A substantial proportion of symptomatic patients with urinary tract infections (UTIs) have negative or low-count urine cultures despite clinically compatible presentations. Biofilm-associated bacterial aggregation and the presence of viable bacteria not recovered by standard culture may contribute to this diagnostic gap. Flow cytometry (FACS) can detect these viable cells, but culturability remains essential for species identification and antimicrobial susceptibility testing. We evaluated whether low-concentration dithiothreitol (DTT) can safely disperse urinary biofilm-associated aggregates and improve bacterial recovery from urine samples. Seventy-two clinical urine samples were processed in parallel (untreated vs 0.1% DTT-treated). Viability was quantified using SYTO9/PI-based flow cytometry with bead-normalized bacterial counts. Culturability was assessed on chromogenic media. Comparative analyses included paired statistics, Bland-Altman agreement, and correlation between viable and culturable fractions. DTT (0.1%) did not alter bacterial viability (P = 0.47), confirming its non-toxicity at this concentration. In contrast, DTT significantly increased CFU recovery (median: 10 to 5.5 × 10³ CFU/mL; P < 0.0001), converting several previously negative or low-count samples into positive cultures. Agreement between viable and culturable fractions improved after DTT treatment, primarily through reduced systematic bias in Bland-Altman analysis, while correlation coefficients remained similar (r: 0.44 vs 0.45). A persistent discrepancy between FACS-derived viable counts and culture-based counts suggests the presence of viable bacterial populations not fully recovered by standard culture conditions, findings that are consistent with but do not directly demonstrate the presence of VBNC subpopulations. Low-dose DTT is a safe and effective pre-analytical treatment that markedly improves culture sensitivity without compromising bacterial viability. These findings support further investigation of DTT-assisted processing as a potential strategy to reduce culture underestimation in urinary diagnostics and to better characterize viable bacterial populations not detected by standard culture methods.IMPORTANCEStandard urine culture may fail to detect the full spectrum of bacteria present in urinary tract infections, particularly when microorganisms are embedded within mucus, biofilm-like aggregates, or enter a viable but non-culturable state. These hidden bacterial populations may contribute to persistent symptoms or recurrent infections while remaining undetected by routine culture methods. Our findings suggest that pre-treating urine samples with a low concentration of dithiothreitol may help disperse biofilm-like aggregates while preserving bacterial viability. When combined with flow cytometry, this approach may allow a more comprehensive estimation of the viable bacterial fraction and, in some cases, may increase the likelihood of bacterial recovery by culture in samples that would otherwise appear negative. Because the procedure relies on inexpensive reagents and techniques compatible with routine laboratory workflows, it may represent a practical adjunct to conventional diagnostics. Further studies are needed to confirm its clinical impact.

Antimicrobial resistance is a growing global health crisis, with polymicrobial infections caused by Acinetobacter baumannii and Staphylococcus aureus posing a significant healthcare challenge due to their synergistic virulence, multidrug resistance, high incidence, and association with severe outcomes. Accordingly, urgent alternative treatments are needed, with bacteriophage or phage therapy showing considerable promise. Here, we isolated six phage types based on plaque morphology, including small clear plaques (SCP; phiAR002 and phiAR004), medium clear plaques (MCP; phiAR010 and phiAR011), and hazy-halo plaques (HHP; phiAR014 and phiAR015). These phages demonstrate the ability to lyse a broad spectrum of several carbapenem-resistant A. baumannii strains collected from secondary and tertiary care hospitals in Thailand, highlighting their suitability for therapeutic applications. To broaden host range and enhance bactericidal activity, phage cocktails comprising the halo-forming phage (phiAR014) in combination with either SCP (phiAR002) or MCP (phiAR010), which are genetically and mechanistically distinct, were formulated and demonstrated synergistic effects, suppressing bacterial growth for up to 36 h. These relatively small myophages, SCP and MCP, possess larger genomes (around 165-188 kbp) compared with HHP (around 106 kbp), exhibit a longer latent period (30 min versus 20 min), and produce a greater number of progenies, yielding 678-882 PFU per infected cell compared with 255 PFU per infected cell for HHP. To demonstrate their therapeutic potential, the phage cocktails were further assessed in co-cultures of A. baumannii and S. aureus under both planktonic and biofilm conditions. Cocktails combining HHP with SCP or MCP exhibited significant synergistic antibacterial activity at MOIs of 10 and 100, with effects evident within 10 h and sustained for 24 h. Relative to individual phages and uninfected controls, the cocktails achieved greater bacterial reduction, decreasing A. baumannii by approximately 2 