Specific bla_CTX-M-8/IncI1 Plasmid Transfer among Genetically Diverse Escherichia coli Isolates between Humans and Chickens

ABSTRACT

We investigated the genetic backbones of 14 bla_CTX-M-8-positive Escherichia coli isolates recovered from human stool samples and chicken meat. All isolates carried IncI1 plasmids with bla_CTX-M-8 (bla_CTX-M-8/IncI1), and most (9/14) belonged to a specific genetic lineage, namely, plasmid sequence type 113 (pST113). The genetic contexts of the nine bla_CTX-M-8/IncI1 pST113 plasmids were similar, regardless of the source. These results suggest the probable local transfer of bla_CTX-M-8/IncI1 between humans and chickens with genetically diverse E. coli.

TEXT

Escherichia coli isolates harboring CTX-M-type extended-spectrum β-lactamase (ESBL) genes have become a global concern because they are widely disseminated in clinical settings, livestock, healthy humans, companion animals, and wild animals (1). CTX-M-type ESBL-producing E. coli isolates in livestock/retail meat need special attention because food contamination could be a major cause of their transfer to humans (2, 3). To assess local transmission of the CTX-M-type ESBL gene between livestock/retail meat and humans in Japan, we analyzed the genetic backbones of CTX-M-8-producing E. coli, since its spread is still expected to be limited, at least in Japan (4–6). As described below, our findings suggest the possible horizontal transfer of plasmids of specific genetic lineages bearing the CTX-M-8 β-lactamase gene between humans and chicken meat.

We collected CTX-M-type ESBL-producing E. coli from several sources, such as ill patients, healthy people handling food (including employees of retail meat shops and meat producers), and retail foods (chicken meat, beef, and pork) (4, 7, 8). The isolates from ill patients were collected from hospitals spread across Japan, while those from healthy people handling food and retail foods were collected in Aichi Prefecture, Japan (4, 7, 8). Only 14 CTX-M-8-producing E. coli isolates were identified. Six were from stool samples from healthy food handlers, and eight were from imported chicken meat from Brazil (Table 1). All isolates were resistant to cefotaxime but susceptible to ceftazidime, imipenem, gentamicin, and fosfomycin (Table 1). We performed whole-genome sequencing (WGS) analysis of 14 CTX-M-8-producing E. coli isolates with the MiSeq platform and an A5-miseq assembler to investigate their genetic backbones (9). Multilocus sequence typing (MLST) was performed by transferring the WGS data to the MLST 1.8 server (10), and the presence of antibiotic resistance genes was confirmed by transferring the WGS data to the ResFinder 2.1 server (11). MLST showed highly diverse backbones; 14 isolates were classified into 12 different sequence types (STs), although strain ST131 was found in both human stool samples and chicken meat and ST1144 was found in stool samples from two different people (Table 1). The susceptibility-testing results and carriage of antibiotic resistance genes were quite consistent (Table 1). The replicon types of plasmids carried by 14 CTX-M-8 producers were confirmed by transferring the WGS data to the PlasmidFinder 1.3 server (12). The numbers of plasmids carried by CTX-M-8 producers were estimated by S1 nuclease pulsed-field gel electrophoresis (PFGE) analysis (13) and simple agarose gel electrophoresis of plasmids extracted with the Plasmid Miniprep System (Promega) (Table 1). All 14 CTX-M-8 producers had IncI1 plasmids, as well as several plasmids with different incompatibility groups (Table 1).

TABLE 1.

