Characterization of the Plasmidome Encoding Carbapenemase and Mechanisms for Dissemination of Carbapenem-Resistant Enterobacteriaceae

Global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens human health by limiting the efficacy of antibiotics even against common bacterial infections. Carbapenem resistance, mainly due to carbapenemase, is generally encoded on plasmids and is spread across bacterial species by conjugation. Most CRE epidemiological studies have analyzed whole genomes or only contigs of CRE isolates. Here, plasmidome analysis on 230 CRE isolates carrying blaIMP was performed to shed light into the dissemination of a single carbapenemase gene in Osaka, Japan. The predominant dissemination of blaIMP-6 by the pKPI-6 plasmid among genetically distinct isolates was revealed, as well as the emergences of pKPI-6 derivatives that acquired advantages for further disseminations. Underlying vast clonal dissemination of a carbapenemase-encoding plasmid, heteroresistance was found in CRE offspring, which was generated by the transcriptional regulation of blaIMP-6, stabilization of blaIMP-6 through chromosomal integration, or broadened antimicrobial resistance due to a single point mutation in blaIMP-6.

was found in CRE offspring, which was generated by the transcriptional regulation of bla IMP-6 , stabilization of bla IMP-6 through chromosomal integration, or broadened antimicrobial resistance due to a single point mutation in bla IMP-6 . KEYWORDS Enterobacteriaceae, IMP-1, IMP-6, carbapenem resistance, carbapenemase, chromosomal integration, heteroresistance, plasmid analysis, plasmid dynamics, plasmidome T he rapid global dissemination of multidrug-resistant Enterobacteriaceae threatens health care systems worldwide (1). Carbapenem-resistant Enterobacteriaceae (CRE) are of major concern because alternative treatment options are limited (2). Carbapenem resistance is primarily conferred by carbapenemases that hydrolyze carbapenem (3). KPC, NDM, and OXA-48 are the most commonly detected carbapenemases (3). Carbapenemase genes are generally plasmid encoded and are frequently transmitted across species (4). Therefore, genetic tracking of plasmids encoding carbapenemase genes has allowed the monitoring of the spread of CRE isolates. For example, structural similarities among plasmids from isolates obtained in a single hospital outbreak allowed elucidating links between patients carrying the isolates (5)(6)(7), and plasmid data accumulated globally revealed the worldwide spread of an epidemic plasmid carrying bla KPC. (8). However, most regional surveillance studies compared the whole genomes or only contigs of CRE isolates without analyzing the clonality of the spreading carbapenemase-encoding plasmids, and few studies have comprehensively analyzed carbapenemase-encoding plasmids broadly spreading in a certain region (9).
We previously conducted a surveillance study of CRE in 1,507 patients from 43 hospitals in northern Osaka (population, 1,170,000; area, 307 km 2 ), Japan (10), and we reported that 12% of the patients carried CRE and 95% of CRE isolates harbored bla IMP-6 , the predominant carbapenemase in Japan. The predominance of this particular carbapenemase gene might have resulted from vigorous horizontal spreading of a specific plasmid carrying bla IMP-6 in this region. The aim of the present study was to analyze the plasmidome transmitting carbapenemase genes in order to unveil the mechanisms for their regional dissemination.

RESULTS
Dissemination of pKPI-6. All bla IMP -positive CRE isolates of Escherichia coli (n ϭ 135) and Klebsiella pneumoniae (n ϭ 95) were classified into seven groups based on the results of S1-PFGE followed by Southern blotting hybridization with probes for the bla IMP and repA genes encoded on the IncN-type plasmid pKPI-6, sporadically reported as a plasmid carrying bla IMP-6 (11) (Fig. 1). Ninety-nine of the 135 E. coli isolates (73%) and 88 of the 95 K. pneumonia isolates (93%) carried plasmids classified as group pKPI-6 based on plasmid size and replicon type (see Fig. S1 in the supplemental material). Next, we compared the similarity between pKPI-6 and 39 representative plasmids categorized as group pKPI-6 based on whole-genome sequencing (WGS) data using Illumina HiSeq 3000 or Illumina MiSeq (see Fig. S1). The overall sequence identity was 99% Ϯ 0.28%, and the sequence coverage was 98% Ϯ 4.0% (mean Ϯ the standard deviation). The complete sequences of three plasmids were previously confirmed as clonal with pKPI-6 using a combination of PacBio RsII, Illumina HiSeq 3000, and Southern blot methods (12). These analyses confirmed that pKPI-6 was the predominant plasmid responsible for the transmission of bla IMP-6 in the study area (187 of 230 [81.3%] bla IMP -positive CRE isolates).
