Characterization of Third-Generation-Cephalosporin-Resistant Shiga Toxin-Producing Strains of Escherichia coli O157:H7 in Japan

Abstract

We isolated Shiga toxin-producing Escherichia coli O157:H7 strains resistant to third-generation cephalosporins. The resistant strains harbored bla_CMY-2, a plasmid-mediated AmpC β-lactamase. Genotyping of isolates revealed the possible spread of this problematic bacterium. Results suggested the importance of the investigation and surveillance of enterobacteria with plasmids harboring bla_CMY-2.

TEXT

Shiga toxin-producing Escherichia coli (STEC) is a recently emerged pathogen of importance. It causes fatal infections, such as hemolytic-uremic syndrome (HUS) and hemorrhagic colitis. Among STEC serotypes, O157:H7 is the most important (1, 2).

β-Lactam resistance among Enterobacteriaceae has emerged worldwide. Broad-spectrum β-lactamases, such as extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases, are found in many species of Enterobacteriaceae, including E. coli, Klebsiella pneumonia, Salmonella enterica, and Shigella spp. (3, 4).

STEC infections caused by cephalosporin-resistant isolates have been reported in Europe and Japan. For example, a large outbreak of STEC O104:H4 harboring bla_CTX-M-15 occurred in Germany in 2011 (5). In addition, STEC O26:H11 with plasmid-encoded bla_CTX-M-18 was isolated from a human infection in Japan (6). However, only few reports have described the detection of cephalosporin resistance among STEC O157 strains. In the United States, STEC O157 with bla_CMY has been detected in human isolates (7), and cephalosporin-resistant STEC O157 strains were isolated from bovine feces in Japan, although the resistance genes were not identified (8).

Between 1996 and 2011, we collected 2,167 STEC O157:H7 and O157:HNM strains associated with infections in Osaka Prefecture, Japan, and examined their drug susceptibility using the disk diffusion method (9). Among these, seven isolates exhibited β-lactam resistance, including third-generation cephalosporins, but were susceptible to meropenem. The isolates were isolated from five independent diarrhea patients and two asymptomatic family members of a patient between 2006 and 2007 (Table 1). The common source of infection in each case and the epidemiological link between the five symptomatic patients was not detected. All strains were identified as O157:H7 with antiserum, and their production of Stx1 and Stx2 was confirmed using a verotoxin E. coli reversed passive latex agglutination (VTEC-RPLA) assay (Denka Seiken, Tokyo, Japan). The results of the biochemical characterization indicated the typical phenotype of sorbitol-negative and β-glucuronidase-negative STEC O157.

TABLE 1.

MICs of β-lactams and plasmid genotypes of third-generation-cephalosporin-resistant strains of E. coli O157:H7 and transconjugants

Strain	Source	Year	MIC (μg/ml) for:						β-Lactamase	CMY-2-positive plasmid size (kbp)	Integron	Replicon type(s)	Plasmid MLST (IncI1)
			AMP	FOX	CTX	CAZ	ATM	IPM					repI1	ardA	trbA	sogS	pilL	ST
C600			8	8	≤0.5	≤0.5	≤1	≤0.5	none	none	none	none	NDa
18H093	Patient	2006	>32	>32	32	>32	32	≤0.5	CMY-2	ND	intI1b	IncI1, Frep,FIB	ND
C600/18H093c			>32	>32	16	32	8	≤0.5	CMY-2	110	intI1	IncI1	1	5	15	11	3	NAd
19H131	Patient	2007	>32	>32	16	16	16	≤0.5	CMY-2	ND	none	IncI1, Frep,FIB	ND
C600/19H131c			>32	32	8	16	4	≤0.5	CMY-2	95	none	IncI1	4	5	15	11	3	55
19H180	Patient	2007	>32	>32	16	32	8	≤0.5	CMY-2	ND	none	IncI1, Frep,FIB	ND
									TEM-1
C600/19H180c			>32	32	8	16	4	≤0.5	CMY-2	95	none	IncI1	4	5	15	11	3	55
19H187	Carrier	2007	>32	>32	8	16	8	≤0.5	CMY-2	ND	none	IncI1, Frep,FIB	ND
									TEM-1
C600/19H187c			>32	>32	8	16	4	≤0.5	CMY-2	95	none	IncI1	4	5	15	11	3	55
19H188	Carrier	2007	>32	>32	8	16	8	≤0.5	CMY-2	ND	none	IncI1, Frep,FIB	ND
									TEM-1
C600/19H188c			>32	32	8	16	4	≤0.5	CMY-2	95	none	IncI1	4	5	15	11	3	55
19H252	Patient	2007	>32	>32	16	32	8	≤0.5	CMY-2	ND	none	IncI1, Frep,FIB	ND
C600/19H252c			>32	32	8	16	4	≤0.5	CMY-2	95	none	IncI1	4	5	15	11	3	55
19H311	Patient	2007	>32	>32	16	32	8	≤0.5	CMY-2	ND	none	IncI1, Frep,FIB	ND
C600 19H311c			>32	32	8	16	4	≤0.5	CMY-2	95	none	IncI1	4	5	15	11	3	55

^aND, not determined.

