ColiSeq: a multiplex amplicon assay that provides strain level resolution of Escherichia coli directly from clinical specimens.

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 × 105 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.

Pan-Enterovirus Characterization Reveals Cryptic Circulation of Clinically Relevant Subtypes in Arizona Wastewater.

Background: Most seasonally circulating enteroviruses result in asymptomatic or mildly symptomatic infections. In rare cases, however, infection with some subtypes can result in paralysis or death. Of the 300 subtypes known, only poliovirus is reportable, limiting our understanding of the distribution of other enteroviruses that can cause clinical disease. Objective: The overarching objectives of this study were to: 1) describe the distribution of enteroviruses in Arizona during the late summer and fall of 2022, the time of year when they are thought to be most abundant, and 2) demonstrate the utility of viral pan-assay approaches for semi-agnostic discovery that can be followed up by more targeted assays and phylogenomics. Methods: This study utilizes pooled nasal samples collected from school-aged children and long-term care facility residents, and wastewater from multiple locations in Arizona during July-October of 2022. We used PCR to amplify and sequence a region common to all enteroviruses, followed by species-level bioinformatic characterization using the QIIME 2 platform. For Enterovirus-D68 (EV-D68), detection was carried out using RT-qPCR, followed by confirmation using near-complete whole EV-D68 genome sequencing using a newly designed tiled amplicon approach. Results: In the late summer and early fall of 2022, multiple enterovirus species were identified in Arizona wastewater, with Coxsackievirus A6, EV-D68, and Coxsackievirus A19 composing 86% of the characterized reads sequenced. While EV-D68 was not identified in pooled human nasal samples, and the only reported acute flaccid myelitis case in Arizona did not test positive for the virus, an in-depth analysis of EV-D68 in wastewater revealed that the virus was circulating from August through mid-October. A phylogenetic analysis on this relatively limited dataset revealed just a few importations into the state, with a single clade indicating local circulation. Significance: This study further supports the utility of wastewater-based epidemiology to identify potential public health threats. Our further investigations into EV-D68 shows how these data might help inform healthcare diagnoses for children presenting with concerning neurological symptoms.

A local-scale One Health genomic surveillance of Clostridioides difficile demonstrates highly related strains from humans, canines, and the environment.

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.

Coccidioidomycosis in Northern Arizona: an Investigation of the Host, Pathogen, and Environment Using a Disease Triangle Approach.

Coccidioides immitis and Coccidioides posadasii are the etiological agents of coccidioidomycosis (Valley fever [VF]). Disease manifestation ranges from mild pneumonia to chronic or extrapulmonary infection. If diagnosis is delayed, the risk of severe disease increases. In this report, we investigated the intersection of pathogen, host, and environment for VF cases in Northern Arizona (NAZ), where the risk of acquiring the disease is much lower than in Southern Arizona. We investigated reported cases and assessed pathogen origin by comparing genomes of NAZ clinical isolates to isolates from other regions. Lastly, we surveyed regional soils for presence of Coccidioides. We found that cases of VF increased in NAZ in 2019, and Coccidioides NAZ isolates are assigned to Arizona populations using phylogenetic inference. Importantly, we detected Coccidioides DNA in NAZ soil. Given recent climate modeling of the disease that predicts that cases will continue to increase throughout the region, and the evidence presented in this report, we propose that disease awareness outreach to clinicians throughout the western United States is crucial for improving patient outcomes, and further environmental sampling across the western U.S. is warranted. IMPORTANCE Our work is the first description of the Valley fever disease triangle in Northern Arizona, which addresses the host, the pathogen, and the environmental source in the region. Our data suggest that the prevalence of diagnosed cases rose in 2019 in this region, and some severe cases necessitate hospitalization. We present the first evidence of Coccidioides spp. in Northern Arizona soils, suggesting that the pathogen is maintained in the local environment. Until disease prevention is an achievable option via vaccination, we predict that incidence of Valley fever will rise in the area. Therefore, enhanced awareness of and surveillance for coccidioidomycosis is vital to community health in Northern Arizona.

Differential Thermotolerance Adaptation between Species of Coccidioides.

Coccidioidomycosis, or Valley fever, is caused by two species of dimorphic fungi. Based on molecular phylogenetic evidence, the genus Coccidioides contains two reciprocally monophyletic species: C. immitis and C. posadasii. However, phenotypic variation between species has not been deeply investigated. We therefore explored differences in growth rate under various conditions. A collection of 39 C. posadasii and 46 C. immitis isolates, representing the full geographical range of the two species, was screened for mycelial growth rate at 37 °C and 28 °C on solid media. The radial growth rate was measured for 16 days on yeast extract agar. A linear mixed effect model was used to compare the growth rate of C. posadasii and C. immitis at 37 °C and 28 °C, respectively. C. posadasii grew significantly faster at 37 °C, when compared to C. immitis; whereas both species had similar growth rates at 28 °C. These results indicate thermotolerance differs between these two species. As the ecological niche has not been well-described for Coccidioides spp., and disease variability between species has not been shown, the evolutionary pressure underlying the adaptation is unclear. However, this research reveals the first significant phenotypic difference between the two species that directly applies to ecological research.

