Intestinal Persistence of Colonizing Escherichia coli Strains, Especially ST131-H30, in Relation to Bacterial and Host Factors.

BACKGROUND: Superior gut colonization may underlie the pandemic emergence of the resistance-associated H30 subclone of Escherichia coli sequence type 131 (ST131-H30). Little is known about the associated host and bacterial characteristics, or the comparative persistence of non-ST131 intestinal E. coli. METHODS: Generic and fluoroquinolone-resistant E. coli isolates from volunteers' serial fecal samples underwent clonal analysis and extensive polymerase chain reaction (PCR)-based characterization (phylogroup, selected sequence types, virulence genes). Kaplan-Meier survival analysis and Cox proportional hazards survival analysis using penalized regression (a machine-learning method) were used to identify correlates of strain persistence. RESULTS: Screening of 2005 subjects at the Minneapolis VA Medical Center identified 222 subjects (117 veterans, 105 human and animal household members) for longitudinal fecal surveillance. Analysis of their 585 unique-by-subject fecal E. coli strains identified multiple epidemiological, ecological, and bacterial correlates of strain persistence. ST131-H30, a strong univariable correlate of persistence, was superseded in multivariable analysis by outpatient status, fluoroquinolone resistance, and diverse (predominantly iron uptake-related) virulence genes. CONCLUSIONS: ST131-H30 exhibits exceptional intestinal persistence, possibly due to a combination of fluoroquinolone resistance and virulence factors, which may be primarily colonization factors. This identifies both likely contributors to the ST131-H30 pandemic and potential targets for interventions against it.

Activity of plazomicin against carbapenem-intermediate or -resistant Escherichia coli isolates from the United States and international sites in relation to clonal background, resistance genes, co-resistance, and region.

BACKGROUND: Emerging carbapenem resistance in Escherichia coli, including sequence type 131 (ST131), threatens therapeutic efficacy. Plazomicin (PLZ), a semisynthetic aminoglycoside approved by the FDA in 2018, overcomes the most common aminoglycoside resistance mechanisms and maintains activity against many carbapenem-intermediate or -resistant (CIR) E. coli strains. OBJECTIVES: To assess plazomicin susceptibility among CIR E. coli in relation to region and multiple bacterial characteristics. METHODS: We determined broth microdilution MICs for plazomicin and 11 comparators against 343 CIR clinical E. coli isolates, then compared susceptibility results by bacterial characteristics and region. The collection comprised 203 US isolates (2002-17) and 141 isolates from 17 countries in Europe, Latin America, and the Asia-West Pacific region (2003-17). Isolates were characterized for phylogenetic group, resistance-associated sequence types (STs) and subsets thereof, and relevant ß-lactamase-encoding genes. RESULTS: Plazomicin exhibited the highest percentage susceptible (89%) after tigecycline (99%). The percentage susceptible to plazomicin varied significantly by phylogroup (63%, group B1; versus >93%, others) and ST131 subclone (92%, H30Rx; versus 87%-89%, H30R1 and non-H30), but not ST. It also varied by resistance genotype [higher with Klebsiella pneumoniae carbapenemase (KPC), lower with metallo-ß-lactamases], global region [highest for Latin America (94%), lowest for Asia-West Pacific (69%)], and US region (80%, South, versus 96%-100%, others). Although reduced susceptibility to comparators often predicted reduced susceptibility to plazomicin, even among comparator-intermediate or -resistant isolates the plazomicin-susceptible fraction was ≥77%, except for amikacin (53%). CONCLUSIONS: The likely utility of plazomicin against CIR E. coli is high overall, but varies with region and multiple bacterial characteristics.

Molecular Characteristics, Ecology, and Zoonotic Potential of Escherichia coli Strains That Cause Hemorrhagic Pneumonia in Animals.