Characteristics of 14 E. coli isolates carrying bla_CTX-M-8^a

Sample	Source	ST	CTX resistance transfer by conjugation	Plasmid replicon type	No. of plasmids	MIC (μg/ml)								Antimicrobial resistance gene(s)
						CTX	CAZ	IPM	GM	TC	CP	FOM	CIP
HU23	Human stool sample	131	Yes	IncI1, IncFIB, IncFIC (FII)	3	8	0.5	0.25	0.5	0.5	4	0.5	0.25	blaCTX-M-8
HU447	Human stool sample	1144	Yes	IncI1, Col156, IncFIC (FII), IncFIB, IncQ1	5	4	2	0.06	1	64	4	0.5	≤0.03	blaCTX-M-8, blaTEM-1B, strA/B, sul2, tetA, dfrA8
HU476	Human stool sample	1144	Yes	IncI1, Col156, IncFIC (FII), IncFIB, IncQ1	5	8	0.5	0.12	0.5	64	8	0.5	≤0.03	blaCTX-M-8, blaTEM-1B, strA/B, sul2, tetA, dfrA8
HU485	Human stool sample	23	Yes	IncI1, ColpVC, IncFIB	3	8	0.5	0.25	0.5	1	4	1	≤0.03	blaCTX-M-8
HU493	Human stool sample	1170	Yes	IncI1, IncFIB, IncFIA, IncFIC (FII)	3	8	1	0.25	1	1	4	1	0.12	blaCTX-M-8
HU590	Human stool sample	2526	Yes	IncI1, IncFIB (K)	2	8	1	0.25	0.5	64	8	1	0.25	blaCTX-M-8, aadA5, sul2, tetA, dfrA17
CH11	Chicken meat	345	Yes	IncI1, IncFII, IncFIB, IncQ1, IncN	5	8	0.5	0.12	0.5	64	4	0.5	0.25	blaCTX-M-8, blaTEM-1B, aadA1, strA/B, sul1, sul2, tetA, dfrA1
CH41	Chicken meat	351	Yes	IncI1, IncQ1, IncFII, IncFIB	3	8	1	0.25	0.5	64	4	0.5	0.25	blaCTX-M-8, aadA1, strA/B, sul1, sul2, tetA, dfrA1
CH42	Chicken meat	88	Yes	IncI1, IncX1, IncFIC (FII), IncFIB	4	8	1	0.25	0.5	128	8	1	0.25	blaCTX-M-8, blaTEM-1B, tetA
CH49b	Chicken meat	224	Yes	IncI1, IncX4, ColpVC, IncFII, IncY, IncX1, IncFIB	7	8	1	0.25	1	128	64	1	32	blaCTX-M-8, aadA1, aadA2, aph(3′)-Ia, cmlA1, sul3, tetA, dfrA12
CH56	Chicken meat	131	No	IncI1, IncFIB, IncFIC(FII), IncX1	4	8	0.5	0.25	0.5	0.5	4	0.5	0.25	blaCTX-M-8, blaTEM-1B
CH110	Chicken meat	4576	Yes	IncI1, IncFII, IncQ1, IncFIB	4	8	0.5	0.25	0.5	32	4	1	≤0.03	blaCTX-M-8, blaTEM-1B, aadA1, strA/B, sul1, sul2, tetA, dfrA1
CH365	Chicken meat	602	Yes	IncI1, Col156, IncFIB, IncFIC (FII)	4	8	1	0.25	0.5	0.5	4	1	≤0.03	blaCTX-M-8
CH407	Chicken meat	101	Yes	IncI1, IncFIB, IncFIC(FII)	2	8	0.5	0.12	0.5	1	8	1	0.25	blaCTX-M-8

^aAbbreviations: CTX, cefotaxime; CAZ, ceftazidime; IPM, imipenem; GM, gentamicin; TC, tetracycline; CP, chloramphenicol; FOM, fosfomycin; CIP, ciprofloxacin.

^bStrain CH49 has amino acid substitutions S83L and D87N in GyrA and S80I in ParC, which confer ciprofloxacin resistance.