log₁₀ CFU/mL compared with single phages (and ~5 log₁₀ versus controls) and S. aureus by ~2 log₁₀ CFU/mL. Moreover, the cocktails significantly enhanced biofilm inhibition and eradication compared to single-phage treatments, demonstrating synergistic interactions. These findings indicate that halo phages represent promising therapeutic candidates with potent antibacterial and anti-biofilm activity against polymicrobial infections.IMPORTANCEPhages are promising therapeutic agents due to their host specificity and ability to penetrate biofilms via structural and enzymatic mechanisms. The halo-plaque phenotype, indicative of depolymerase activity, facilitates the degradation of extracellular polysaccharides and cell wall components, thereby enhancing infectivity and biofilm penetration. Based on phenotypic and genomic characterization, halo-forming phage cocktails were developed and demonstrated effective suppression of polymicrobial growth and disruption of established biofilms in A. baumannii and S. aureus co-culture models. These cocktails exhibited synergistic inhibition of planktonic growth, biofilm formation, and biofilm eradication. Phage replication and lysis of A. baumannii may promote the release of putative depolymerases and/or cell wall hydrolases that could potentially contribute to matrix degradation; however, further investigation is required to elucidate the precise mechanisms and enzymatic functions involved. Together, these findings would pave the way for advancing halo-forming phages and support the targeted engineering of tail fiber or spike proteins for therapeutic applications in multidrug-resistant polymicrobial infections.

This study aimed to characterize early-life changes in the fecal microbiome and resistome of calves. Fecal samples were collected from 49 Holstein heifers born and raised at a large organic dairy in Texas without antimicrobial drug exposures. Samples were collected from five age groups: early pre-weaning at 2-3 days old (Pre 1), late pre-weaning at 5 weeks old (Pre 2), prior to weaning at 12-13 weeks old (Pre 3), post-weaning in group hutches at 12-13 weeks old (Post 1), and later post-weaning at 13-14 weeks old (Post 2). Fecal samples were analyzed using 16S rRNA gene sequencing to characterize microbial communities and target-enriched shotgun sequencing to characterize antimicrobial resistance genes in the resistome. Richness of microbial communities increased as calves aged through the Pre 1, 2, and 3 samplings, before plateauing in the Post 1 and 2 groups. Diversity also increased in the Pre 1 and 2 groups, remaining similar thereafter. In contrast, resistome richness and diversity decreased during early life and then stabilized at around 5 weeks of age (Pre 2). Changes in microbial community structures were dramatic during the first 12 weeks, largely due to a significant decrease in the relative abundance (RA) of Pseudomonadota (Proteobacteria) and an increase in the RA of Bacillota (Firmicutes) and Bacteroidota. The resistome changed with an increased RA of tetracycline resistance genes, while drug and biocide resistance genes decreased. The apparent stabilization of microbial community features after 12 weeks of age may reflect a period when gut microbiome structure begins to establish greater stability.IMPORTANCEEarly-life development of the gut microbiome can have lasting effects on animal health, immune maturation, and productivity. Using 16S rRNA gene sequencing together with target-enriched metagenomic sequencing, we provide an in-depth characterization of the fecal microbiome and resistome of antibiotic-naïve dairy calves during early life. We demonstrate that microbiome diversity increased with age while resistome diversity decreased, revealing distinct temporal trajectories and suggesting ecological succession as a potential driver of resistance gene dynamics independent of antimicrobial drug exposure. Major resistome features appeared to stabilize earlier than overall microbiome structure, highlighting critical windows in early development when resistance gene composition may be most dynamic. These findings establish an important baseline for interpreting microbiome-resistome interactions and for evaluating how management practices and antimicrobial exposures may influence calf health and antimicrobial resistance ecology in dairy production systems.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a complex etiology. Emerging evidence implicates gut microbiota dysbiosis in ALS pathology via the gut-brain axis, yet the specific integrative profile of the gut microbiome, virome, and metabolome, particularly in Chinese patients, remains incompletely characterized. Although global diversity indices showed no significant differences, taxonomic analysis revealed distinct compositional shifts. The ALS microbiome was characterized by a significant depletion of beneficial anti-inflammatory genera, specifically Akkermansia and Faecalibacterium, and an expansion of opportunistic pathogens such as Escherichia and oral-associated taxa (e.g., Streptococcus). We also observed a specific alteration in the gut virome, with viral genera including Puppervirus and Donellivirus enriched in ALS patients. Functionally, the ALS microbiome exhibited a marked upregulation of pathways involved in