Hemorrhagic pneumonia (HP) is a rare but highly lethal disease, mainly of dogs and cats, caused by hemolytic Escherichia coli strains that contain cnf1 (encoding cytotoxic necrotizing factor 1). After encountering fatal HP in two dogs, we used contemporary molecular methods, including multilocus sequence typing and whole-genome sequencing, to compare the corresponding case isolates with published HP clinical isolates and newly obtained fecal E. coli isolates from 20 humans and animals in the index HP case household. We also compared the aggregated HP clinical isolates, which represented 13 discrete strains, by pulsotype with a large, private pulsotype library of diverse-source E. coli. The HP clinical isolates represented a narrow range of phylogenetic group B2 lineages (mainly sequence types 12 and 127), O types (mainly O4 and O6), and H types (mainly H5 and H31), but diverse fimH alleles (type-1 fimbriae adhesin). Their extensive, highly conserved virulence genotypes, which qualified as extraintestinal pathogenic E. coli (ExPEC), encoded diverse adhesins, toxins, iron uptake systems, and protectins. Household surveillance identified multiple HP-like fecal strains, plus abundant between-host strain sharing, including of the household's index HP strain. The pulsotype library search identified, for five HP clinical strains, same-pulsotype human and animal fecal and clinical (predominantly urine) isolates, from diverse locales and time periods. Thus, E. coli strains that cause HP derive from a narrow range of ExPEC lineages within phylogroup B2, contain multiple virulence genes other than cnf1, are shared extensively between hosts, and likely function in nature mainly as intestinal colonizers and uropathogens. IMPORTANCE This study clarifies the clonal background and extensive virulence genotypes of the E. coli strains that cause hemorrhagic pneumonia in domestic animals (mainly dogs and cats), shows that such strains circulate among animals and humans, identifies a substantial intestinal colonization component to their lifestyle, and extends their known clinical manifestations to include bacteremia and urinary tract infection. The findings place these strains better into context vis-à-vis current understandings of E. coli phylogeny, ecology, and pathogenesis; identify questions for future research; and may prove relevant for surveillance and prevention efforts.

Comparative activity of plazomicin against extended-spectrum cephalosporin-resistant Escherichia coli clinical isolates (2012-2017) in relation to phylogenetic background, sequence type 131 subclones, blaCTX-M genotype, and resistance to comparator agents.

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCREC) are a growing threat. Leading ESCREC lineages include sequence type ST131, especially its (blaCTX-M-15-associated) H30Rx subclone and (blaCTX-M-27-associated) C1-M27 subset within the H30R1 subclone. The comparative activity against such strains of alternative antimicrobial agents, including the recently developed aminoglycoside plazomicin, is undefined, so was investigated here. We assessed plazomicin and 11 comparators for activity against 216 well-characterized ESCREC isolates (Minnesota, 2012-2017) and then compared broth microdilution MICs with phylogenetic and clonal background, beta-lactamase genotype (blaCTX-M; group 1 and 9 variants), and co-resistance. Percent susceptible was > 99% for plazomicin, meropenem, imipenem, and tigecycline; 96-98% for amikacin and ertapenem; and ≤ 75% for the remaining comparators. For most comparators, MICs varied significantly in relation to multiple bacterial characteristics, in agent-specific patterns. By contrast, for plazomicin, the only bacterial characteristic significantly associated with MICs was ST131 subclone: plazomicin MICs were lowest among O16 ST131 isolates and highest among ST131-H30R1 C1-M27 subclone isolates. Additionally, plazomicin MICs varied significantly in relation to resistance vs. susceptibility to comparator agents only for amikacin and levofloxacin. For most study agents, antimicrobial activity against ESCREC varied extensively in relation to multiple bacterial characteristics, including clonal background, whereas for plazomicin, it varied only by ST131 subclone (C1-M27 isolates least susceptible, O16 isolates most susceptible). These findings support plazomicin as a reliable alternative for treating ESCREC infections and urge continued attention to the C1-M27 ST131 subclone.

Global molecular epidemiology of carbapenem-resistant Escherichia coli (2002-2017).

The emergence of carbapenem-resistant (CR) Escherichia coli obliges an assessment of such strains' molecular epidemiology. Accordingly, we characterized in detail a globally distributed collection of CR E. coli isolates, then explored for associations between geographical origin and bacterial traits, and between different bacterial traits. We used established PCR-based assays and broth microdilution MIC determinations to characterize 343 global CR (i.e., non-susceptible to ≥ 1 carbapenem) extraintestinal E. coli isolates (2002-2017) for diverse molecular traits-including phylogroups, sequence types (STs), beta-lactamase genes, and 51 virulence genes-and susceptibility to 12 relevant antimicrobial agents. The study population was tremendously diverse according to all assessed variables. Nonetheless, certain geographically aligned, unifying themes emerged. These included an association of an Asia/West Pacific origin with non-B2/D/F phylogroups and STs, lower molecularly inferred virulence, more extensive resistance, and specific resistance genes (notably, metallo-beta-lactamases). Likewise, U.S. isolates from the central region, vs. other regions, were more virulent-appearing and more often from phylogroup B2 and ST131, but less extensively resistant and more often carbapenemase-gene negative. The global CR E. coli population is highly diverse according to multiple characteristics and varies significantly by geographical region. This predictably will pose challenges for prevention and management, and obliges ongoing surveillance.

