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

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.

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.