Anthrax is an endemic and undercharacterized zoonotic disease in the Middle East, including Jordan. Between 2018 and 2020, we conducted a comprehensive investigation of 13 confirmed anthrax outbreaks in Jordan, analyzing 822 samples from animal farm environments, including carcasses, asymptomatic livestock, and abiotic environmental surfaces. All samples were tested by qPCR targeting the chromosomal marker (ba177) and the plasmid marker pXO1 (pag). Among carcass samples, 75/195 (38.5%) were ba177-positive, of which 81% harbored the pXO1. Of specimens from live-animals and from environmental surfaces, 218/627 (35%) were positive by qPCR, likely reflecting environmental contamination during active outbreak periods. Serological analysis using anti-protective antigen (PA) ELISA revealed a high seroprevalence of 53% (75/141) among asymptomatic animals, indicating widespread sub-clinical exposure to B. anthracis antigens and previously undocumented endemicity. An integrated approach combining qPCR with ELISA demonstrated that 11% of seropositive animals with paired swab testing also yielded swab samples that were positive by qPCR, suggesting environment-to-host transition. Molecular strain typing using canonical SNP (canSNP) analysis identified the rare sublineage C.USA.A1055 within lineage C.Br.A1005 across two distinct outbreaks, suggesting the environmental persistence of this endemic lineage. Overall, our findings provide the first systematic molecular and serological surveillance baseline data for Jordan, demonstrating a complex genomic persistence and subclinical exposure landscape. This study suggests the need for enhanced surveillance strategies under the One Health framework to mitigate the risk of anthrax endemicity.

Anthrax is an endemic and persistent zoonotic disease in Pakistan. This fact emphasizes the need for surveillance in high-risk endemic regions. The aim of this study was to assess the role of wool and hair of ruminants and canines in the transmission of Bacillus anthracis, the causative agent of anthrax, and to evaluate the exposure to anthrax in asymptomatic animals and humans. Samples from five districts were analyzed using microscopic methods, culture methods (on both PLET and blood agars) and PCR. Additionally, to check the exposure, ELISA was performed on animals and human samples. Interestingly, 114/441 (25.85%) samples exhibited Gram-positive rods and characteristic spore-forming bacilli. Culturing on selective media (PLET) and blood agar resulted in characteristic growth in 42 (9.52%) of samples. These 42 samples were confirmed for B. anthracis by PCR. The ELISA findings indicated exposure to anthrax in both animals and humans. The exposure rate in human samples varied across exposure groups, and the highest exposure was detected in humans exposed to the sheep (33%) and goats (33%), indicating zoonotic potential by small ruminants. The high degree of spore contamination indicated that the sole use of conventional microscopy may lead to an overestimation of anthrax prevalence. For accurate detection of anthrax, the present study suggests the need to integrate bacterial culture with molecular methods, e.g., PCR. These results highlighted the role of small ruminants as potential reservoirs and the need for strict biosecurity measures to mitigate the transmission of anthrax in endemic regions.

Streptococcus vaginalis is a recently identified bacterial species closely related to Streptococcus anginosus. It has been isolated from the human urogenital tract. We report the complete genome sequence of S. vaginalis UMB8616 (=ATCC TSD-371 = CCUG 77169 = DSM 115471) isolated from the bladder of a human female with urge urinary incontinence.

