Increased extended-spectrum β-lactamase–producing E. coli in Japan resulted mainly from a clade containing this gene.

Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extendedspectrum-␤-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n ϭ 12), B (n ϭ 12), and C, including subclades C1-M27 (n ϭ 16), C1-nM27 (n ϭ 20), C2 (n ϭ 17), and other C (n ϭ 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n ϭ 54), B (n ϭ 23), and C (n ϭ 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with bla CTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.

High levels of fecal bacteria are a concern for the aquatic environment, and identifying sources of those bacteria is important for mitigating fecal pollution and preventing waterborne disease. Escherichia coli has been used as an indicator of fecal pollution, however less success has been achieved using this organism for library-independent microbial source tracking. In this study, using next-generation sequencing technology we sequenced the whole genomes of 22 E. coli isolates from known sources (9 from humans, 2 from cows, 6 from pigs, and 5 from chickens) and identified candidate host-specific genomic regions. Specificity testing on the candidate regions was performed using 30 E. coli isolates from each source. Finally, we identified 4 human-, 2 cow-, 3 pig-, and 4 chicken-specific genetic markers useful for source tracking. We also found that a combination of multiplex PCR and dual index sequencing is effective for detecting multiple genetic markers in multiple isolates at one time. This technique was applied to investigating identified genetic markers in 549 E. coli isolates obtained from the Yamato River, Japan. Results indicate that humans constitute a major source of water contamination in the river. However, further work must include isolates obtained from geographically diverse animal hosts to make this method more reliable.

Klebsiella pneumoniae is a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study of K. pneumoniae clinical isolates in hospital patients. Whole-genome sequence (WGS) data reveals a diverse pathogen population, including other species within the K. pneumoniae species complex (18%). Several infections were caused by K. variicola/K. pneumoniae hybrids, one of which shows evidence of nosocomial transmission. A wide range of antimicrobial resistance (AMR) phenotypes are observed, and diverse genetic mechanisms identified (mainly plasmid-borne genes). ESBLs are correlated with presence of other acquired AMR genes (median n = 10). Bacterial genomic features associated with nosocomial onset are ESBLs (OR 2.34, p = 0.015) and rhamnose-positive capsules (OR 3.12, p < 0.001). Virulence plasmid-encoded features (aerobactin, hypermucoidy) are observed at low-prevalence (<3%), mostly in community-onset cases. WGS-confirmed nosocomial transmission is implicated in just 10% of cases, but strongly associated with ESBLs (OR 21, p < 1 × 10−11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate that K. pneumoniae infections in hospitalised patients are due largely to opportunistic infections with diverse strains, with an additional burden from nosocomially-transmitted AMR strains and community-acquired hypervirulent strains.

Contamination of surface waters by antimicrobial-resistant bacteria and pathogenic bacteria is a great concern. In this study, 531 Escherichia coli isolates obtained from the Yamato River in Japan were evaluated phenotypically for resistance to 25 antimicrobials. Seventy-six isolates (14.3%) were multidrug resistant (MDR), 66 (12.4%) were nonsusceptible to one or two classes of agents, and 389 (73.3%) were susceptible. We performed whole-genome sequencing of selected strains by using Illumina technology. In total, the genome sequences of 155 strains were analyzed for antibiotic resistance determinants and phylogenetic characteristics. More than 50 different resistance determinants, including acquired resistance genes and chromosomal resistance mutations, were detected. Among the sequenced MDR strains (n ϭ 66), sequence type 155 (ST155) complex (n ϭ 9), ST10 complex (n ϭ 9), and ST69 complex (n ϭ 7) were prevalent. Among extraintestinal pathogenic E. coli (ExPEC) strains (n ϭ 58), clinically important clonal groups, namely, ST95 complex (n ϭ 18), ST127 complex (n ϭ 8), ST12 complex (n ϭ 6), ST14 complex (n ϭ 6), and ST131 complex (n ϭ 6), were prevalent, demonstrating the clonal distribution of environmental ExPEC strains. Typing of the fimH (type 1 fimbrial adhesin) gene revealed that ST131 complex strains carried fimH22 or fimH41, and no strains belonging to the fimH30 subgroup were detected. Fine-scale phylogenetic analysis and virulence gene content analysis of strains belonging to the ST95 complex (one of the major clonal ExPEC groups causing community-onset infections) revealed no significant differences between environmental and clinical strains. The results indicate contamination of surface waters by E. coli strains belonging to clinically important clonal groups.IMPORTANCE The prevalence of antimicrobial-resistant and pathogenic E. coli strains in surface waters is a concern because surface waters are used as sources for drinking water, irrigation, and recreational purposes. In this study, MDR and ExPEC strains in river water were characterized by genomic sequencing and analysis. We detected more than 50 resistance determinants and identified clonal groups specific to MDR and ExPEC strains. This study showed contamination of surface waters by E. coli strains belonging to clinically important clonal groups. Overall, this study advances our understanding of environmental MDR and ExPEC strains.KEYWORDS antimicrobial resistance, Escherichia coli, ExPEC, whole-genome sequencing E scherichia coli is a commensal member of the gastrointestinal tract of humans and other warm-blooded animals. Consequently, E. coli, as well as Enterococcus spp., has long been used as an indicator of fecal contamination to assess the microbial quality of surface waters. Although most E. coli strains are harmless, some strains are pathogenic