Bringing Physical Exam Skills Back from the Dead.

Physical examination education begins early for medical learners. A hindrance to physical exam competency is lack of exposure to pathology in standardized patient settings. This research focuses on improving medical education through the utilization of cadavers that have undergone a soft-embalming technique: the Thiel method. Three scenarios were created in four Thiel cadavers: anterior cruciate ligament (ACL) tear, posterior cruciate ligament (PCL) tear, and sham incision. Students were asked to diagnose ACL tears using the Lachman exam. A total of 54 learners participated in the study. Post-surveys indicated most learners: (1) prefer to use standardized patients (SPs) and soft-embalmed cadavers in their physical examination courses, (2) increased their confidence in performing the Lachman exam on real patients, and (3) enhanced their Lachman technique. SPs ultimately cannot volitionally reproduce the physical exam findings of ACL deficiency. Consequently, learners cannot accurately identify positive versus negative examination findings. Thiel-embalmed cadavers are a valuable resource for physical examination education. (Journal of Surgical Orthopaedic Advances 30(2):112-115, 2021).

Despite Predominance of Uropathogenic/Extraintestinal Pathotypes Among Travel-acquired Extended-spectrum ß-Lactamase-producing Escherichia coli, the Most Commonly Associated Clinical Manifestation Is Travelers' Diarrhea.

BACKGROUND: One-third of the 100 million travelers to the tropics annually acquire extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-PE), with undefined clinical consequences. METHODS: Symptoms suggesting Enterobacteriaceae infections were recorded prospectively among 430 Finnish travelers, 90 (21%) of whom acquired ESBL-PE abroad. ESBL-PE isolates underwent polymerase chain reaction-based detection of diarrheagenic Escherichia coli (DEC) pathotypes (enteroaggregative E. coli [EAEC], enteropathogenic E. coli [EPEC], enterotoxigenic E. coli [ETEC], enteroinvasive E. coli, and Shiga toxin-producing E. coli), and extraintestinal pathogenic/uropathogenic E. coli (ExPEC/UPEC). Laboratory-confirmed ESBL-PE infections were surveyed 5 years before and after travel. RESULTS: Among the 90 ESBL-PE carriers, manifestations of Enterobacteriaceae infection included travelers' diarrhea (TD) (75/90 subjects) and urinary tract infection (UTI) (3/90). The carriers had 96 ESBL-producing E. coli isolates, 51% exhibiting a molecular pathotype: 13 (14%) were DEC (10 EAEC, 2 EPEC, 1 ETEC) (12 associated with TD) and 39 (41%) ExPEC/UPEC (none associated with UTI). Of ESBL-PE, 3 (3%) were ExPEC/UPEC-EAEC hybrids (2 associated with diarrhea, none with UTI). Potential ESBL-PE infections were detected in 15 of 90 subjects (17%). The 10-year medical record survey identified 4 laboratory-confirmed ESBL-PE infections among the 430 travelers, all in subjects who screened ESBL-PE negative after returning home from their index journeys but had traveled abroad before their infection episodes. CONCLUSIONS: Half of all travel-acquired ESBL-producing E. coli strains qualified molecularly as pathogens. Extraintestinal and uropathogenic pathotypes outnumbered enteric pathotypes (41% vs 14%), yet the latter correlated more closely with symptomatic infection (0% vs 92%). Despite more ESBL-PE strains qualifying as ExPEC/UPEC than DEC, travel-acquired ESBL-PE are more often associated with TD than UTI.

Activity of Cefiderocol, Ceftazidime-Avibactam, and Eravacycline against Carbapenem-Resistant Escherichia coli Isolates from the United States and International Sites in Relation to Clonal Background, Resistance Genes, Coresistance, and Region.