Abstract In type 1 diabetes (T1D), insulin-producing β cells are destroyed by an autoimmune response driven by pro-inflammatory cytokines, including interferons. β-cell cytokine signaling is mediated in part by post-translational modifications, such as phosphorylation and acetylation. However, the role of other post-translational modifications in β-cell cytokine signaling represents an important knowledge gap. In the context of autoimmune diseases, lysine carbamylation has gained attention for its role in pathogenesis. Here, we investigate the role of carbamylation in T1D. We found that pancreatic islet cells from the T1D model, non-obese diabetic (NOD) mice, exhibit 11% carbamylation-positive cells, whereas non-diabetic CD1 mice have only 5%. Proteomics analysis of the MIN6 insulin-producing cell line treated with a cocktail of three pro-inflammatory cytokines IFNγ + IL-1β + TNFα identified 284 carbamylated peptides from 222 proteins impacted by the cytokine treatment. Integration of carbamylation and acetylation provided a deep view of the cytokine-regulated PTMs and potential points of interplay. A functional-enrichment analysis revealed that carbamylation was enriched in pathways related to autoimmune diseases, metabolism, DNA replication, and protein translation. Moreover, functional testing demonstrated that carbamylation inhibits the glycolytic enzyme aldolase A and the insulin-processing enzyme carboxypeptidase E, identifying a possible role for cytokine-induced β-cell dysfunction. In summary, protein carbamylation is elevated in islets from NOD mice, and pro-inflammatory cytokine treatment regulates protein carbamylation in MIN6 cells. These data identify carbamylation as a potential regulatory mechanism for β-cell metabolism and insulin production in the context of islet inflammation.

Despite increased recognition of the diverse resident microbiome of the urinary tract (i.e., the urobiome) in postmenopausal women, the roles and functions of these microbes remain largely unknown. Further empirical research is needed to understand the physiology, interactions, and antibiotic resistance evolution of urobiome members with pathogenic potential. However, experimental work relies on viable, culturable isolates. Standard urine culturing practices are designed for identifying a narrow set of known urinary microbes, and are thus poorly suited for cultivating taxa from the resident urobiome. Here we expand the urine culturing toolkit to reliably recover diverse urobiome taxa for downstream empirical research. Urine samples collected from postmenopausal women with recurrent urinary tract infections were shipped at ambient temperature to a central point for culturing. Microbial viability was maintained using boric acid preservative tubes during multi–day transport of sample aliquots. Selective media incubated under specialized conditions were used to promote recovery of diverse urobiome members, including fastidious taxa. Under 5% CO 2 –enriched atmospheric conditions and with longer incubation times, we leveraged a chromogenic agar (UTIC) to further differentiate isolates based on colony color and morphology. We evaluated the workflow for its ability to isolate and characterize urobiome taxa, as determined by morphological differentiation and taxonomic identification. Across 108 urine samples, 6.3 ± 3.2 distinct isolates were recovered, with no detectable relationship between sample shipment duration and isolate richness. On chromogenic agar, colony growth and color intensity was improved with CO 2 –enriched atmospheric conditions and extended incubation times. We identified diverse taxa that are typically underrepresented in standard diagnostic culture and provide novel morphological characterizations for members of the genera Actinotignum, Aerococcus, Facklamia, Lactobacillus, Latilactobacillus, Limosilactobacillus, and Streptococcus species, which have not been previously described on UTIC chromogenic agar. Using this novel workflow, we recovered a diverse collection of urobiome isolates from urine samples shipped over multiple days. We also demonstrated the utility of a chromogenic agar for the visual differentiation of key urobiome taxa. While sequencing approaches have enhanced our understanding of urobiome composition, culturing is needed to investigate microbial interactions, virulence mechanisms, and antimicrobial susceptibility. This protocol adds to the growing toolkit for the cultivation of diverse urobiome isolates needed to support downstream empirical studies and advance urinary tract infection research.

ABSTRACT Background Urinary tract infections (UTIs) represent a major public health concern, increasingly complicated by rising antibiotic resistance, diminishing treatment efficacy, and increasing prevalence of recurrence. The urinary tract microbiome (urobiome) remains poorly characterized, despite its potential role in UTIs. Results To provide a comprehensive overview of the urobiome, we here integrated seven publicly available shotgun metagenomics studies, linking microbial composition to clinical infection status or diagnostics. Community-level analyses revealed distinct urobiome clusters, defined by one predominant bacterial taxon. Genome-resolved metagenomics allowed recovery of high-quality metagenome-assembled genomes (MAGs), enabling phylogenetic reconstruction, prediction of pathogenic potential, and profiling of antimicrobial resistance genes across multiple taxa. We then analyzed the pangenome of the clinically significant Escherichia coli species and found that its genomic variation is driven more by phylotype than isolation source or UTI status, supporting a model of opportunistic infection. Conclusions Taken together, our analyses represent a systematic, cross-study view of the urobiome that emphasizes the ecological complexity of the urobiome and the importance of integrating functional and phylogenetic information when studying UTIs.