Wastewater is considered a major source of antibiotic-resistant bacteria released into the environment. Here, we characterized carbapenemase-producing (CPE) in wastewater by whole-genome analysis. Wastewater samples ( = 40) were collected from municipal wastewater treatment plants and hospital wastewater in Japan and Taiwan. Samples were screened for CPE using selective media, and the obtained isolates were sequenced using an Illumina MiSeq. The isolates ( = 45) included the following microorganisms: ( = 12), ( = 10), complex ( = 10), ( = 8), ( = 2), ( = 1), ( = 1), and ( = 1). Among the 45 isolates, 38 harbored at least one carbapenemase-encoding gene. Of these, the (, , and) genes were found in 29 isolates. The genes were situated in novel class 1 integrons, but the integron structures were different between the Japanese (In1439 with and In1440 with) and Taiwanese (In1441 with and In1442 with) isolates. Other carbapenemase-encoding genes (, ,, , and) were found in one to three isolates. Notably, class 1 integrons previously reported among clinical isolates obtained in the same regions as the present study, namely, In477 with and In73 with, were found among the Japanese and Taiwanese isolates, respectively. The results indicate that CPE with various carbapenemase-encoding genes in different genetic contexts were present in biologically treated wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Contamination of environmental waters by extended-spectrum-␤-lactamase (ESBL)-producing Escherichia coli (ESBLEC) is of great concern. Wastewater treatment plants (WWTPs) and hospitals release large amounts of ESBLEC into the environment. In the present study, we isolated ESBLEC strains from wastewater collected from a WWTP and a hospital in Japan and performed whole-genome sequencing to characterize these strains. Genomic analysis of 54 strains (32 from the WWTP and 22 from hospital wastewater) revealed the occurrence of clinically important clonal groups with extraintestinal pathogenic E. coli status in the WWTP and hospital wastewater. Fine-scale phylogenetic analysis was performed to further characterize 15 sequence type 131 (ST131) complex strains (11 from the WWTP and 4 from hospital wastewater). These ST131 complex strains were comprised of the following different subgroups: clade A (n ϭ 2), C1-M27 (n ϭ 8), and C1 (non-C1-M27) (n ϭ 1) for strains from the WWTP and clade A (n ϭ 2), C1-M27 (n ϭ 1), and C1 (non-C1-M27) (n ϭ 1) for strains from hospital wastewater. The results indicate that ESBLEC strains belonging to clinically important lineages, including the C1-M27 clade, may disseminate into the environment through wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

The occurrence of pathogenic Escherichia coli in environmental waters increases the risk of waterborne disease. In this study, 14 virulence genes in 669 E. coli isolates (549 isolates from the Yamato River in Japan, and 30 isolates from each of the following hosts: humans, cows, pigs, and chickens) were simultaneously quantified by multiplex PCR and dual index sequencing to determine the prevalence of potentially pathogenic E. coli. Among the 549 environmental isolates, 64 (12%) were classified as extraintestinal pathogenic E. coli (ExPEC) while eight (1.5%) were classified as intestinal pathogenic E. coli (InPEC). Only ExPEC-associated genes were detected in human isolates and pig isolates, and 11 (37%) and five (17%) isolates were classified as ExPEC, respectively. A high proportion (63%) of cow isolates possessed Shiga-toxin genes (stx1 or stx2) and they were classified as Shiga toxin-producing E. coli (STEC) or enterohemorrhagic E. coli (EHEC). Among the chicken isolates, 14 (47%) possessed iutA, which is an ExPEC-associated gene. This method can determine the sequences as well as the presence/absence of virulence genes. By comparing the sequences of virulence genes, we determined that sequences of iutA were different among sources and may be useful for discriminating isolates, although further studies including larger numbers of isolates are needed. Results indicate that humans are a likely source of ExPEC strains in the river.