Emerging carbapenem resistance in Escherichia coli, including sequence type 131 (ST131), the leading cause of extraintestinal E. coli infections globally, threatens therapeutic efficacy. Accordingly, we determined broth microdilution MICs for three distinctive newer agents, i.e., cefiderocol (CFDC), ceftazidime-avibactam (CZA), and eravacycline (ERV), plus 11 comparators, against 343 carbapenem-resistant (CR) clinical E. coli isolates, then compared susceptibility results with bacterial characteristics and region. The collection comprised 203 U.S. isolates (2002 to 2017) and 141 isolates from 17 countries in Europe, Latin America, and the Asia-West Pacific region (2003 to 2017). Isolates were characterized for phylogenetic group, resistance-associated sequence types (STs) and subsets thereof, and relevant beta-lactamase-encoding genes. CFDC, CZA, and ERV exhibited the highest percent susceptible (82% to 98%) after tigecycline (TGC) (99%); avibactam improved CZA's activity over that of CAZ (11% susceptible). Percent susceptible varied by phylogroup and ST for CFDC and CZA (greatest in phylogroups B2, D, and F, and in ST131, ST405, and ST648). Susceptibility also varied by resistance genotype, being higher with the Klebsiella pneumoniae carbapenemase (KPC) for CZA, lower with metallo-beta-lactamases for CFDC and CZA, and higher with the beta-lactamase CTX-M for ERV. Percent susceptible also varied by global region for CZA (lower in Asia-Pacific) and by U.S. region for ERV (lower in the South and Southeast). Although resistance to comparators often predicted reduced susceptibility to a primary agent (especially CFDC and CZA), even among comparator-resistant isolates the primary-agent-susceptible fraction usually exceeded 50%. These findings clarify the likely utility of CFDC, CZA, and ERV against CR E. coli in relation to multiple bacterial characteristics and geographical region.

Activity of Imipenem-Relebactam against Carbapenem-Resistant Escherichia coli Isolates from the United States in Relation to Clonal Background, Resistance Genes, Coresistance, and Region.

Imipenem-relebactam (I-R) is a recently developed carbapenem-beta-lactamase inhibitor combination agent that can overcome carbapenem resistance, which has now emerged in Escherichia coli, including sequence type 131 (ST131) and its fluoroquinolone-resistant H30R subclone, the leading cause of extraintestinal E. coli infections globally. To clarify the likely utility of I-R for carbapenem-resistant (CR) E. coli infections in the United States, we characterized 203 recent CR clinical E. coli isolates from across the United States (years 2002 to 2017) for phylogroup, clonal group (including ST131, H30R, and the CTX-M-15-associated H30Rx subset within H30R), relevant beta-lactamase genes, and broth microdilution MICs for I-R and 11 comparator agents. Overall, I-R was highly active (89% susceptible), more so than all comparators except tigecycline and colistin (both 99% susceptible). I-R's activity varied significantly in relation to phylogroup, clonal background, resistance genotype, and region. It was greatest among phylogroup B2, ST131-H30R, H30Rx, Klebsiella pneumoniae carbapenemase (KPC)-positive, and northeast U.S. isolates and lowest among phylogroup C, New Delhi metallo-ß-lactamase (NDM)-positive, and southeast U.S. isolates. Relebactam improved imipenem's activity against CR isolates within each phylogroup-especially groups A, B1, and B2-and particularly against isolates containing KPC. I-R remained substantially active against isolates coresistant to comparator agents, albeit somewhat less so than against the corresponding susceptible isolates. These findings suggest that I-R should be useful for treating most CR E. coli infections in the United States, largely independent of coresistance, although this likely will vary in relation to the local prevalence of specific E. coli lineages and carbapenem resistance mechanisms.

In Silico Genotyping of Escherichia coli Isolates for Extraintestinal Virulence Genes by Use of Whole-Genome Sequencing Data.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause in humans of urinary tract infection and bacteremia. The previously published web tool VirulenceFinder (http://cge.cbs.dtu.dk/services/VirulenceFinder/) uses whole-genome sequencing (WGS) data for in silico characterization of E. coli isolates and enables researchers and clinical health personnel to quickly extract and interpret virulence-relevant information from WGS data. In this study, 38 ExPEC-associated virulence genes were added to the existing E. coli VirulenceFinder database. In total, 14,441 alleles were downloaded. A total of 1,890 distinct alleles were added to the database after removal of redundant sequences and analysis of the remaining alleles for open reading frames (ORFs). The database now contains 139 genes-of which 44 are related to ExPEC-and 2,826 corresponding alleles. Construction of the database included validation against 27 primer pairs from previous studies, a search for serotype-specific P fimbriae papA alleles, and a BLASTn confirmation of seven genes (etsC, iucC, kpsE, neuC, sitA, tcpC, and terC) not covered by the primers. The augmented database was evaluated using (i) a panel of nine control strains and (ii) 288 human-source E. coli strains classified by PCR as ExPEC and non-ExPEC. We observed very high concordance (average, 93.4%) between PCR and WGS findings, but WGS identified more alleles. In conclusion, the addition of 38 ExPEC-associated genes and the associated alleles to the E. coli VirulenceFinder database allows for a more complete characterization of E. coli isolates based on WGS data, which has become increasingly important considering the plasticity of the E. coli genome.