Barring occasional isolations of organisms coharboring different carbapenemase genes (13,14), few studies have shown the coexistence of two identical carbapenemase genes on different plasmids within an isolate (15). WGS revealed that isolate E119 carried pKPI-6 and an IncF-type plasmid (pEC743_1) that had a bla IMP-6 cassette from pKPI-6 integrated (49) (see Fig. S3B and C). Characterization of IncF plasmids encoding bla IMP-6 . In addition to the K. pneumoniae isolates carrying group non-IncN KP plasmids, E. coli isolates carrying plasmids without IncN replicon were found in a single hospital (hospital D; Fig. 1A). WGS of these isolates revealed that they harbored nearly identical bla IMP-6 -encoding plasmids with an IncFIA-type replicon (categorized as group IncF) ( Fig. 2A; see also Table S1). These plasmids were generated by integration of a cassette carrying bla IMP-6 on pKPI-6 into another IncF plasmid at IS26. This IncF plasmid (pEC302/04; Fig. 2B) has been reported to transmit antimicrobial resistance since 1965 (16).
The MICs of meropenem for the E. coli isolates carrying group IncF plasmids were low compared to those of E. coli isolates harboring other bla IMP-6 -encoding plasmids, such as pKPI-6 (see Fig. S4). Mutations or deletions in the porin (OmpF) gene in E. coli have been reported to enhance resistance to ␤-lactams (17). However, all E. coli isolates carrying group IncF plasmids had a premature termination codon within ompF, whereas the other isolates carried wild-type ompF (Table 1; see also Table S2). MICs of meropenem were low for these group IncF plasmid-carrying isolates, despite them being OmpF deficient. To investigate carbapenem resistance in the same genetic background, plasmids from representative isolates in each bla IMP-6 carriage group were transformed into the E. coli TOP10 strain and MICs for the transformants were determined. Transformant T305 carrying pE305_IMP6 single of group IncF from E. coli isolate E305 was more susceptible to meropenem than transformants carrying bla IMP-6harboring plasmids of groups ( Table 2). The transcription of bla IMP-6 in the pE305_IMP6 single transformant was significantly lower than that in the pKPI-6 transformant (see Fig. S5A), although the plasmid copy numbers in the bacterial cells were comparable (see Fig. S5B). These results indicated that the lower MICs of meropenem in E. coli isolates carrying group IncF plasmids were due to the reduced transcription of bla IMP-6 .
WGS of E305 and E318 revealed the complete sequence of pE318_IMP6; however, it failed to determine the complete sequence of pE305_IMP6. Therefore, to analyze the structure of pE305_IMP6, we used a combination of WGS, Southern blotting, and qPCR analysis. The length and depth of each contig of pE305_IMP6 deduced from WGS are shown in the de novo assembly graphs generated using the Bandage software (19) in Fig. 3A. The total length of pE305_IMP6 deduced from WGS data were ϳ149 kbp.    In addition to showing high similarity to each other, the region containing bla IMP-6 bracketed by a set of IS26 was identical to a part of pKPI-6. Block arrows indicate confirmed or putative open reading frames (ORFs), and their orientations. Arrow size is proportional to the predicted ORF length. The color code is as follows: red, carbapenem resistance gene; yellow, other antimicrobial resistance gene; light blue, conjugative transfer gene; blue, mobile element; and purple, toxin-antitoxin. Putative, hypothetical, or unknown genes are represented as gray arrows. The gray-shaded area indicates regions with high identity between the two sequences. Accession numbers of the plasmids are indicated in brackets. (B) Ancestor of plasmid pE301_IMP6. The backbone of plasmid pE301_IMP6 which is representative of the plasmids in group IncF, corresponded to the structure of plasmid pEC302_04 reported in Malaysia in 2004.