A broth-mating conjugation experiment was performed to transfer the cefotaxime resistance phenotype of 14 CTX-M-8 producers to E. coli J53 (azide resistant), and 13 conjugants were selected on Luria-Bertani (LB) agar plates containing sodium azide (150 μg/ml) and cefotaxime (1 μg/ml) (Table 1). Further, plasmids were extracted from 14 CTX-M-8 producers and introduced into the E. coli DH10B strain by electroporation. Fourteen cefotaxime-resistant E. coli DH10B transformants were selected on LB agar plates containing cefotaxime (1 μg/ml) (Table 2). As expected, bla_CTX-M-8 was detected in these cefotaxime-resistant conjugants and transformants. The plasmids were extracted from 14 E. coli DH10B transformants with the Qiagen Plasmid Midi kit and subjected to PFGE. DNA bands corresponding to the plasmids were extracted and used as a DNA template for WGS analysis as described above. The assembled contigs derived from the plasmids were transferred to the PlasmidFinder 1.3 and ResFinder 2.1 servers to investigate the replicon types and presence of antibiotic resistance genes, respectively (11), and plasmid MLST was performed through the pMLST 1.4 server (12). Although the sizes of the 14 plasmids, which were estimated by S1 nuclease PFGE analysis of 14 cefotaxime-resistant E. coli DH10B transformants, varied from 82 to 105 kbp, these plasmids were assigned to the IncI1 group and carried bla_CTX-M-8 as the only antibiotic resistance gene (Table 2). The 14 IncI1 plasmids were assigned to five plasmid STs (pSTs); 9 were pST113, 1 was pST114, 2 were pST131, 1 was pST132, and 1 was pST235 (newly assigned) (Table 2). IncI1 pST113 plasmids were dominant in E. coli isolates from humans (3/6) and retail chicken meat (6/8) (Table 2). These results indicated the possibility that the bla_CTX-M-8 spread in E. coli in Japan was mainly due to the horizontal transfer of IncI1 plasmids belonging to a specific genetic lineage, such as pST113, regardless of their sources, rather than due to the distribution of a clonal E. coli strain producing CTX-M-8. To date, CTX-M-8-producing E. coli isolates have been found in Germany (14), French Guiana (15), Tunisia (16), Kenya (17), Spain (18), and Brazil (19–21), and IncI1 pST113 plasmids harboring bla_CTX-M-8 have been reported (22, 23). Preferential carriage of bla_CTX-M-8/IncI1 pST113 plasmids has also been reported in CTX-M-8-producing Enterobacteriaceae isolates, including E. coli and Salmonella spp. in Germany, whose carriage may be related to contaminated food (14). The carriage of IncI1 plasmids pST114, pST131, and pST132 is lower than that of IncI1 pST113 in this study, and these plasmids were found in CTX-M-8 producers from both humans and poultry in Brazil (22, 23). Worldwide dissemination of the bla_CTX-M-8 gene might also be mediated by specific IncI1 plasmids such as pST113 and less-well-known plasmids pST114, pST131, and pST132.

TABLE 2.

Characteristics of 14 E. coli transformants carrying IncI1 plasmids with bla_CTX-M-8

Transformant	Plasmid replicon type	Plasmid ST	Approximate plasmid size estimated by S1 PFGE	MIC (μg/ml) of cefotaxime	β-Lactamase gene
E. coli(pHU23)	IncI1	pST113	91,831 bpa	8	blaCTX-M-8
E. coli(pHU447)	IncI1	pST131	90 kb	4	blaCTX-M-8
E. coli(pHU476)	IncI1	pST131	91 kb	8	blaCTX-M-8
E. coli(pHU485)	IncI1	pST114	82 kb	8	blaCTX-M-8
E. coli(pHU493)	IncI1	pST113	94 kb	8	blaCTX-M-8
E. coli(pHU590)	IncI1	pST113	88 kb	8	blaCTX-M-8
E. coli(pCH11)	IncI1	pST113	101,377 bpa	8	blaCTX-M-8
E. coli(pCH41)	IncI1	pST113	87 kb	8	blaCTX-M-8
E. coli(pCH42)	IncI1	pST113	86 kb	8	blaCTX-M-8
E. coli(pCH49)	IncI1	pST113	84 kb	8	blaCTX-M-8
E. coli(pCH56)	IncI1	pST113	87 kb	8	blaCTX-M-8
E. coli(pCH110)	IncI1	pST235	92 kb	8	blaCTX-M-8
E. coli(pCH365)	IncI1	pST132	105 kb	8	blaCTX-M-8
E. coli(pCH407)	IncI1	pST113	83 kb	8	blaCTX-M-8
E. coli DH10B				0.06

^aPlasmid size was determined by WGS analysis, gap-closing PCR, and subsequent Sanger sequencing.

To further evaluate the genetic backbones of bla_CTX-M-8/IncI1 plasmids from humans and chicken meat, we determined the complete nucleotide sequences of representative bla_CTX-M-8/IncI1 pST113 plasmids, pHU23 from humans, and pCH11 from chicken meat by gap-closing PCR and Sanger sequencing based on the draft sequences of these plasmids. The plasmid sequences were submitted to the Microbial Genome Annotation Pipeline (http://www.migap.org) for annotations. Figure 1A was prepared on the basis of the complete sequences of pHU23 and pCH11 with Easyfig (24). The backbones of the plasmids, including the protein-coding genes traA to traY responsible for plasmid transfer and the protein-coding genes pilI to pilV responsible for pilus formation, were identical, and both had no antibiotic resistance gene, except for bla_CTX-M-8 (Fig. 1A). The nucleotide sequence of the pHU23 plasmid showed 97% query coverage and >99% nucleotide identity to that of pCH11. Both plasmids were slightly different in terms of the presence or absence of several putative transposase and integrase genes and hypothetical protein genes (Fig. 1A). Comparison of 14 IncI1/bla_CTX-M-8 plasmids (pHU23 and pCH11 with complete sequences and 12 plasmids with draft sequences) was performed on the basis of the complete sequence of the pHU23 plasmid with BRIG software (25), and nine bla_CTX-M-8/IncI1 pST113 plasmids showed high similarity, regardless of the source (Fig. 1B).