L-ascorbate (vitamin C) degradation and fatty acid biosynthesis, suggesting a microbial contribution to systemic oxidative stress. Metabolomic analysis corroborated these findings, identifying 271 differentially expressed metabolites. ALS patients showed elevated levels of inflammatory lipids (e.g., LysoPC) and metabolic intermediates of the tricarboxylic acid (TCA) cycle, alongside a downregulation of antioxidants. Integrative analysis highlighted profound dysregulation in porphyrin metabolism, oxidative phosphorylation, and energy homeostasis. Our findings demonstrate that ALS is associated with a specific dysbiotic gut ecosystem characterized by the loss of protective commensals, unique viral signatures, and functional metabolic reprogramming that exacerbates host oxidative stress and energy deficits. These results provide new insights into gut-brain interactions and highlight microbial antioxidant depletion as a potential therapeutic target.IMPORTANCEAmyotrophic lateral sclerosis (ALS) is a devastating disease with no cure. While gut bacteria are known to influence brain health, we still do not understand exactly how they contribute to ALS progression. In this study, we used advanced DNA sequencing and chemical analysis to deeply examine the gut ecosystem of ALS patients. Beyond just cataloging which bacteria are present, we discovered what they are doing: the ALS microbiome actively breaks down vitamin C (a critical antioxidant) and disrupts energy metabolism. We also found a loss of protective bacteria that maintain the gut barrier. These findings are significant because they suggest that the gut microbiome in ALS patients may be actively fueling the disease by depleting the body's antioxidant reserves. This points to a new potential treatment strategy: targeting these specific bacterial functions or replenishing specific metabolites to protect motor neurons.

This work describes our software pipeline Cas-CRISPR Automated Design and Evaluation (CasCADE) that allows for in silico design of Cas-CRISPR detection assays. We demonstrate the viability of our design process in the lab and report 15 successful designs across nine diverse target organisms. The rapid time to answer afforded by CasCADE, combined with the superior specificity and sensitivity offered by emergent CRISPR detection assays compared to traditional PCR methods, makes this a timely contribution to pandemic preparedness and biosurveillance interests.

Escherichia coli is a major cause of hospital-acquired infections in China, including urinary tract, bloodstream, and intestinal infections. Given the rising prevalence of antibiotic-resistant E. coli, phages are increasingly regarded as promising alternatives to conventional antibiotics. Henuyfy11N was isolated using the double-layer agar method and characterized via transmission electron microscopy in this study. Biological assays included stability under varying pH and temperature, UV sensitivity, host range, optimal multiplicity of infection, adsorption rate, and one-step growth curve. In vitro lytic activity against extended-spectrum β-lactamase (ESBL)-producing E. coli and biofilm eradication capacity was assessed. Whole-genome sequencing enabled phylogenetic, synteny (the analysis of conserved blocks of genetic sequence between different genomes), and functional annotation analyses. In vivo, the therapeutic efficacy was evaluated in a mouse infection model. Phage Henuyfy11N has not yet been classified. It demonstrated high lytic activity, a short latent period, and a burst size of 57.1 PFU/cell. The phage remained stable across a broad pH range (3-11) and temperatures up to 70℃. Its circular double-stranded DNA genome (41,103 bp, G + C% 50.88) contains 54 open reading frames, with no tRNA, virulence, or antibiotic resistance genes. Genomic and phylogenetic analyses revealed close relatedness to phage BUCT789. Henuyfy11N effectively lysed ESBL-producing E. coli, disrupted biofilms, and significantly improved survival in the mouse infection model. Henuyfy11N shows high host specificity, efficient lytic activity, rapid replication, and a safe genomic profile, demonstrating some potential as a therapeutic agent against ESBL-producing E. coli infections.IMPORTANCEThe widespread use of antibiotics has led to increasing antibiotic resistance, which is a growing global health concern. Therefore, the development of novel antimicrobial therapy that can cure drug-resistant bacteria-induced infections is imperative. Phages are of increasing interest as natural enemies of bacteria, with clear advantages in antibacterial applications. In this study, by using extended-spectrum β-lactamase (ESBL)-producing Escherichia coli 2025011N as a host, we successfully isolated and purified Escherichia phage Henuyfy11N and conducted a series of experiments to verify its genomic character and biological character. Our findings revealed that the phage exhibited excellent tolerance to a broad spectrum of pH and wide temperature range. Phage Henuyfy11N was effective in disrupting mature biofilm, and no genes for virulence, lysogenic, integrase, or AMRs were found in the genome. Besides, Henuyfy11N showed promising antibacterial effects in vivo and in vitro, indicating potential as a therapeutic agent against ESBL-producing E. coli infections.