A global to local genomics analysis of Clostridioides difficile ST1/RT027 identifies cryptic transmission events in a northern Arizona healthcare network.

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.

Domestic canines do not display evidence of gut microbial dysbiosis in the presence of Clostridioides (Clostridium) difficile, despite cellular susceptibility to its toxins.

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.

Phylogenetic Analysis of Francisella tularensis Group A.II Isolates from 5 Patients with Tularemia, Arizona, USA, 2015-2017.

We examined 5 tularemia cases in Arizona, USA, during 2015-2017. All were caused by Francisella tularensis group A.II. Genetically similar isolates were found across large spatial and temporal distances, suggesting that group A.II strains are dispersed across long distances by wind and exhibit low replication rates in the environment.

Tick-Borne Relapsing Fever Outbreak Among a High School Football Team at an Outdoor Education Camping Trip, Arizona, 2014.

During August 2014, five high school students who had attended an outdoor education camp were hospitalized with a febrile illness, prompting further investigation. Ten total cases of tick-borne relapsing fever (TBRF) were identified-six cases confirmed by culture or visualization of spirochetes on blood smear and four probable cases with compatible symptoms (attack rate: 23%). All patients had slept in the campsite's only cabin. Before the camp, a professional pest control company had rodent proofed the cabin, but no acaricides had been applied. Cabin inspection after the camp found rodents and Ornithodoros ticks, the vector of TBRF. Blood samples from a chipmunk trapped near the cabin and from patients contained Borrelia hermsii with identical gene sequences (100% over 630 base pairs). Health departments in TBRF endemic areas should consider educating cabin owners and pest control companies to apply acaricides during or following rodent proofing, because ticks that lack rodents for a blood meal might feed on humans.

Hypervirulent emm59 Clone in Invasive Group A Streptococcus Outbreak, Southwestern United States.

The hyper-virulent emm59 genotype of invasive group A Streptococcus was identified in northern Arizona in 2015. Eighteen isolates belonging to a genomic cluster grouped most closely with recently identified isolates in New Mexico. The continued transmission of emm59 in the southwestern United States poses a public health concern.

Whole genome SNP typing to investigate methicillin-resistant Staphylococcus aureus carriage in a health-care provider as the source of multiple surgical site infections.

BACKGROUND: Prevention of nosocomial transmission of infections is a central responsibility in the healthcare environment, and accurate identification of transmission events presents the first challenge. Phylogenetic analysis based on whole genome sequencing provides a high-resolution approach for accurately relating isolates to one another, allowing precise identification or exclusion of transmission events and sources for nearly all cases. We sequenced 24 methicillin-resistant Staphylococcus aureus (MRSA) genomes to retrospectively investigate a suspected point source of three surgical site infections (SSIs) that occurred over a one-year period. The source of transmission was believed to be a surgical team member colonized with MRSA, involved in all surgeries preceding the SSI cases, who was subsequently decolonized. Genetic relatedness among isolates was determined using whole genome single nucleotide polymorphism (SNP) data. RESULTS: Whole genome SNP typing (WGST) revealed 283 informative SNPs between the surgical team member's isolate and the closest SSI isolate. The second isolate was 286 and the third was thousands of SNPs different, indicating the nasal carriage strain from the surgical team member was not the source of the SSIs. Given the mutation rates estimated for S. aureus, none of the SSI isolates share a common ancestor within the past 16 years, further discounting any common point source for these infections. The decolonization procedures and resources spent on the point source infection control could have been prevented if WGST was performed at the time of the suspected transmission, instead of retrospectively. CONCLUSIONS: Whole genome sequence analysis is an ideal method to exclude isolates involved in transmission events and nosocomial outbreaks, and coupling this method with epidemiological data can determine if a transmission event occurred. These methods promise to direct infection control resources more appropriately.

Pulmonary Embolism Mimicking Pneumonia in a HIV Patient.

Recent studies have shown an increased risk of arterial and venous vascular diseases in HIV patients, pulmonary thromboembolism being one of them. HIV-infected individuals may have procoagulants predisposing them to thromboembolism. Patients with thromboembolism may have a clinical presentation mimicking common opportunistic infections. It is important to consider pulmonary embolism in the differential of HIV patients with fever, cough, and dyspnea, particularly in those with well-controlled HIV infection.