Accessory Traits and Phylogenetic Background Predict Escherichia coli Extraintestinal Virulence Better Than Does Ecological Source.

Background: The distinguishing characteristics of extraintestinal pathogenic Escherichia coli (ExPEC) strains are incompletely defined. Methods: We characterized 292 diverse-source human Escherichia coli isolates (116 from fecal specimens, 79 from urine specimens [of which 39 were from patients with cystitis and 40 were from patients with pyelonephritis], and 97 from blood specimens) for phylogenetic group, sequence type complex (STc), and 49 putative extraintestinal pathogenic E. coli (ExPEC)-associated virulence genes. We then assessed these traits and ecological source as predictors of illness severity in a murine sepsis model. Results: The study isolates exhibited a broad range of virulence in mice. Most of the studied bacterial characteristics corresponded significantly with experimental virulence, as did ecological source and established molecular definitions of ExPEC and uropathogenic E. coli (UPEC). Multivariable modeling identified the following bacterial traits as independent predictors of illness severity both overall and among the fecal and clinical (ie, urine and blood) isolates separately: fyuA (yersiniabactin receptor), kpsM K1 (K1 capsule), and kpsM II (group 2 capsules). Molecular UPEC status predicted virulence independently only among fecal isolates. Neither ecological source (ie, clinical vs fecal) nor molecular ExPEC status added predictive power to these traits, which accounted collectively for up to 49% of the observed variation in virulence. Conclusions: Among human-source E. coli isolates, specific accessory traits and phylogenetic/clonal backgrounds predict experimental virulence in a murine sepsis model better than does ecological source.

Rapid Emergence, Subsidence, and Molecular Detection of Escherichia coli Sequence Type 1193-fimH64, a New Disseminated Multidrug-Resistant Commensal and Extraintestinal Pathogen.

Escherichia coli sequence type 1193 (ST1193) is an emerging multidrug-resistant pathogen. We performed longitudinal and cross-sectional surveillance for ST1193 among clinical and fecal E. coli isolates from Minneapolis Veterans Affairs Medical Center (VAMC) patients and their household members, other Minnesota centers, and national VAMCs and compared these ST1193 isolates with archival human and canine ST1193 isolates from Australia (2008). We also developed and extensively validated a novel multiplex PCR assay for ST1193 and its characteristic fimH64 (type 1 fimbrial adhesin) allele. We found that ST1193-H64 (where "H64" refers to a phylogenetic subdivision within ST1193 that is characterized by the fimH64 allele), which was uniformly fluoroquinolone resistant, appeared to emerge in the United States in a geographically staggered fashion beginning around 2011. Its prevalence among clinical and fecal E. coli isolates at the Minneapolis VAMC rose rapidly beginning in 2013, peaked in early 2017, and then plateaued or declined. In comparison with other ST14 complex (STc14) isolates, ST1193-H64 isolates were more extensively multidrug resistant, whereas their virulence genotypes were less extensive but included (uniquely) K1 capsule and fimH64 Pulsed-field gel electrophoresis separated ST1193-H64 isolates from other STc14 isolates and showed genetic commonality between archival Australian versus recent U.S. isolates, fecal versus clinical isolates, and human versus canine isolates. Three main ST1193 pulsotypes differed significantly in resistance profiles and capsular types; emergent pulsotype 2123 was associated with trimethoprim-sulfamethoxazole resistance and K1 (versus K5) capsule. These findings clarify ST1193-H64's temporal prevalence trends as a fluoroquinolone-resistant pathogen and commensal; document clonal subsets with distinctive geographic, temporal, resistance, and virulence gene associations; and establish a new laboratory tool for rapid and simple detection of ST1193-H64.