However, according to Southern blotting results, pE318_IMP6 and pE305_IMP6 were ϳ145 and ϳ200 kbp in size, respectively (Fig. 3B). Based on the depth of each contig, the copy number of each contig was predicted as follows: Contig3, 1 copy; Contig2 and Contig5, 6 copies; Contig1 and Contig6, 3 copies; and Contig4, 5 copies (Fig. 3A). Therefore, pE305_IMP6 was predicted to have an ϳ19-kbp repeat region consisting of triplication of Contig1 and Contig6, sextuplication of Contig2 and Contig5, and quintuplication of Contig4 (Fig. 3C). Except for the repeat region, pE305_IMP6 and pE318_IMP6 exhibited high sequence similarity (identity, 99.27%; coverage, 100%) (Fig. 3D). The bla IMP-6 gene was located on Contig6 and was predicted to be triplicated. qPCR analysis corroborated that pE305_IMP6 carried three copies of bla IMP-6 , whereas pE318_IMP6 harbored a single copy (see Fig. S5C). bla IMP-6 transcription was significantly higher in isolate E305 than in isolate E318 (Fig. 3E), even though the bla IMP-6carrier plasmid copy numbers in the cells of these isolates were not significantly different (see Fig. S5D). Triplication of bla IMP-6 in tandem resulted in a higher transcription level in E305 and thus a higher level of resistance to meropenem.
Subculture of the clonal isolate E305 in broth medium revealed a mixture of subpopulations of bacteria carrying a plasmid with multiple bla IMP-6 copies (which represented the majority) and bacteria carrying a plasmid with a single bla IMP-6 copy. In Southern blotting analyses for bla IMP-6 , a faint band at ϳ145 kbp was observed in addition to the major band at ϳ200 kbp (Fig. 3B). It was also found that T305 (a a Groups correspond to those in Fig. 1 transformant of pE305_IMP6 single extracted from E305) carried an ϳ145-kbp plasmid without bla IMP-6 amplification due to recA deficiency in the recipient E. coli TOP10 strain (see Fig. S5E) (20). qPCR analysis confirmed that T305 carried one bla IMP-6 copy on its plasmid (see Fig. S5F). These results indicated the existence of a subpopulation carrying a Groups correspond to those presented in Fig. 1   a plasmid with one bla IMP-6 copy within E. coli isolate E305, whereas the majority of the population carried a plasmid harboring three copies of bla IMP-6 . Comparison of CRE isolates carrying pKPI-6 with those carrying other groups of plasmids harboring bla IMP-6 . bla CTX-M-2 , which is an ESBL gene located distant from bla IMP-6 on pKPI-6, compensated for the narrow range of hydrolysis of ␤-lactams by IMP-6 (11,18). However, these two ␤-lactamase genes were not always transferred together from pKPI-6 to another plasmid. Plasmids categorized as group non-IncN KP and group IncF did not carry ESBL genes (see Table S3) and rarely conferred resistance to penicillins, in contrast to pKPI-6, which confers broad resistance to ␤-lactams (Fig. 1). We next measured the conjugation efficiency of representative plasmids in each group ( Table 2). pKPI-6 plasmids and group IncN plasmids, which had the entire pKPI-6 plasmid incorporated, showed a higher conjugation efficiency than group non-IncN KP/IncF plasmids. These characteristics may have facilitated the vast horizontal dissemination of pKPI-6 in the study area.
Compared with the chromosomal diversity among E. coli isolates bearing pKPI-6, K. pneumoniae isolates carrying pKPI-6 exhibited higher clonality as indicated by pulsedfield gel electrophoresis with XbaI (XbaI-PFGE) analysis (Fig. 1). This may be explained by the presence of the kikA gene on pKPI-6, the product of which reportedly promotes cell death of K. pneumoniae following conjugation (21). The conjugation efficiency of pKPI-6 into K. pneumoniae ATCC 13883 was considerably lower than that into E. coli TUM3456 (3.3 ϫ 10 Ϫ4 and 3.7 ϫ 10 Ϫ1 , respectively). Maybe only "kikA-resistant" K. pneumoniae are able to acquire pKPI-6, leading to clonal similarity among the K. pneumoniae isolates bearing pKPI-6.

DISCUSSION
IMP-producing Enterobacteriaceae have been reported sporadically on a global basis (2). IMP-4-producing Enterobacteriaceae are endemic to Australia (22), and IMP-1, -4, and -8 producers have been occasionally detected in China (23). Our study revealed the exclusive dissemination of IMP-6 producers (95% of CRE isolates) in northern Osaka, Japan, consistent with findings in previous studies (11,24,25). By analyzing the plasmidome transmitting bla IMP , we clarified the relationships between bla IMP -harboring isolates that seemed diverse based on XbaI-PFGE analysis or comparison of short-read WGS results.
