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Escherichia coli is a diverse pathogen, causing a range of disease in humans, from self-limiting diarrhea to urinary tract infections (UTIs). Uropathogenic E. coli (UPEC) is the most frequently observed uropathogen in UTIs, a common disease in high-income countries, incurring billions of dollars yearly in treatment costs. Although E. coli is easily grown and identified in the clinical laboratory, genotyping the pathogen is more complicated, yet critical for reducing the incidence of disease. These goals can be achieved through whole-genome sequencing of E. coli isolates, but this approach is relatively slow and typically requires culturing the pathogen in the laboratory. To genotype E. coli rapidly and inexpensively directly from clinical samples, including but not limited to urine, we developed and validated a multiplex amplicon sequencing assay, called ColiSeq. The assay consists of targets designed for E. coli species confirmation, high resolution genotyping, and mixture deconvolution. To demonstrate its utility, we screened the ColiSeq assay against 230 clinical urine samples collected from a hospital system in Flagstaff, Arizona, USA. A limit of detection analysis demonstrated the ability of ColiSeq to identify E. coli at a concentration of ~2 genomic equivalent (GEs)/mL and to generate high-resolution genotyping at a concentration of 1 × 10 5 GEs/mL. The results of this study suggest that ColiSeq could be a valuable method to understand the source of UPEC strains and guide infection mitigation efforts. As sequence-based diagnostics become accepted in the clinical laboratory, workflows such as ColiSeq will provide actionable information to improve patient outcomes.IMPORTANCEUrinary tract infections (UTIs), caused primarily by Escherichia coli , create an enormous health care burden in the United States and other high-income countries. The early detection of E. coli from clinical samples, including urine, is important to target therapy and prevent further patient complications. Additionally, understanding the source of E. coli exposure will help with future mitigation efforts. In this study, we developed, tested, and validated an amplicon sequencing assay focused on direct detection of E. coli from urine. The resulting sequence data were demonstrated to provide strain level resolution of the pathogen, not only confirming the presence of E. coli , which can focus treatment efforts, but also providing data needed for source attribution and contact tracing. This assay will generate inexpensive, rapid, and reproducible data that can be deployed by public health agencies to track, diagnose, and potentially mitigate future UTIs caused by E. coli ., Competing Interests: The authors declare no conflict of interest.

Although infections caused by Clostridioides difficile have historically been attributed to hospital acquisition, growing evidence supports the role of community acquisition in C. difficile infection (CDI). Symptoms of CDI can range from mild, self-resolving diarrhoea to toxic megacolon, pseudomembranous colitis, and death. In this study, we sampled C. difficile from clinical, environmental, and canine reservoirs in Flagstaff, Arizona, USA, to understand the distribution and transmission of the pathogen in a One Health framework; Flagstaff is a medium-sized, geographically isolated city with a single hospital system, making it an ideal site to characterize genomic overlap between sequenced C. difficile isolates across reservoirs. An analysis of 562 genomes from Flagstaff isolates identified 65 sequence types (STs), with eight STs being found across all three reservoirs and another nine found across two reservoirs. A screen of toxin genes in the pathogenicity locus identified nine STs where all isolates lost the toxin genes needed for CDI manifestation ( tcdB , tcdA ), demonstrating the widespread distribution of non-toxigenic C. difficile (NTCD) isolates in all three reservoirs; 15 NTCD genomes were sequenced from symptomatic, clinical samples, including two from mixed infections that contained both tcdB+ and tcdB - isolates. A comparative single nucleotide polymorphism (SNP) analysis of clinically derived isolates identified 78 genomes falling within clusters separated by ≤2 SNPs, indicating that ~19 % of clinical isolates are associated with potential healthcare-associated transmission clusters; only symptomatic cases were sampled in this study, and we did not sample asymptomatic transmission. Using this same SNP threshold, we identified genomic overlap between canine and soil isolates, as well as putative transmission between environmental and human reservoirs. The core genome of isolates sequenced in this study plus a representative set of public C. difficile genomes ( n =136), was 2690 coding region sequences, which constitutes ~70 % of an individual C. difficile genome; this number is significantly higher than has been published in some other studies, suggesting that genome data quality is important in understanding the minimal number of genes needed by C. difficile . This study demonstrates the close genomic overlap among isolates sampled across reservoirs, which was facilitated by maximizing the genomic search space used for comprehensive identification of potential transmission events. Understanding the distribution of toxigenic and non-toxigenic C. difficile across reservoirs has implications for surveillance sampling strategies, characterizing routes of infections, and implementing mitigation measures to limit human infection.