Host and microbial factors in kidney transplant recipients with Escherichia coli acute pyelonephritis or asymptomatic bacteriuria: a prospective study using whole-genome sequencing.

BACKGROUND: Urinary tract infection is the most common infection among kidney transplant recipients (KTRs). Many transplant physicians fear that host compromise will allow low-virulence strains to cause pyelonephritis in KTRs, so they often treat asymptomatic bacteriuria with antibiotics. Identification of the host/microbe factors that determine the clinical presentation (i.e. pyelonephritis versus asymptomatic bacteriuria) once an Escherichia coli strain enters a KTRs bladder could inform management decisions. METHODS: We prospectively collected all E. coli isolates causing either pyelonephritis or asymptomatic bacteriuria in KTRs at our institution (December 2012-June 2015). Whole-genome sequencing was used to assess bacterial characteristics (carriage of 48 virulence genes and phylogenetic and clonal background). Host parameters were also collected. RESULTS: We analysed 72 bacteriuria episodes in 54 KTRs (53 pyelonephritis, 19 asymptomatic bacteriuria). The pyelonephritis and asymptomatic bacteriuria isolates exhibited a similar total virulence gene count per isolate [median 18 (range 5-33) and 18 (5-30), respectively; P = 0.57] and for individual virulence genes differed significantly only for the prevalence of the pap operon (pyelonephritis 39%,versus asymptomatic bacteriuria 0%; P = 0.002). No other significant between-group differences were apparent for 86 other bacterial and host variables. CONCLUSIONS: Our findings suggest that bacterial adherence plays a role in the pathogenesis of pyelonephritis in KTRs despite significantly altered host urinary tract anatomy and weakened immunity. Whether KTRs might benefit from targeted therapies (e.g. vaccination or inhibitors of fimbrial adhesion) has yet to be studied.

Paediatric window falls and interannual temperature variability in the Seattle metro area.

We explore whether the timing and burden of paediatric window fall injuries in the Seattle area have changed with higher temperatures and increased air conditioning (AC) prevalence. Using hospital trauma registry records from 2005 to 2017, along with population estimates from the National Center for Health Statistics, we calculate trauma incidence rates from paediatric window falls. Using local temperature data, we explore the relationship between temperature increases over time and the seasonality and rate of incidents. Window falls are a substantial cause of injury among children, with an annual trauma incidence rate among children age 0-8 of 10.4 per 100 000 boys and 5.9 per 100 000 girls in our sample. We find a mildly positive trend in the crude rate of window falls over the study period, increasing further when a population-level adjustment for AC prevalence is included. We do not find evidence of earlier injury timing during the calendar year.

Survey of US wastewater for carbapenem-resistant Enterobacteriaceae.

A survey for antibiotic-resistant (AR) Escherichia coli in wastewater was undertaken by collecting samples from primary clarifiers and secondary effluents from seven geographically dispersed US wastewater treatment plants (WWTPs). Samples were collected at each WWTP in cool and summer months and cultured using selective media. The resulting isolates were characterized for resistance to imipenem, ciprofloxacin, cefotaxime, and ceftazidime, presence of carbapenemase and extended-spectrum beta-lactamase (ESBL) genes, and phylogroups and sequence types (STs). In total, 322 AR E. coli isolates were identified, of which 65 were imipenem-resistant. Of the 65 carbapenem-resistant E. coli (CREC) isolates, 62% were positive for more than one and 31% were positive for two or more of carbapenemase and ESBL genes targeted. The most commonly detected carbapenemase gene was blaVIM (n = 36), followed by blaKPC (n = 2). A widespread dispersal of carbapenem-resistant STs and other clinically significant AR STs observed in the present study suggested the plausible release of these strains into the environment. The occurrence of CREC in wastewater is a potential concern because this matrix may serve as a reservoir for gene exchange and thereby increase the risk of AR bacteria (including CR) being disseminated into the environment and thence back to humans.

Extensive Genetic Commonality among Wildlife, Wastewater, Community, and Nosocomial Isolates of Escherichia coli Sequence Type 131 (H30R1 and H30Rx Subclones) That Carry blaCTX-M-27 or blaCTX-M-15.