The present study revealed predominant dissemination of pKPI-6 in the study area, which may have resulted in the emergence of diverse derivatives. Group IncF plasmids possessed similar genomic structures, consisting of the globally disseminated IncF plasmid and a bla IMP-6 cassette cointegrated on the pKPI-6 genome, without accompaniment of bla CTX-M-2 (Fig. 2). Our analysis revealed that bla IMP-6 transcription was lower from group IncF plasmid (pE305_IMP6 single ) than from pKPI-6 in E. coli cells of the same genetic background (see Fig. S5A). Low carbapenemase gene transcription is considered one of the reasons for reduced resistance to meropenem (26). Therefore, CRE isolates carrying group IncF plasmids might have a reduced fitness cost for the carriage of bla IMP-6 , leading to further environmental dissemination of bla IMP-6 (27).
Unlike for other plasmids in group IncF, the complete sequence of pE305_IMP6 could not be obtained by long-read or short-read sequencing because of a signature 19-kbp repeat sequence unit. Based on combined WGS, Southern blotting, and qPCR data, we proposed a hypothetical structure of pE305_IMP-6 (Fig. 3C). Our results indicated that, despite its clonal origin, CRE isolate E305 comprised two different populations: a major population carrying pE305_IMP-6 with multiple bla IMP-6 copies and a minor population carrying pE305_IMP-6 single with a single bla IMP-6 copy ( Fig. 3B; see also Fig. S5E and F). Moreover, the amplification of bla IMP-6 on the IncF plasmid enhanced the transcription of bla IMP-6 ( Fig. 3E), resulting in increased resistance to meropenem (Table 3). These results are consistent with previous studies reporting higher resistance to carbapenem through amplification of bla OXA-58 (28) and bla NDM-1 (20). All E. coli isolates carrying group IncF plasmids were found to possess ompF with a premature termination codon (see Table S2). When an isolate producing wild-type OmpF carries this plasmid with a single copy of bla IMP-6 , the isolate is difficult to detect due to weaker resistance to meropenem. However, when an isolate with a porin mutation acquires a group IncF plasmid with multiple bla IMP-6 copies, it may abruptly exhibit strong resistance to meropenem without any direct trace of horizontal transfer. These types of plasmids may act as "hidden transmitters" of bla IMP-6 .
Moreover, we demonstrated chromosomal integration of group IncF plasmids in some E. coli isolates. Carbapenemase genes have been reported to be transmitted primarily through plasmid conjugation (4), and chromosomal integration has been reported in a limited number of strains (29). In our study, 3 of 135 E. coli isolates (2.2%) exhibited chromosomal integration of bla IMP-6 , which presumably occurred during the vast horizontal spread of pKPI-6. Compared to bla IMP-6 on plasmids, chromosomal bla IMP-6 was not readily transmissible to another patient. However, these isolates may stably possess bla IMP-6 within a patient and not lose carbapenem resistance through the elimination of plasmids harboring bla IMP-6 .
In the early 1990s, some unique metallo-␤-lactamases were reported in Japan (30, 31), followed by the identification of IMP-1 (32). Since then, these ␤-lactamases have The genomic structure of pE105_IMP1 (group IMP1) was compared to plasmids pKPI-6 and pE013_IMP6 (group pKPI-6) obtained from K. pneumoniae isolate E013. Differences between pE105_IMP1 and pE013_IMP6 are visually extended at the bottom. The color code is the same as that described in the legend of Fig. 2. (B) Schematic chart of homologous recombination. The 713-bp region of plasmid pE013_IMP6 was removed by homologous recombination at the 32-bp region. been frequently identified in Japan (33). The single amino acid variant, IMP-6, was identified in 2001 (18). IMP-1 producers have disseminated mainly in eastern Japan, including Tokyo (24,34), whereas IMP-6 producers have been almost exclusively found in western Japan, including Osaka (7,10,11,25). Consistent with these findings, in the present study only one K. pneumoniae isolate carrying bla IMP-1 , E105, was isolated in hospital A, where CRE carrying pKPI-6 were dominant. The patient carrying CRE isolate E105 was hospitalized for 512 days with other inpatients carrying CRE with pKPI-6, and the isolate showed ϳ83% similarity with a cluster of K. pneumoniae isolates carrying pKPI-6 in the XbaI-PFGE phylogeny (Fig. 1B). In addition, WGS of the plasmids revealed that a 714-bp region bracketed by 32-bp homologous regions was the only difference between pE105_IMP1 and pE013_IMP6 (Fig. 5A). This very small fragment appeared to have been removed by homologous recombination in pE105_IMP1 (Fig. 5B). Our results suggest that bla IMP-6 had disseminated via the transmission of pKPI-6, and spontaneous mutation may have generated the bla IMP-1 -encoding plasmid providing broader antimicrobial resistance, resulting in increased fitness in the clinical setting.