FIG 1.

(A) Genetic comparison of the pHU23 plasmid (GenBank accession no. AP017892) with the pCH11 plasmid (GenBank accession no. AP017893). The open reading frames are represented by arrows and color coded according to their functions. bla_CTX-M-8 is red. Insertion sequences ΔIS10 and IS26 are yellow and blue, respectively. The tra region is green. Yellow shading indicates regions with high genetic identity. IR_L, left inverted repeat; IR_R, right inverted repeat. (B) Comparison of 14 bla_CTX-M-8/IncI1plasmids by using BRIG software. The comparison was performed on the basis of the pHU23 plasmid (91,831 bp), whose nucleotide sequences were completely determined. Color coding is based on pST types as follows: green, pST113; yellow, pST131; orange, pST114; blue, pST235; cyan, pST132. Plasmids marked with the symbol # were completely sequenced, while those with draft sequences are not marked.

In addition, a common IS10 element with a partially truncated 3′ end was upstream of the bla_CTX-M-8 gene, although its location slightly differed between pHU23 and pCH11 (Fig. 1A). The bla_CTX-M-8 gene was flanked by two IS26 elements. Although the WGS analyses of the remaining 12 IncI1 plasmids could not determine the extended genetic region around bla_CTX-M-8, considering the corresponding regions of these plasmids, the genetic region around bla_CTX-M-8 of pCH56 was identical to that of pHU23 with 916-bp ΔΙS10, while those around bla_CTX-M-8 of pHU447, pHU476, pHU485, pHU493, pHU590, pCH41, pCH42, pCH49, pCH110, and pCH407 were identical to that of pCH11 with 929-bp ΔΙS10 (Fig. 1A). The genetic context around bla_CTX-M-8 in the pCH365 plasmid could not be categorized because the terminal end of the contigs carrying bla_CTX-M-8 neighbored the middle of the IS10 element. However, the assembled 2,158-bp sequence of the contigs was the same as that of pHU23 and pCH11. Therefore, the DNA sequence around bla_CTX-M-8 showed low diversity among the 14 IncI1 plasmids analyzed, as well as low overall diversity (Fig. 1B), indicating that the E. coli isolates from healthy individuals and retail chicken meat had bla_CTX-M-8/IncI1 plasmids with almost the same sequences. These results can potentially explain the possible horizontal transfer of bla_CTX-M-8/IncI1 plasmids with specific genetic lineages between humans and retail chicken meat.

In conclusion, this study is the first to identify and evaluate the genetic relatedness of CTX-M-8-producing E. coli derived from different origins (i.e., humans and retail chicken meat), and we revealed the possible horizontal transfer of bla_CTX-M-8/IncI1 plasmids with a specific genetic lineage, such as pST113. In Japan, CTX-M-8-producing E. coli has been mainly found in retail chicken meat imported from Brazil (4, 26) but has rarely been found in other sources such as patients in clinical settings and livestock (5, 6, 8). Our findings suggest that carriage of CTX-M-8-producing E. coli in humans might be attributed to the horizontal transfer of bla_CTX-M-8/IncI1 harbored by genetically diverse E. coli lineages through imported chicken meat. The food handlers analyzed in this study might have acquired CTX-M-8-producing E. coli and/or its bla_CTX-M-8/IncI1 plasmids by handling chicken meat. The carriage of antibiotic resistance genes by E. coli in retail meat should be regularly and carefully monitored to prevent their further dissemination to humans.

Accession number(s).

The complete nucleotide sequences of pHU23 from healthy humans and pCH11 from chicken meat were deposited in the DDBJ database under accession numbers AP017892 and AP017893, respectively.