Facklamia species are Gram-positive, catalase-negative cocci within the family Aerococcaceae. Historically misidentified as streptococci or enterococci due to phenotypic similarity and conventional biochemical testing limitations, their clinical significance remains incompletely defined. This narrative review synthesizes the current literature on the microbiology, epidemiology, clinical manifestations, diagnostic challenges, and antimicrobial susceptibilities of Facklamia infections, with emphasis on urogynecologic relevance. A structured literature search identified peer-reviewed reports of microbiologically confirmed human infection published through December 2025. These reports indicate global distribution and a broad clinical spectrum. Invasive infections were reported most often in older adults and individuals with structural abnormalities, chronic comorbidities, or recent surgical interventions; however, cases in otherwise healthy patients are described. Emerging microbiome data suggest that F. hominis may be enriched in adult females with lower urinary tract symptoms, supporting the possibility that the female urogenital tract functions as a potential reservoir and site of pathogenic activity. Antimicrobial susceptibility patterns vary, with documented resistance to penicillin, macrolides, clindamycin, and tetracyclines, and susceptibility to cephalosporins, vancomycin, and linezolid. Overall, Facklamia species should be recognized as underdiagnosed opportunistic pathogens with both invasive and urogynecologic relevance. Increased awareness, improved diagnostic identification, and systematic susceptibility profiling are needed to clarify their pathogenic role and guide appropriate antimicrobial therapy.

Abstract Urinary tract infection (UTI) remains the most common infectious complication among individuals with neurogenic lower urinary tract dysfunction (NLUTD) due to spinal cord injury or disease (SCI/D). Despite widespread reliance on microbiological and symptom-based criteria for UTI diagnosis, significant ambiguity persists—especially in distinguishing clinically meaningful change from normal variability in urinary analysis results. This uncertainty contributes to overdiagnosis, inappropriate antibiotic use, and antimicrobial resistance. The present study seeks to operationalize “normal variability” of the urinary microbiome (urobiome) among adults with SCI/D. Using repeated samples collected from asymptomatic individuals over time, we analyzed inter- and intra-individual microbial composition to determine stability and fluctuation under baseline conditions. We observed wide intra- and inter-individual variability, substantial overlap between asymptomatic and pre-symptomatic states, and a consistent predominance of genera conventionally labeled as “uropathogens” even in the absence of symptoms. These findings suggest that assumptions drawn from cross-sectional studies—linking particular taxa or diversity values to health or disease—are not supported within individuals over time, at least in people with NLUTD. This study provides a foundation for distinguishing expected variation from those potentially related to infection, supporting development of precision-based diagnostic thresholds. Results offer critical insight into the ecological dynamics of the urobiome among people with NLUTD who are asymptomatic, establishes a methodological precedent for urobiome-informed clinical decision-making in SCI/D populations, and provides a foundation for distinguishing expected variation from those potentially related to infection, supporting development of precision-based diagnostic thresholds. By identifying personalized baselines and patterns of change, we aim to support research designed to obtain actionable information from the urobiome to enhance the accuracy and stewardship of UTI diagnosis and treatment in this high-risk population.