Foodborne illness caused by Salmonella remains a major public health concern. Among >2,500 known Salmonella serovars, S. Infantis ranks among the top 10 serovars causing human illness. The significance of S. Infantis is further amplified by its ability to harbor and disseminate antimicrobial resistance (AMR) genes mediated by pESI-like megaplasmid. Therefore, understanding the population dynamics and assessing the risk of AMR gene carriage in S. Infantis isolates collected from diverse sources and geographic locations are critical. The findings from this analysis help identify high-risk reservoirs and track current AMR trends, enabling the development of streamlined treatment strategies and data-driven interventions aimed at limiting the global spread of multidrug-resistant S. Infantis.

ABSTRACT Urinary tract infections (UTIs) are diagnosed based on symptoms and confirmed by urine culture, despite its limitations in sensitivity. False-negative cultures can lead to inappropriate antimicrobial use or urosepsis in high-risk patients. Next-generation sequencing (NGS)-based metagenomics offers a comprehensive and precise alternative but is rarely applied clinically. We developed and validated BIOTIA-ID, a clinical-grade NGS-based diagnostic pipeline for pathogen detection in urine. Remnant clinical and spiked urine samples underwent extraction, metagenomic library preparation, and Illumina NextSeq 550 sequencing. We trained and applied a bioinformatic pipeline that uses machine learning to identify pathogens and resistance markers. BIOTIA-DX was intentionally designed and trained to increase stringency and reduce false positive detection of urogenital commensals or opportunistic microbes present at colonization levels. Internal controls ensured standardized, high-stringency results. The assay was validated on 1,470 urine specimens evaluating over 14.5k analytes. The clinical validation achieved a 97.2% sensitivity and 99.6% specificity with a limit of detection (LoD) of &lt;15,000 CFU/mL for most bacterial species and &lt;5,000 CFU/mL for fungal species. Discordant results were reconciled by target-specific qPCR or 16S Sanger sequencing, and 87% of the NGS results were concordant with the comparator. A subset of 332 clinical specimens was tested and validated for antimicrobial resistance (AMR). sul and blaSHV genes were commonly associated with Escherichia coli and Klebsiella pneumoniae , while cfxA was found in Prevotella and Pseudomonas spp. detected by BIOTIA-ID. Overall, these data demonstrate that BIOTIA-ID is a comprehensive, highly accurate end-to-end diagnostic assay with notable advantages over current culture-based diagnostics. IMPORTANCE Urinary tract infections (UTIs) are among the most common infections, yet current diagnostic methods, including urine culture, often fail to detect pathogens accurately, leading to delayed treatment and inappropriate antimicrobial use. Clinical metagenomics offers a powerful alternative, especially in complicated cases. BIOTIA-ID is a validated, clinical-grade next-generation sequencing (NGS)-based assay that provides highly accurate pathogen identification and antimicrobial resistance profiling. By incorporating machine learning and stringent quality controls, BIOTIA-ID minimizes false positives and enhances diagnostic precision. Our study demonstrates its superior performance over culture, with potential to improve UTI diagnostics, guide targeted therapy, and support antimicrobial stewardship. The implementation of urine metagenomic diagnostics could support recurrent and complicated UTI patient management, providing a more reliable alternative to traditional methods.

