates demonstrated positive amplification in all 24 confirmed Salmonella strains, while a strain initially identified by conventional bacteriology as Salmonella but subsequently confirmed by whole-genome sequencing as Proteus mirabilis yielded a negative result. ConclusionsThe hilA gene represents a highly conserved and reliable molecular target for the detection of Salmonella spp. The developed TaqMan real-time PCR assay demonstrated high analytical sensitivity, excellent specificity, and broad serovar coverage, supporting its application in laboratory detection of Salmonella, food safety monitoring, and epidemiological surveillance.

Municipal wastewater constitutes a major reservoir of unutilized reactive nitrogen, representing a significant opportunity for biological valorization. The biopolymer cyanophycin is promising as a means of nitrogen capture and recovery, but current production strategies are not optimized for the physicochemical constraints of municipal wastewater systems. Here, we engineered the naturally competent soil bacterium Acinetobacter baylyi ADP1 ISx to synthesize cyanophycin from carbon and nitrogen sources prevalent in municipal wastewater and over a range of wastewater-relevant temperatures. To overcome the recurring problem of arginine availability limiting cyanophycin synthesis, we engineered an arginine-producing strain (AP1) which accumulated cyanophycin when grown on acetate and ammonium (19% CDW), nitrate (9% CDW), or urea (29% CDW) and without arginine supplementation. During this work, we observed that conditions associated with reduced cell fitness correlated with increased intracellular cyanophycin content. As temperature strongly influences cell growth but cannot be realistically modulated in wastewater contexts, we investigated the potential of induced fructose-auxotrophy to modulate cell growth independently from temperature. This intervention, accomplished with a single knockout (gap), expanded the effective range of cyanophycin accumulation from 12 C up to 30 C. Collectively, these results establish the relevance of arginine-producing strains for cyanophycin biosynthesis and position A. baylyi as a promising chassis for continued development under real-world wastewater conditions.

Soil harbours a wide diversity of microbes responsible for essential functions, such as depolymerizing the C and N in organic matter through the production of exoenzymes. Some of these exoenzymes are universal, whereas others are specific to certain microbes. We hypothesized that higher microbial alpha diversity is associated with greater depolymerization capacity, specifically for protein and cellulose depolymerization, which will result in more N being mineralized. We therefore diluted two soil microbial communities, one from a forest soil and one from an agricultural soil, to create a diversity gradient. After nine weeks, we transferred these communities to a synthetic soil in which microbial necromass was the only nitrogen source. Before the transfer and two weeks after, we quantified protease, deaminase and {beta}-glucosidase potential activity, characterized the bacterial and fungal communities, and measured the quantity of nitrogen mineralized. The dilution had very little effect on the processes measured, with no clear trend. For identical alpha diversity values, some communities had high process rates, while other not. It appeared that these communities varied widely, a side effect of the dilution approach, and that this variation was significantly linked to process rates. This shows that community composition (beta diversity) is more strongly related to enzymatic potential and mineralization than species richness (alpha diversity) following necromass addition. In conclusion, the relationship between diversity and depolymerization of microbial necromass is not simply a matter of a linear decrease along with diversity but is rather linked to how reduced diversity results in more stochastic microbial communities. Highlights- Community composition (beta diversity) influence more microbial necromass depolymerization than species richness - Abundance of specific microbes explained ammonification and nitrification processes - Mineralization rates is different between crop and forest soil