Escherichia coli sequence type 131 (ST131) is currently one of the leading causes of multidrug-resistant extraintestinal infections globally. Here, we analyzed the phenotypic and genotypic characteristics of 169 ST131 isolates from various sources (wildlife, wastewater, companion animals, community, and hospitals) to determine whether wildlife and the environment share similar strains with humans, supporting transmission of ST131 between different ecological niches. Susceptibility to 32 antimicrobials was tested by disc diffusion and broth microdilution. Antibiotic resistance genes, integrons, plasmid replicons, 52 virulence genes, and fimH-based subtypes were detected by PCR and DNA sequencing. Genomic relatedness was determined by pulsed-field gel electrophoresis (PFGE). The genetic context and plasmid versus chromosomal location of extended-spectrum beta-lactamase and AmpC beta-lactamase genes was determined by PCR and probe hybridization, respectively. The 169 ST131 study isolates segregated predominantly into blaCTX-M-15H30Rx (60%) and blaCTX-M-27H30R1 (25%) subclones. Within each subclone, isolates from different source groups were categorized into distinct PFGE clusters; genotypic characteristics were fairly well conserved within each major PFGE cluster. Irrespective of source, the blaCTX-M-15H30Rx isolates typically exhibited virotype A (89%), an F2:A1:B- replicon (84%), and a 1.7-kb class 1 integron (92%) and had diverse structures upstream of the blaCTX-M region. In contrast, the blaCTX-M-27H30R1 isolates typically exhibited virotype C (86%), an F1:A2:B20 replicon (76%), and a conserved IS26-ΔISEcp1-blaCTX-M-like structure. Despite considerable overall genetic diversity, our data demonstrate significant commonality between E. coli ST131 isolates from diverse environments, supporting transmission between different sources, including humans, environment, and wildlife.

Clinical and Molecular Correlates of Escherichia coli Bloodstream Infection from Two Geographically Diverse Centers in Rochester, Minnesota, and Singapore.

Escherichia coli bacteremia is caused mainly by sequence type complex 131 (STc131) and two clades within its fluoroquinolone-resistance-associated H30 subclone, H30R1 and H30Rx. We examined clinical and molecular correlates of E. coli bacteremia in two geographically distinct centers. We retrospectively studied 251 unique E. coli bloodstream isolates from 246 patients (48 from the Mayo Clinic, Rochester, MN [MN], and 198 from Tan Tock Seng Hospital, Singapore [SG]), from October 2013 through March 2014. Isolates underwent PCR for phylogroup, STc, blaCTX-M type, and virulence gene profiles, and medical records were reviewed. Although STc131 accounted for 25 to 27% of all E. coli bacteremia isolates at each site, its extended-spectrum-ß-lactamase (ESBL)-associated H30Rx clade was more prominent in SG than in MN (15% versus 4%; P = 0.04). In SG only, patients with STc131 (versus other E. coli STc isolates) were more likely to receive inactive initial antibiotics (odds ratio, 2.8; P = 0.005); this was true specifically for patients with H30Rx (odds ratio, 7.0; P = 0.005). H30Rx comprised 16% of community-onset bacteremia episodes in SG but none in MN. In SG, virulence scores were higher for H30Rx than for H30R1, non-H30 STc131, and non-STc131 isolates (P < 0.02 for all comparisons). At neither site did mortality differ by clonal status. The ESBL-associated H30Rx clade was more prevalent and more often of community onset in SG, where it predicted inactive empirical treatment. The clonal distribution varies geographically and has potentially important clinical implications. Rapid susceptibility testing and clonal diagnostics for H30/H30Rx might facilitate earlier prescribing of active therapy.

Genomic Analysis of Multidrug-Resistant Escherichia coli from North Carolina Community Hospitals: Ongoing Circulation of CTX-M-Producing ST131-H30Rx and ST131-H30R1 Strains.