ABSTRACT Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a leading cause of bacteremia, yet the genetic basis for its success in this hostile environment remains poorly defined. In this study, we employed transposon-directed insertion site sequencing (TraDIS) to map the fitness landscape of CA-MRSA strain USA300 JE2 through a genome-wide screening in fresh, immunocompetent human blood. We identified 76 genes required for fitness, including genes involved in respiratory and central carbon metabolisms, heme detoxification, and de novo purine biosynthesis. As validation of fitness genes, competition assays confirmed that individual disruption of purA , purB , fbp , hssR , or aroA2 significantly reduced bacterial fitness in blood. Conversely, inactivation of specific regulators, such as the saeRS two-component system, the alternative sigma factor σ B , and adhesins, including fnbA and clfA , conferred a competitive advantage. These findings provide a genome-scale map of S. aureus fitness requirements in a physiologically relevant blood model, offering a platform for further investigation of bacterial adaptation to the intravascular environment. IMPORTANCE Understanding how Staphylococcus aureus maintains fitness in the human bloodstream is essential for explaining its success as an invasive pathogen. This study provides a comprehensive, genome-wide definition of the genes that enable S. aureus to remain competitive in blood, revealing the key physiological requirements for adaptation to this challenging environment. By identifying genetic functions whose disruption impairs fitness, our findings highlight the specific pathways that sustain adaptation and competitiveness under host-imposed stress. Extending previous genome-scale investigations conducted in other infection niches, this study emphasizes the importance of physiological context in shaping bacterial fitness and identifies conserved cross-fitness determinants shared among S. aureus lineages. These insights advance our current understanding of how S. aureus adapts to the bloodstream and strengthen the foundation for future functional and comparative studies on staphylococcal pathophysiology.

Vaginal infections are closely associated with human papillomavirus (HPV) infection, yet research on vaginal microecology and HPV mRNA expression in pregnant women remains limited. This study aimed to explore the prevalence of vaginal microecological disorders and their relationship with HPV infection among pregnant women. A cross-sectional study was conducted involving 630 pregnant women who received prenatal care at Fujian Maternity and Child Health Hospital from 2023 to 2024. All participants underwent both the Aptima HPV E6/E7 mRNA assay and vaginal microecological examinations. Clinical characteristics, virological results, and vaginal microecological alterations were analyzed. Of the 630 participants, 241 (38.3%) exhibited vaginal microecology disorders, with aerobic vaginitis (AV, 12.7%) being the most common subtype. A total of 97 (15.4%) participants had received HPV vaccination. The overall high-risk (HR)-HPV E6/E7 mRNA positivity rate was 28.6 % , including 4.8 % for HPV16/18/45 and 23.8 % for the other 11 high-risk genotypes. Age-adjusted multivariate logistic regression analysis identified the following independent risk factors for HR-HPV E6/E7 mRNA positivity: bacterial vaginosis (BV) (aOR = 2.42, 95% CI = 1.25-4.70, P = 0.009), Nugent score ≥7 (aOR= 2.35, 95% CI = 1.21-4.57, P = 0.012), leukocyte esterase positivity (aOR = 1.49, 95% CI = 1.03-2.15, P = 0.036), and lack of HPV vaccination (aOR = 1.99, 95% CI = 1.27-3.12, P = 0.003). Vaginal microecological disorders-particularly BV and leukocyte esterase-are independent risk factors for HR-HPV E6/E7 mRNA positivity. To optimize the management of HPV-infected pregnant individuals, enhanced focus on the prevention and targeted treatment of BV is warranted.IMPORTANCEThis study addresses the limited evidence linking vaginal microecology to high-risk human papillomavirus (HR-HPV) infection in pregnancy. Bacterial vaginosis, high Nugent score, and leukocyte esterase positivity were independently associated with HPV infection, whereas HPV vaccination offered protection. These results underscore the value of integrating vaginal microecological assessment and HPV testing into routine prenatal care.