This study investigated the genomic and biochemical characteristics of two amylolytic microbial strains, NATIVEDY160T (= JE7B6T = NRRL B-68523T) and NATIVEDY161T (= JL13D9T, = NRRL B-68524T) isolated from the rumen of healthy Holstein dairy cattle. Both strains are obligately anaerobic, non-motile, Gram positive, catalase-negative, and oxidase-negative. Morphologically, NATIVEDY160T grows in long coccoid chains while NATIVEDY161T grows in short chains or pairs. NATIVEDY160T can catabolize amygdalin, esculin/ferric citrate, and starch, compared to NATIVEDY161T which utilizes amygdalin, arbutin, esculin/ferric citrate, glycogen, and D-maltose as determined by API 50 CH carbon panels. Starch degradation ability was verified for both strains, but neither showed cellulolytic activity as confirmed by starch agar and Congo red agar assays, respectively. HPLC analysis revealed that lactate was the primary end product of both strains carbohydrate fermentation, while strain NATIVEDY161T also produced small amounts of acetate. 16S rRNA sequences from both strains cluster with the Oscillospiraceae (formerly Ruminococcaceae) lineage Ruminococcus species, but average nucleotide identity of either strain compared to closely related Ruminococcus members was under the species threshold (95%). Genomic, phylogenetic, and phenotypic interrogation support NATIVEDY160T and NATIVEDY161T as novel species. Each strain was isolated from the rumen of dairy cows located within the central valley of southern California, which has a rich history of Dutch and Basque dairy farm ownership and is still the case today in the region. In recognition of the contributions and heritage of the central and southern California dairy industry, the names Ruminococcus hollandia and Ruminococcus vasco are proposed with NATIVEDY160T and NATIVEDY161T as their respective type strains.

Filamentous phages infecting Gram-positive bacteria remain largely unexplored. Notably, only two filamentous phages, B5 and Philemon infecting Propionibacterium freudenreichii, have been described to date in the phage-rich dairy ecosystem. Although both were genomically characterized, only B5 was confirmed to be an infective filamentous single-stranded DNA phage. The aim of this study was to isolate and characterize new filamentous phages from Swiss-type cheese to investigate their diversity, structural features, host specificity, and potential adaptation to the dairy environment. Thirty raw and pasteurized milk cheeses from France were screened for phages infecting P. freudenreichii strains. Eleven phages were isolated, nine of which displayed a filamentous morphology. Named MINOG1 to MINOG9, these filamentous phages exhibited genomic features typical of this morphotype, including small single-stranded DNA genomes with collinear genes organized into functional modules. Comparison with B5 and Philemon revealed sequence divergence ranging from 0.1% to 7%. These phages also exhibited a diverse host range. To further explore phage-P. freudenreichii interactions, we screened the genomes of the strains used in this study, as well as additional genomes retrieved from the NCBI database, for CRISPR spacers predicted to target these filamentous phages. Numerous strains contained CRISPR spacers showing 79 to 100% identity to genomic regions of these phages. Two P. freudenreichii strains displayed markedly different phage resistance levels despite exact spacer-protospacer matches with phages B5, MINOG1, MINOG2, and MINOG8. Conversely, several strains were resistant to nearly all tested phages despite lacking CRISPR spacers targeting them suggesting the presence of additional defense systems in P. freudenreichii. IMPORTANCEFilamentous phages can play important roles in bacterial ecology by modulating host physiology, population dynamics, and bacterial adaptation to specific environments. However, filamentous phages infecting Gram-positive bacteria remain among the least explored bacterial viruses, and their diversity, ecology, and interactions with their hosts are still poorly understood. This knowledge gap is particularly relevant in dairy ecosystems, where phages are abundant and can influence microbial communities and fermentation processes. In characterizing nine new filamentous phages infecting Propionibacterium freudenreichii from Swiss-type cheeses, this study expands the known diversity of filamentous phages associated with Gram-positive bacteria and provides new insights into phage-host interactions and bacterial defense strategies in dairy-associated bacteria.

Hand eczema has been described as having an increased prevalence in persons with increased frequency of hand washing. This study investigated the differences in the hand microbiome of persons with and without a history of eczema and secondly the sensitivity of these microbes to commercial liquid soap as a potential trigger for eczema flares. The study identified Staphylococcus to be the most populus genus on the hands in both groups, but the distribution of species was different. Additionally, there was no difference in the number of soaps that produced zones of inhibition but there were some differences in the overall sensitivity to the different soaps tested. Overall, it was determined that liquid soap can cause bactericidal effects on some species of the commensal microbiome, but further work is required to determine if this could be the cause of hand eczema.