Clostridioides difficile is a pathogen often associated with hospital-acquired infection or antimicrobial-induced disease; however, increasing evidence indicates infections can result from community or environmental sources. Most genomic sequencing of C. difficile has focused on clinical strains, although evidence is growing that C. difficile spores are widespread in soil and water in the environment. In this study, we sequenced 38 genomes collected from soil and water isolates in Flagstaff (AZ, USA) and Slovenia in an effort targeted towards environmental surveillance of C. difficile . At the average nucleotide identity (ANI) level, the genomes were divergent to C. difficile at a threshold consistent with different species. A phylogenetic analysis of these divergent genomes together with Clostridioides genomes available in public repositories confirmed the presence of three previously described, cryptic Clostridioide s species and added two additional clades. One of the cryptic species (C-III) was almost entirely composed of Arizona and Slovenia genomes, and contained distinct sub-groups from each region (evidenced by SNP and gene-content differences). A comparative genomics analysis identified multiple unique coding sequences per clade, which can serve as markers for subsequent environmental surveys of these cryptic species. Homologues to the C. difficile toxin genes, tcdA and tcdB , were found in cryptic species genomes, although they were not part of the typical pathogenicity locus observed in C. difficile , and in silico PCR suggested that some would not amplify with widely used PCR diagnostic tests. We also identified gene homologues in the binary toxin cluster, including some present on phage and, for what is believed to be the first time, on a plasmid. All isolates were obtained from environmental samples, so the function and disease potential of these toxin homologues is currently unknown. Enzymatic profiles of a subset of cryptic isolates ( n =5) demonstrated differences, suggesting that these isolates contain substantial metabolic diversity. Antimicrobial resistance (AMR) was observed across a subset of isolates ( n =4), suggesting that AMR mechanisms are intrinsic to the genus, perhaps originating from a shared environmental origin. This study greatly expands our understanding of the genomic diversity of Clostridioides . These results have implications for C. difficile One Health research, for more sensitive C. difficile diagnostics, as well as for understanding the evolutionary history of C. difficile and the development of pathogenesis.

A single-chromosome closed genome of Peptacetobacter ( Clostridium ) hiranonis strain DGF055142 was generated using Illumina MiSeq short reads paired with Oxford Nanopore MinION long reads. This isolate was obtained from a canine in Flagstaff, Arizona, in 2019. Peptacetobacter ( C. ) hiranonis was hypothesized to contribute to canine Clostridium difficile infection resistance., (Copyright © 2021 Stone et al.)

Clostridioides difficile infection (CDI) is an emerging public health threat and C. difficile is the most common cause of antimicrobial-associated diarrhea worldwide and the leading cause of hospital-associated infections in the US, yet the burden of community-acquired infections (CAI) is poorly understood. Characterizing C. difficile isolated from canines is important for understanding the role that canines may play in CAI. In addition, several studies have suggested that canines carry toxigenic C. difficile asymptomatically, which may imply that there are mechanisms responsible for resistance to CDI in canines that could be exploited to help combat human CDI. To assess the virulence potential of canine-derived C. difficile, we tested whether toxins TcdA and TcdB (hereafter toxins) derived from a canine isolate were capable of causing tight junction disruptions to colonic epithelial cells. Additionally, we addressed whether major differences exist between human and canine cells regarding C. difficile pathogenicity by exposing them to identical toxins. We then examined the canine gut microbiome associated with C. difficile carriage using 16S rRNA gene sequencing and searched for deviations from homeostasis as an indicator of CDI. Finally, we queried 16S rRNA gene sequences for bacterial taxa that may be associated with resistance to CDI in canines. Clostridioides difficile isolated from a canine produced toxins that reduced tight junction integrity in both human and canine cells in vitro. However, canine guts were not dysbiotic in the presence of C. difficile. These findings support asymptomatic carriage in canines and, furthermore, suggest that there are features of the gut microbiome and/or a canine-specific immune response that may protect canines against CDI. We identified two biologically relevant bacteria that may aid in CDI resistance in canines: 1) Clostridium hiranonis, which synthesizes secondary bile acids that have been shown to provide resistance to CDI in mice; and 2) Sphingobacterium faecium, which produces sphingophospholipids that may be associated with regulating homeostasis in the canine gut. Our findings suggest that canines may be cryptic reservoirs for C. difficile and, furthermore, that mechanisms of CDI resistance in the canine gut could provide insights into targeted therapeutics for human CDI., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Clostridioides difficile is a ubiquitous, diarrhoeagenic pathogen often associated with healthcare-acquired infections that can cause a range of symptoms from mild, self-limiting disease to toxic megacolon and death. Since the early 2000s, a large proportion of C. difficile cases have been attributed to the ribotype 027 (RT027) lineage, which is associated with sequence type 1 (ST1) in the C. difficile multilocus sequence typing scheme. The spread of ST1 has been attributed, in part, to resistance to fluoroquinolones used to treat unrelated infections, which creates conditions ideal for C. difficile colonization and proliferation. In this study, we analysed 27 isolates from a healthcare network in northern Arizona, USA, and 1352 publicly available ST1 genomes to place locally sampled isolates into a global context. Whole genome, single nucleotide polymorphism analysis demonstrated that at least six separate introductions of ST1 were observed in healthcare facilities in northern Arizona over an 18-month sampling period. A reconstruction of transmission networks identified potential nosocomial transmission of isolates, which were only identified via whole genome sequence analysis. Antibiotic resistance heterogeneity was observed among ST1 genomes, including variability in resistance profiles among locally sampled ST1 isolates. To investigate why ST1 genomes are so common globally and in northern Arizona, we compared all high-quality C. difficile genomes and identified that ST1 genomes have gained and lost a number of genomic regions compared to all other C. difficile genomes; analyses of other toxigenic C. difficile sequence types demonstrate that this loss may be anomalous and could be related to niche specialization. These results suggest that a combination of antimicrobial resistance and gain and loss of specific genes may explain the prominent association of this sequence type with C. difficile infection cases worldwide. The degree of genetic variability in ST1 suggests that classifying all ST1 genomes into a quinolone-resistant hypervirulent clone category may not be appropriate. Whole genome sequencing of clinical C. difficile isolates provides a high-resolution surveillance strategy for monitoring persistence and transmission of C. difficile and for assessing the performance of infection prevention and control strategies.