Urinary tract infections represent one of the most prevalent bacterial diseases, yet current diagnostic and research methodologies are hampered by inadequate culture media that fail to replicate the bladder biochemical environment. Conventional artificial urine formulations contain undefined components, lack essential nutrients, or inadequately support urinary microbiome (urobiome) growth. To address these limitations, we developed SimUrine, a fully defined synthetic urine medium that aims to replicate human bladder chemistry while supporting diverse microbial growth requirements. SimUrine was systematically developed through iterative optimization of multi-purpose artificial urine, incorporating defined concentrations of carbon sources, vitamins, trace elements, and amino acids within physiologically relevant ranges. The modular design enables component substitution without complete reformulation, facilitating customization for culturomics, antimicrobial susceptibility testing, and microbial ecology studies, while reducing batch-to-batch variability associated with authentic urine. Performance evaluation demonstrated SimUrine's capability to support the growth of fastidious urobiome members, including Lactobacillus species, Aerococcus urinae, and Corynebacterium riegelii, which fail to proliferate in conventional minimal media. Physicochemical characterization confirmed that SimUrine formulation exhibits properties within normal human urine ranges for density, conductivity, osmolarity, and viscosity, ensuring physiological relevance. Clinical applications revealed reduced antibiotic susceptibility compared to standard media, suggesting a more accurate representation of in vivo conditions. Co-culture experiments using Escherichia coli and Enterococcus faecalis demonstrated previously unobserved microbial interactions, highlighting SimUrine's utility for investigating urobiome dynamics. SimUrine represents a significant advancement in urobiome research methodology, providing a standardized, reproducible platform for investigating the urobiome under physiologically relevant conditions, potentially improving fundamental understanding and clinical diagnostic approaches.IMPORTANCEUrinary tract infections (UTIs) affect millions globally, yet current research and diagnostic methods rely on inadequate culture media that fail to replicate the bladder's unique biochemical environment. This fundamental limitation has hindered accurate UTI research and potentially compromised clinical treatment decisions. SimUrine addresses this critical gap as the first fully defined synthetic urine medium that mimics human bladder chemistry while supporting the growth of diverse urinary microbes. The breakthrough enables the cultivation of urobiome organisms in a minimal medium that resembles natural conditions, revealing novel microbial interactions that influence urinary health. Crucially, SimUrine demonstrates different antimicrobial susceptibility patterns compared to standard clinical media, suggesting current testing protocols may inaccurately predict treatment outcomes. This standardized, reproducible platform eliminates the variability of authentic urine samples while maintaining physiological relevance, potentially transforming urobiome research methodology and providing a new tool for the study of UTIs worldwide.

Evolutionary conservation has been considered a hallmark of essential basic functions in cells. Therefore, the study of evolutionarily conserved post-translational modifications (PTMs) can provide insight into their role in protein function. In this context, mass spectrometry can identify and quantify thousands of PTM sites. However, a major bottleneck lies in analyzing the large amounts of data collected by the mass spectrometer. Here we address the need for a protein sequence alignment tool for multiple PTMs across several species. We developed a tool named PTMOverlay that takes peptide identification output files and overlays PTM sites onto multiple protein sequence alignments. Examining 31 bacteria isolates, we combined their protein sequences with select PTM types, including acetylation, phosphorylation, monomethylation, dimethylation, and trimethylation. The tool revealed a variety of conserved modification sites on the bacterial central carbon metabolism. Further structural analysis revealed possible interactions between methylated arginine and lysine residues with phosphothreonine/serine sites on the homodimer interface of enolase. Overall, this tool can parse large amounts of mass spectrometry data and allows for more informed and efficient selection of sites for future studies of protein function.

Objective:To determine whether urine neutrophil gelatinase-associated lipocalin (uNGAL) or urobiome alterations can differentiate urinary tract infections (UTI) from asymptomatic bacteriuria (ASB). Methods:Female 8-week-old C57BL/6 mice were instilled with either Escherichia coli CFT073 (UTI model, n=12), E. coli 83972 (ASB model, n=12), or saline (control, n=3). uNGAL was measured daily for 3 days post-instillation. Urobiome composition was assessed pre- and post-instillation using 16S rRNA sequencing. At day 3, kidneys were harvested for culture. Comparisons were made across groups for uNGAL levels and urobiome diversity. Results:Baseline β diversity did not differ between groups. Post-instillation, β diversity significantly differed across groups (p=0.01), driven by increased relative abundance of E. coli in UTI mice compared to ASB mice. Median uNGAL levels increased significantly in both UTI and ASB groups relative to controls, but no significant difference was observed between UTI and ASB groups. Conclusion:Introduction of a uropathogenic E. coli strain reduced urobiome diversity, while a non-uropathogenic strain did not, suggesting strain-specific effects on microbial ecology. Bladder instillation itself also altered the urobiome. Elevated uNGAL levels were observed in both UTI and ASB models, indicating that while uNGAL reflects bacterial exposure, it does not distinguish between uropathogenic and non-uropathogenic E. coli. These findings highlight urobiome analysis as a potential tool for differentiating UTI from ASB, whereas uNGAL alone is insufficient.