^bintI1, class 1 integron.

^cTransconjugant (recipient/donor).

^dNot applicable in the Plasmid MLST database (http://pubmlst.org/plasmid/).

The susceptibility of the isolates to ampicillin (AMP), cefoxitin (FOX), cefotaxime (CTX), ceftazidime (CAZ), aztreonam (ATM), imipenem (IPM), gentamicin (GEN), amikacin (AMK), minocycline (MIN), nalidixic acid (NAL), ciprofloxacin (CIP), trimethoprim-sulfamethoxazole (SXT), and fosfomycin (FOF) was determined by the broth microdilution method using dry plate Eiken (Eiken Chemical Co., Ltd., Tokyo, Japan), and the results were interpreted as described by the Clinical and Laboratory Standards Institute (9). ESBL and AmpC β-lactamase production were determined by the ESBL confirmatory test (9) and the double-disk synergy test with 3-aminophenylboronic acid (boronic acid test [10]), respectively. As shown in Table 1, the MICs of β-lactams, including penicillins, cephalosporins, and ATM, were high, with the exception of IPM. In addition, these isolates were susceptible to meropenem and gave negative results in the ESBL confirmatory test but positive results in the boronic acid test. These results suggested that the β-lactam-resistant strains produced AmpC β-lactamase but not ESBL or carbapenemase. In contrast, the strains were susceptible to aminoglycosides, FOF, MIN, NAL, CIP, and SXT (data not shown).

Genes belonging to the CIT (bla_CMY-2-related genes) group were detected in seven STEC O157:H7 strains by multiplex PCR screening for plasmid-mediated AmpC β-lactamase genes, as described previously (11). The entire coding regions of CIT-like β-lactamase genes harbored in the isolates were amplified with primers CMY21-120F (5′-GGCCCGGACACCTTTTTG-3′) and CMY21-1324R (5′-CCTGGGCCTCATCGTCAG-3′) using standard PCR conditions and sequenced with an ABI 3130 genetic analyzer (Life Technologies, Carlsbad, CA). DNA sequences were identical to the bla_CMY-2 gene, a plasmid-mediated AmpC β-lactamase, in the GenBank database (accession no. X91840) as determined using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi [11]). In addition, bla_TEM-1 penicillinase genes were detected and identified in three isolates from the same family members by PCR using the primers TEM-19F (5′-AAAGGGCCTCGTGATACGC-3′) and TEM-1077R (5′-AGTTACCAATGCTTAATCAGTGAGGC-3′) and sequencing.

We performed insertion element (IS)-typing to compare the strains harboring bla_CMY-2 with other cephalosporin-susceptible STEC O157 human isolates. For IS-typing, a multiplex PCR-based typing method for STEC O157 (12) was performed using the IS-printing system (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's instructions. The results for the seven STEC O157:H7 and 506 randomly selected STEC O157 strains from cephalosporin-susceptible STEC O157 human isolates in Osaka between 1996 and 2011 were converted to binary profiles and visualized using the minimum spanning tree algorithm with BioNumerics software version 6.5 (Applied Maths, Kortrijk, Belgium). The results of the minimum spanning tree analysis indicated that the seven strains harboring bla_CMY-2 formed a large cluster (Fig. 1). The IS type of three isolates from the family members was identical. We also confirmed by pulsed-field gel electrophoresis using XbaI and BlnI restriction enzymes (data not shown) that these strains showed the same patterns. In contrast, the other four isolates showed different patterns with double- or triple-locus variations. In addition, strains harboring the same IS type, 19H131, 19H252, or 19H311 (Fig. 1, arrows 2, 6, and 7), but not encoding the bla_CMY-2 gene were detected in STEC O157 strains isolated in Osaka between 1996 and 2011.

FIG 1.

Minimum spanning tree analysis of IS profiles. Circle size indicates the number of isolates. The number of locus mismatches between the IS type profiles was used as the distance. The O157:H7 strains harboring bla_CMY-2 are indicated as 1 (18H093), 2 (19H131), 3 (19H180), 4 (19H187), 5 (19H188), 6 (19H252), and 7 (19H311).