This multi-institutional surveillance study uncovered the clonal dissemination of a plasmid encoding a specific carbapenemase IMP-6 and demonstrated that a seemingly clonal horizontal dissemination of CRE isolates had embraced heterogeneous minor subpopulations, which exhibited broadened antimicrobial resistance, stable carriage of bla IMP-6 through chromosomal integration, or heteroresistance related to covert bla IMP transmission. Such diverse gene adaptations might also be common among CRE isolates carrying other carbapenemase genes. By multifaceted analysis of the plasmidome, this study revealed the vast regional dissemination of a carbapenemase-encoding plasmid, along with the presence of diverse derivatives that would ensure and facilitate the dissemination of carbapenemase genes in various environments, resulting in serious complications in clinical settings.

CRE isolates and PFGE phylogenetic analysis.
We performed a CRE surveillance study of 1,507 patients hospitalized in 43 hospitals located in northern Osaka between December 2015 and January 2016 (10). In the present study, we analyzed 230 CRE isolates carrying bla IMP obtained in the surveillance study, including 135 E. coli isolates and 95 K. pneumoniae isolates. All isolates were subjected to XbaI-digested PFGE for phylogenetic analysis (35). Dendrograms were generated from PFGE patterns by the UPGMA method using BioNumerics software (version 6.6; Applied Maths NV, Sint-Martens-Latem, Belgium).
Classification of bla IMP carriage by PFGE and Southern blotting. The size and replicon type of bla IMP -harboring plasmids were determined by S1-nuclease-digested PFGE followed by Southern hybridization (S1 nuclease was obtained from TaKaRa Bio, Shiga, Japan). S1-PFGE and Southern blot hybridization for the bla IMP-6 and repA genes encoded on the IncN-type plasmid were performed as described in our previous study (12). The sizes of bla IMP -encoding plasmids were determined using BioNumerics software (version 7.5; Applied Maths NV). The modes of bla IMP carriage were classified into seven groups based on the sizes and replicon types of the plasmids carrying bla IMP . The groups and their associated characteristics are as follows: group pKPI-6, a pKPI-6-like bla IMP-6 -encoding plasmid (ϳ50 kbp, encoding repA for IncN plasmid); group IncN, a bla IMP-6 -encoding plasmid (not ϳ50 kbp, encoding repA for IncN plasmid); group non-IncN KP, a bla IMP-6 -encoding plasmid (without repA for IncN plasmid) harbored by K. pneumoniae isolates; group IncF, a bla IMP-6 -encoding plasmid (without repA for IncN plasmid) harbored by E. coli isolates; group double bla IMP-6 , multiple plasmids with bla IMP-6 harbored by a single isolate; group chromosome, chromosomal bla IMP-6 ; group non-typeable, a bla IMP-6 -encoding plasmid of unknown size; group IMP1, a bla IMP-1 -carrier plasmid.
Isolates classified as chromosomal bla IMP carriers were further analyzed to identify the location of bla IMP . In brief, I-CeuI endonuclease-digested PFGE followed by Southern blotting using probes for bla IMP-6 and 16S rRNA genes was performed to confirm the location of the bla IMP gene in three E. coli isolates-E138, E300, and E302-as previously described (29).
Antimicrobial susceptibility testing. Susceptibility to ampicillin, ampicillin/sulbactam, piperacillintazobactam, piperacillin, cefotaxime, cefepime, imipenem, and meropenem was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute document M100-S28 (36). MICs of meropenem were determined using Etest (bioMérieux, Marcy l'Etoile, France), following the manufacturer's instructions. E. coli ATCC 25922 was used as a control strain.