To compare the bladder microbiomes of participants with and without a gynecological cancer at the time of diagnostic surgery to determine their suitability as control participants in urobiome research studies. IRB-approved clinical sample of continent adult female patients undergoing surgery for suspected gynecologic malignancy. Participants contributed clinical data (abstracted from the electronic medical record), and a catheterized urine sample obtained prior to antibiotic administration on the day of surgery. The study urine sample underwent DNA sequencing following amplification of hyper-variable region 4 (V4) of the bacterial 16S rRNA gene. The sequences were processed, annotated, and decontaminated. Alpha (within-sample) and beta (between-sample) diversity indices were computed, and the microbiome compositions across all participants were compared. Ninety-two women participated, including 20 with a gynecological cancer diagnosis and a comparison group of 72 without gynecologic cancer detected. The mean age was 53 years old (range 22–92) with a mean BMI of 31 kg/m2 (26–38 kg/m2); the majority (62

Bacillus anthracis, the causative agent of anthrax, is a highly virulent zoonotic pathogen primarily affecting domesticated and wild herbivores. Human exposure to B. anthracis is primarily through contact with infected animals or contaminated animal products. In Pakistan, where livestock vaccines are largely unavailable and infected carcasses are often disposed of improperly, the risk to humans, wildlife and livestock is significant. Currently, the diagnosis of anthrax infections and outbreak tracing necessitates the isolation and culturing of B. anthracis, a process that requires BSL- 3 facilities. In this study, we show that positive identification, genome reconstruction and lineage assignment can be accomplished using bioinformatic analysis of DNA extracted directly from environmental samples that would otherwise provide the starting material for isolation and culturing. This approach does not require laboratory target enrichment as is necessary for other pathogens, due in part to the extremely high bacterial load in the bloodstream in the deceased animals. Using these methods, we greatly expand the knowledge of endemic B. anthracis in Pakistan. We provide the first reference B. anthracis genomes from Pakistan since the 1970s and identify A.Br.014 Aust94 as a minor circulating sublineage alongside the dominant A.Br.047 Vollum. Future work will focus on the limits of detection and will determine if this bioinformatic method can be expanded more broadly for B. anthracis or other pathogens to replace typical culture- based methods.

Urinary tract infection (UTI) treatment is a growing public health concern owing to increasing antimicrobial resistance. Phage therapy, an alternative or adjunctive treatment to antibiotics, has the potential to address this challenge. However, clinical use of phage therapy is hindered by knowledge gaps and inconsistent reporting. The objective was to review the current state of phage therapy for UTIs and highlight research priorities that can optimize phage clinical efficacy. Current literature on UTI phage therapy was examined, focusing on the lack of standardized phage susceptibility testing, phage characterization, and microbiological assessments during and after treatment. Critical areas requiring further investigation include appropriate phage dosing, optimal routes of administration, and the dynamics of phage–host and phage–patient interactions. The influence of the urinary microbiome, including endogenous phages, on treatment outcomes also needs to be better understood. Suggested data collection and reporting standards should be developed and implemented to improve clinical impact of studies examining phage therapy for UTI. Randomized clinical trials are needed to establish efficacy and determine the best practices for clinical use. Phage therapy is a promising alternative to antibiotics for managing UTIs, especially in the face of rising antimicrobial resistance. To fully realize its potential, however, future research must focus on standardized protocols, dosing strategies, and the role of the urinary microbiome, with an emphasis on rigorously conducted clinical trials. These steps are essential for integrating phage therapy into mainstream UTI treatment regimens.

(Abstracted from Urogynecology (Phila) 2025;31(7):650–659) Microbiota regulate many processes in the body. Resiliency refers to how well microbiota can return to a baseline state after disruption, and understanding resilience is necessary to determine how to modulate microbiota to optimize healing and prevent disease.