Escherichia coli sequence type 131 (ST131) predominates globally among multidrug-resistant (MDR) E. coli strains. We used whole-genome sequencing (WGS) to investigate 63 MDR E. coli isolates from 7 North Carolina community hospitals (2010 to 2015). Of these, 39 (62%) represented ST131, including 37 (95%) from the ST131-H30R subclone: 10 (27%) from its H30R1 subset and 27 (69%) from its H30Rx subset. ST131 core genomes differed by a median of 15 (range, 0 to 490) single-nucleotide variants (SNVs) overall versus only 7 within H30R1 (range, 3 to 12 SNVs) and 11 within H30Rx (range, 0 to 21). The four isolates with identical core genomes were all H30Rx. Epidemiological and clinical characteristics did not vary significantly by strain type, but many patients with MDR E. coli or H30Rx infection were critically ill and had poor outcomes. H30Rx isolates characteristically exhibited fluoroquinolone resistance and CTX-M-15 production, had a high prevalence of trimethoprim-sulfamethoxazole resistance (89%), sul1 (89%), and dfrA17 (85%), and were enriched for specific virulence traits, and all qualified as extraintestinal pathogenic E. coli The high overall prevalence of CTX-M-15 appeared to be possibly attributable to its association with the ST131-H30Rx subclone and IncF[F2:A1:B-] plasmids. Some phylogenetically clustered non-ST131 MDR E. coli isolates also had distinctive serotypes/fimH types, fluoroquinolone mutations, CTX-M variants, and IncF types. Thus, WGS analysis of our community hospital source MDR E. coli isolates suggested ongoing circulation and differentiation of E. coli ST131 subclones, with clonal segregation of CTX-M variants, other resistance genes, Inc-type plasmids, and virulence genes.

Rapid and Specific Detection of the Escherichia coli Sequence Type 648 Complex within Phylogroup F.

The Escherichia coli sequence type 648 complex (STc648) is an emerging lineage within phylogroup F-formerly included within phylogroup D-that is associated with multidrug resistance. Here, we designed and validated a novel multiplex PCR-based assay for STc648 that took advantage of (i) four distinctive single-nucleotide polymorphisms in icd allele 96 and gyrB allele 87, two of the multilocus sequence typing alleles that define ST648; and (ii) the typical absence within STc648 of uidA, an E. coli-specific gene encoding ß-glucuronidase. Within a diverse 212-strain validation set that included 109 STs other than STc648, from phylogroups A, B1, B2, C, D, E, and F, the assay exhibited 100% sensitivity (95% confidence interval [CI], 82% to 100%) and specificity (95% CI, 98% to 100%). It functioned similarly well in two distant laboratories that used boiled lysates or DNAzol-purified DNA as the template DNA. Thus, this novel multiplex PCR-based assay should enable any laboratory equipped for diagnostic PCR to rapidly, accurately, and economically screen E. coli isolates for membership in STc648.

Phylogenetic Backgrounds and Virulence-Associated Traits of Escherichia coli Isolates from Surface Waters and Diverse Animals in Minnesota and Wisconsin.

Possible external reservoirs for extraintestinal pathogenic Escherichia coli (ExPEC) strains that cause infections in humans are poorly defined. Because of the tremendous human health importance of ExPEC infections, we assessed surface waters and domesticated and wild animals in Minnesota and Wisconsin as potential reservoirs of ExPEC of human health relevance. We characterized 595 E. coli isolates (obtained from 1999 to 2002; 280 from seven surface water sites, 315 from feces of 13 wild and domesticated animal species) for phylogroup and virulence genotype, including inferred ExPEC status, by using multiplex PCR-based methods. We also compared the pulsed-field gel electrophoresis (PFGE) profiles of the isolates with a large private PFGE profile library. We found a predominance of non-ExPEC strains (95% and 93% among water and animal isolates, respectively), which were mainly from phylogroups A and B1, plus a minority of ExPEC strains (5% and 7% among water isolates and animal isolates, respectively), predominantly from phylogroup B2. The ExPEC strains, although significantly associated with cats, dogs, and turkeys, occurred in several additional animal species (goat, horse, chicken, pig) and were distributed broadly across all surface water sites. Virulence gene content among the animal source ExPEC isolates segregated significantly in relation to host species, following established patterns. PFGE analysis indicated that 11 study isolates closely matched (94% to 100% profile similarity) reference human clinical and fecal isolates. These findings imply what probably is a low but non-zero risk to humans from environmental and animal source E. coli isolates, especially those from specific human-associated animal species.IMPORTANCE Our detection of potentially pathogenic strains that may pose a health threat to humans among E. coli isolates from surface waters and wild and domesticated animals suggests a need for heightened attention to these reservoirs as possible sources for human acquisition of disease-causing E. coli Although cats, dogs, and turkeys were especially high-prevalence sources, the presence of such strains in other animal species and at all sampled water sites suggests that this potential risk may be widespread.