Plasmids of the isolates were extracted using a NucleoBond Xtra Midi kit (Clontech, Heidelberg, Germany) according to the manufacturer's instructions and visualized using agarose gel electrophoresis (Fig. 2A). Plasmids isolated from Salmonella enterica serovar Enteritidis L156 and E. coli NR1 were used as DNA size markers. Each strain contained several plasmids, with approximately 90- to 110-kbp plasmids in common. Then, mating experiments with nalidixic acid-resistant E. coli C600 as the recipient were performed to select β-lactam-resistant conjugants on sorbitol MacConkey agar supplemented with cefotaxime (0.5 μg/ml) and nalidixic acid (25 μg/ml) (13). All conjugants with each isolate acquired resistance to β-lactams, except for IPM, and bla_CMY-2 but not to bla_TEM-1 (Table 1). The detected plasmids were approximately 110 kbp in C600/18H093 (recipient/donor) and 95 kbp in others (Fig. 2B).

FIG 2.

Profiling and hybridization of plasmids extracted from the CMY-2-producing E. coli O157:H7 (A) and transconjugant strains (B, C). (A) Lanes: M1, Salmonella Enteritidis L156; M2, E. coli NR1; 1, 18H093; 2, 19H131; 3, 19H180; 4, 19H187; 5, 19H188; 6, 19H252; 7, 19H311; and 8, E. coli C600. (B) Lanes: 1, C600/18H093; 2, C600/19H131; 3, C600/19H180; 4, C600/19H187; 5, C600/19H188; 6, C600/19H252; 7, C600/19H311; and 8, E. coli C600. (C) The plasmids in the agarose gel were hybridized with the bla_CMY-2 probe after being transferred to a nylon membrane.

To identify plasmids harboring the bla_CMY-2 gene among these transconjugants, a Southern hybridization assay with a probe for the internal bla_CMY-2 fragment was carried out, and a PCR digoxigenin (DIG) probe synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany) was used with primers CMY21-399F (5′-TTGAGCTAGGATCGGTTAGTAAGACG-3′) and CMY21-1039R (5′-CATCTCCCAGCCTAATCCCTG-3′) as recommended by the manufacturer. The results demonstrated that the bla_CMY-2 gene existed in 110-and 95-kbp plasmids in C600/18H093 and other transconjugants, respectively (Fig. 2C).

Plasmid genotypes were examined by the detection of integrons, replicon typing, and IncI1 plasmid multilocus sequence typing (IncI1 pMLST) as previously described (14–16). The PCR assay showed that all the conjugants contained a plasmid with the IncI1 replicon (Table 1). The presence of the class 1 integron gene was confirmed only in C600/18H093. As shown in Table 1, all plasmids, except for that found in C600/18H093, showed identical pMLST profiles and were identified as sequence type 55 (ST55) in the global database (http://pubmlst.org/plasmid/). The pMLST profile of C600/18H093, which only differed from the other transconjugants with respect to repI1, did not match any sequence types.

Here, we identified seven STEC O157:H7 isolates from Japan that produced bla_CMY-2 β-lactamase, a plasmid-mediated AmpC β-lactamase. Due to the lack of epidemiological links among patients and the various IS-typing profiles that were observed in the STEC O157:H7 isolates, and with the exception of the closely related strains isolated from the three family members, the infections were considered to be caused by distinct STEC strains in five independent incidences.

Generally, it is not recommended to use antibiotics for the treatment of STEC infections (1). However, it is still necessary to determine the drug susceptibility of STEC strains because therapeutic or prophylactic administration of antibiotics is required in severe situations, for instance, during the German outbreak of ESBL-producing STEC O104:H4 in 2011 (17).

The genotype of the plasmids carrying the bla_CMY-2 gene indicated that all plasmids contained an IncI1 replicon, and six of the strains isolated in 2007 carried a plasmid with ST55. In Taiwan, several isolates of S. enterica serovar Choleraesuis, S. enterica serovar Typhimurium, S. enterica serovar Agona, and S. Enteritidis with ceftriaxone resistance were isolated from patients between 2007 and 2010 and included eight strains that carried bla_CMY-2-harboring IncI1 plasmids (18). In addition, a S. Typhimurium strain isolated in 2010 was found to harbor an IncI1 plasmid of ST55. Notably, our present results suggest that the emergence of O157:H7 strains resistant to β-lactams, including third-generation cephalosporins, may have been caused by the spread of an IncI1 plasmid of ST55 in animals and/or humans.

The data obtained in the present study and those reported for isolates from Taiwan suggest that IncI1 plasmids have high transmissibility across species barriers. Although the sources are presently unknown, these β-lactam-resistant isolates may have emerged by horizontal transfer of similar plasmids containing bla_CMY-2 between enterobacteria and, therefore, may be a threat for the control of not only O157:H7 but also other pathogenic Enterobacteriaceae. For this reason, further investigation and surveillance of enterobacteria with plasmids harboring the bla_CMY-2 gene are strongly recommended to clarify the transmission dynamics of these enterobacteria and design countermeasures for preventing their further spread.