BACKGROUND:Streptococcus anginosus has been found to colonize multiple anatomical sites of the human body, including the oral cavity, gastrointestinal tract, skin, vagina, blood, and urinary tract. It is frequently isolated from catheterized urine samples obtained from adult females most often with lower urinary tract symptoms, but also on occasion from those without symptoms. Our prior genome analysis of 166 S. anginosus genomes identified two distinct groups, one - which we here call group Urinae - exhibits a tropism for the urinary tract. RESULTS:Here we sequenced 100 isolates collected from different urogenital sites, including isolates from urine (both catheterized and voided) samples, urethral swabs, perineal swabs, vaginal swabs, and a foreskin swab. These sequenced isolates, as well as 15 isolates previously sequenced by our group, were collected from 50 unique individuals, with isolates from multiple anatomic sites for 26 of these individuals. The majority (89.57%) of the isolates were representatives of the Urinae group and were found in all sample types. Expanding our prior statistical method, we determined that, for 11 females, the same strain of Urinae was isolated from more than one of the urogenital sites. CONCLUSIONS:The identification of S. anginosus group Urinae strains from all urogenital sites sampled signifies that the tropism of this group is not restricted to the urinary tract. Rather, it seems to be a common constituent of the urogenital tract. The identification of the same strain shared across urogenital sample sites from the same individual suggests that the female urogenital sites in fact have interconnected microbiota.

UTI involves bacterial growth in the disparate environments of the bladder and within uroepithelial cells. The bladder is a low-nutrient environment that nonetheless supports rapid growth. Phylogenetic group B2 Escherichia coli (Ec) is frequently isolated from urinary tract infection (UTI) patients. Non-B2 strains have also been isolated and are often co-isolated with Enterococcus faecalis (Ef). We characterized the interaction between three co-isolated Ec-Ef pairs and other Ec-Ef combinations in a nutrient-rich medium which was intended to emulate the rapid growth condition of the bladder. In this medium, Ef had little effect on Ec growth but resulted in major transcriptome differences. Ef affected the non-B2 and B2 strains differently. For the non-B2 Ec strains, Ef induced transcript for genes whose products degrade ornithine via putrescine to succinate which is subsequently metabolized by the TCA cycle. For a control B2 Ec strain, Ef induced transcripts for growth rate-associated genes of macromolecular synthesis, and for similar metabolic enzymes, except for those that degrade putrescine to succinate. Unexpectedly, Ef induced transcripts for glyoxylate shunt enzymes in both non-B2 and B2 Ec strains. The bacterial disparate and constantly changing environments during UTI suggest the potential for different types of Ec-Ef interactions. Our results provide evidence for nutrient cross-feeding as one type of Ec-Ef interaction.

In this third episode of the Microbiologist in the Clinic series, clinicians and laboratory scientists share their perspectives about a 32 y/o female who has become antibiotic-dependent for her urinary symptoms. Despite escalating methods of antibiotic administration, the patient has persistent and recurrent "UTI" symptoms. Extensive testing has not provided guidance for her treating clinicians. The challenges of this clinical presentation are discussed with evidence for evaluation and treatment.

Polymicrobial or mixed urine cultures of more than one predominant microbe confound clinical urinary tract infection diagnosis. The current College of American Pathologists clinical laboratory standard states that a urine sample cultured with more than two isolates with >10,000 colony forming units/ml is to be considered contaminated. However, the presence of urinary sample bacteria in individuals without urinary symptoms (referred to as asymptomatic bacteriuria) is common especially in older people and in pregnant individuals. Furthermore, the discovery of an indigenous urinary microbiome (urobiome) in healthy humans throughout life from shortly after birth to death conflicts with the long-standing notion that urine derived from sterile filtered blood should be sterile above the urethral sphincter. Polymicrobial infections are not consistent with Koch’s postulates that a single pathogen is causal for disease. In this review, we will discuss current standards of contamination, how to reconcile the sterility of urine with the existence of the urobiome, a history of polymicrobial infections, and why re-examining current practices is essential for the practice of medicine, improving quality of life, and potentially saving lives.

Staphylococcus haemolyticus UMB6531B was isolated from a perineal swab of a female with overactive bladder symptoms and sequenced to better characterize opportunistic pathogens of the female urogenital tract. Here, we present the complete genome sequence, which includes the chromosome as well as two complete plasmid sequences.