Background: Phenotypic antibiotic susceptibility testing (AST) for Helicobacter pylori (H. pylori) has relied on bacterial culture for three decades, requiring 5-7 days to yield results. Genotypic rapid tests can only detect known resistance mutations and fail to reliably identify amoxicillin resistance. To our knowledge, no culture-free rapid phenotypic AST method for H. pylori has been previously reported. Methods: We developed a phenotypic AST method based on fluorescence rapid on-site evaluation (ROSE) technology that completely bypasses bacterial culture. Gastric mucosal biopsy specimens from 40 H. pylori-positive patients were homogenized and co-incubated with an acridine orange/ethidium bromide (AO/EB)-based viability staining reagent and three first-line antibiotics (amoxicillin, clarithromycin, and levofloxacin) at concentrations corresponding to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for H. pylori, at 37C for 1 hour. Fluorescence intensity was measured using a microplate reader. A reduction in fluorescence relative to an antibiotic-free control indicated susceptibility, whereas no significant reduction indicated resistance. Conventional culture-based AST (E-test) served as the reference method. The overall concordance rate, sensitivity, specificity, and Cohen's kappa coefficient were calculated. Results: Fourteen of the 40 samples had unsuccessful culture and were excluded, leaving 26 samples for statistical analysis of each antibiotic. The overall concordance rates between the ROSE method and culture-based AST were 84.6% (22/26) for amoxicillin, 76.9% (20/26) for levofloxacin, and 69.2% (18/26) for clarithromycin. Cohen's kappa coefficients indicated moderate agreement for all three antibiotics ({kappa} = 0.523, 0.539, and 0.412, respectively). Unlike genotypic methods, the ROSE method successfully assessed amoxicillin susceptibility in all 40 patients, a critical first-line antibiotic for which no reliable genetic resistance marker currently exists. The turnaround time was approximately 1 hour (55-65 minutes), compared with 5-7 days for culture-based methods; preliminary estimates indicated a cost reduction of approximately 3,000-5,000 Chinese yuan (CNY) per patient, mainly attributable to the elimination of culture media, prolonged incubation, and repeat clinic visits. Conclusions: This study reports, for the first time, a culture-free 1-hour phenotypic AST for H. pylori. The method enables same-day, susceptibility-guided treatment decisions, addressing an unmet clinical need spanning three decades. Algorithm optimization and a prospective randomized controlled trial are currently underway to further improve diagnostic accuracy and validate clinical utility.

Necrotizing soft tissue infections (NSTIs) carry 10 to 30% mortality. Current empirical antimicrobial guidance derives almost entirely from Western cohorts dominated by Streptococcus pyogenes and aerobic-anaerobic consortia, yet whether this microbial paradigm applies to tropical, high-antimicrobial-pressure settings has not been tested with culture-independent methods. We did a prospective cohort study of 169 patients with intraoperatively confirmed NSTI at a North Indian tertiary center (2021 to 2024). Wound tissue underwent aerobic and anaerobic culture, QIIME2-based 16S rRNA gene amplicon sequencing (V3-V4), and targeted SYBR Green quantitative PCR (qPCR) for Acinetobacter baumannii and S. pyogenes. The wound microbiota was overwhelmingly Gram-negative and polymicrobial, anchored by A. baumannii (culture, 33.7%; metagenomics, 49.1%; qPCR, 37.9%), Escherichia coli (32.0%), and Klebsiella pneumoniae (20.7%); S. pyogenes contributed only 4.7% of culture-positive cases. Polymicrobial wounds had higher Shannon diversity (2.59 versus 2.33; P = 0.048) and discrete community composition (PERMANOVA R2 = 0.511; P = 0.010). Culture-metagenomics agreement ranged from almost perfect for Escherichia ({kappa} = 0.849) to slight for Streptococcus ({kappa} = 0.131). North Indian NSTIs present a microbial picture distinct from the Western paradigm, with implications for empirical therapy.