Whole-genome sequencing and genomic analysis. Genomic DNA for long-and short-read sequencing was extracted by using a DNeasy PowerSoil kit (Qiagen, Hilden, Germany). Short-read sequencing was conducted on an Illumina HiSeq 3000 sequencer using the KAPA library preparation kit (Kapa Biosystems, Woburn, MA) or on an Illumina MiSeq sequencer using the KAPA HyperPlus Library Preparation kit (Kapa Biosystems). Long-read sequencing was conducted on a Nanopore GridION sequencer (Oxford Nanopore Technologies, Oxford, UK) using sn SQK-LSK109 1D ligation sequencing kit and sn EXP-NBD103 native barcoding kit. The reads were assembled and polished using Unicycler (37). In cases where the complete plasmid sequences could not be constructed, sequences were assembled with CANU (version 1.8) (38) or flye (39) and improved using Pilon (40) or Racon (41). The PlasmidFinder (42) and ResFinder (43) databases were used to identify antimicrobial resistance genes and plasmid replicon types, respectively. A detailed analysis of the insertion sequence was performed using ISfinder (44). The sequences were annotated with RASTtk (45), and the genomic structures were compared with EasyFig (46). Plasmids similar to those found in this study were identified using BLAST.
Bacterial conjugation assays were performed using the transformants as donors and the sodium azide-resistant E. coli strain TUM3456 (47) as a recipient. After mixing overnight cultures of donors and recipients at a 1:10 volumetric ratio, the mixture (10 l) was incubated on LB agar for 24 h at 37°C. Transconjugants were selected on LB agar containing cefotaxime (2 g/ml) and sodium azide (150 g/ ml). The conjugation frequency was calculated from the CFU as the number of transconjugants divided by the number of donors plus transconjugants.
Determination of the plasmid copy number per host bacterial cell. DNA of E. coli isolates E305 and E318, and E. coli transformants with plasmids pE188_IMP6 and pE305_IMP6 single (T188 and T305, respectively) was extracted using the DNA minikit (Qiagen). Using qPCR, the copy numbers of the repA2 gene on plasmids pE305_IMP6 and pE318_IMP6 and the bla IMP-6 gene on pE188_IMP6 were compared to the copy number of the rrsA gene encoding 16S rRNA on the chromosome. qPCRs were carried out using Thunderbird SYBR qPCR Mix (Toyobo Life Science, Osaka, Japan) on a LightCycler 96 system (Roche Life Science, Penzberg, Germany). Primers used for this assay are list in Table S4 in the supplemental material. qPCR analysis was performed using data from repeated experiments (n ϭ 6), and the plasmid copy number per cell was calculated from cycle threshold (C T ) values using the comparative C T method (48).
Determination of the copy number of bla IMP-6 per plasmid. Plasmids of E. coli isolates E305 and E318 were extracted using a plasmid miniprep kit (Qiagen). Using qPCR, the copy numbers of the bla IMP-6 gene were compared to those of the repA2 gene on plasmids pE305_IMP6 and pE318_IMP6. qPCRs were carried out using Thunderbird SYBR qPCR Mix on a LightCycler 96 System. Primers used for this assay are listed in Table S4. qPCR analysis was performed using data from repeated experiments (n ϭ 5), and the bla IMP-6 copy number per plasmid was calculated from C T values using the comparative C T method.
Transcription of bla IMP-6 . E. coli isolates E305 and E318, and E. coli transformants T188 and T305 were incubated in LB broth until the optical density at 600 nm reached 0.3 to 0.4. The total RNA was extracted using the RNeasy minikit (Qiagen). RNA was treated with ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo Life Science) to remove contaminating DNA and to reverse transcribe the RNA into cDNA. For quality control, DNase-treated RNA that had not been reverse transcribed was subjected to a DNA contamination test by qPCR. The rrsA gene encoding 16S rRNA served as an endogenous control for normalization. qPCRs were carried out using Thunderbird SYBR qPCR Mix on a LightCycler 96 system. Primers used for this assay are listed in Table S4. qPCR analysis was performed using data from repeated experiments (n ϭ 7), and transcript levels were calculated from C T values using the comparative C T method.
Data availability. The WGS data are available from the DDBJ (DNA Data Bank of Japan) database under accession numbers AB616660, AP019402, AP019405, and AP022349 to AP022369. Raw data of isolate E305 are available at NCBI under accession numbers DRX184368 and DRX182679.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.

ACKNOWLEDGMENTS
We thank Isao Nishi and Akiko Ueda, Osaka University Hospital, for assistance with antimicrobial resistance assays, and we thank Yoshikazu Ishii, Toho University Graduate School of Medicine, for providing E. coli TUM3456.