Detection of CTX-M-64 in Escherichia coli Isolates from Human Patients in China

LETTER

CTX-M-type extended-spectrum β-lactamases (ESBLs) did not become dominant until the early 2000s, when extreme diversification of these enzymes was observed. The evolution of CTX-M-type ESBLs has been based mainly on amino acid substitutions. However, several chimeras of the CTX-M-1 and CTX-M-9 groups have recently been reported (1–3). CTX-M-64, a chimera of the CTX-M-15-like and CTX-M-14 enzymes, was first identified in Shigella sonnei in Japan (1) and was later detected in Escherichia coli isolates from pets in China (4). In the current study, we report two CTX-M-64-producing E. coli isolates from human patients in China.

Both of the isolates (named 5052 and 4136) were collected at the Second Affiliated Hospital of Zhejiang University. Strain 5052 was isolated from the feces of a 63-year-old asymptomatic patient who was undergoing routine physical examination in March 2013. Strain 4136 was isolated from the secretions emerging from a surgical incision of a 51-year-old female patient who was admitted for ascites following cholecystectomy surgery in September 2012. Both isolates were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik GmbH, Bremen, Germany).

To determine their molecular types, pulse-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylogenetic grouping analyses were conducted. Genomic DNA was prepared in agarose blocks and digested with the restriction enzyme XbaI for PFGE as previously reported (5). Seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) were used for MLST (6). Clonal complexes (CC) were compared with those from the website http://mlst.warwick.ac.uk/mlst/dbs/Ecoli. Phylogenetic groups were determined as described previously (7). The two strains demonstrated distinct PFGE profiles, with 5052 belonging to CC38 (phylogenetic group D) and 4136 identified as ST410 (group A). Antimicrobial susceptibility testing and ESBL phenotype detection were carried out using Etest (AB Biodisk, Solna, Sweden), and results were interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI) 2013 guidelines (8). Both isolates were resistant to cephalosporin, as they were for several β-lactamase inhibitors, but differences were observed (Table 1). Additionally, strain 4136 was resistant to imipenem, although amplification of carbapenemases and metallo beta-lactamase was negative, other mechanisms (such as porin loss, altered affinity of the penicillin-binding proteins for carbapenems, and overexpression of the efflux pump) may play a role, which may well interpret the high MICs of amoxicillin-clavulanate and piperacillin-tazobactam for strain 4136.

TABLE 1.

Antimicrobial susceptibility and resistance gene detection of parent isolates (5052 and 4136), an Escherichia coli transconjugant (J4136), an E. coli transformant (J5052), and the recipient E. coli isolate (EC600)

Isolate	MIC (μg/ml)a											ESBL gene(s) detected
	TX	CTX	CAZ	FEP	CFP-SUL	TIM	PIP-TZB	AMCb	AMK	GEN	IPMb
5052	>256	>256	64	32	16	96	3	16	2	1	≤1	blaCTX-M-64, blaCTX-M-14
J5052	>256	>256	>256	24	12	>256	3	16	1	0.75	≤1	blaCTX-M-64
4136	>256	>256	>256	>256	>256	>256	>256	≥32	>256	>256	4	blaCTX-M-64, blaCTX-M-65
J4136	>256	>256	>256	24	16	>256	3	16	>256	>256	≤1	blaCTX-M-64, blaCTX-M-65
EC600	0.125	0.125	0.25	0.125	0.125	8	3	4	1	1	≤1	_c

^aTX, cetirizine; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CFP-SUL, cefoperazone-sulbactam; TIM, ticarcillin-clavulanate; PIP-TZB, piperacillin-tazobactam; AMC, amoxicillin-clavulanate; AMK, amikacin; GEN, gentamicin; IPM, imipenem; ESBL, extended-spectrum β-lactamases.

^bThe MICs of AMC and IPM were detected using the Vitek 2 compact system.

^c–, not detected.

A transconjugant was obtained using strain 4136 with rifampin-resistant E. coli EC600 (LacZ⁻ Nal^r Rif^r) as the recipient, designated J4136, while unsuccessful conjugation attempts using 5052 meant that further transformation into competent E. coli EC600 using the heat shock method was required for this strain. bla_CTX-M-64 was identified in the parent isolates, transconjugant J4136, and transformant J5052. PCR-based replicon typing identified the plasmids from J4136 and J5052 as IncFII (9).

The genetic environment of bla_CTX-M-64 was investigated using both previously reported primers (1) and specifically designed primers (5′-TACTTCACCCAGCCTCAA-3′ and 5′-CCTTACCCAGACAGAGTGC-3′). A region of about 1,900 bp that contains the partial sequences of ISEcp1, bla_CTX-M-64, and orf477 was investigated. ISEcp1 was located 45 bp upstream of bla_CTX-M-64, while orf477 was detected 47 bp downstream. Sequence analysis showed that the region flanking bla_CTX-M-64 was identical to the sequence from an E. coli isolate of animal origin (2).

The first identified CTX-M-64-producing isolate was collected from a diarrheal patient following a trip to China (1), and later, CTX-M-64-, CTX-M-123-, CTX-M-132-, and CTX-M-137-producing isolates were reported in China (2–4). In China, 10.3% of CTX-M-producing Enterobacteriaceae were reported to harbor two or three bla_CTX-M genes (4). The emergence of this alternative mechanism for the diversification of CTX-M-type β-lactamases provides more enzymes that can be transferred by homologous recombination. Furthermore, CTX-M-64 shows enhanced activity against ceftazidime, which may be associated with the distinctive hybrid composition of CTX-M-64 (1). The detection of bla_CTX-M-64 in transformants and transconjugants, suggesting horizontal transfer by plasmids, is of particular concern. The emergence of CTX-M-64 in companion animals and human patients in different regions of China, together with the identical genetic environment of bla_CTX-M-64 from different isolates, indicates dissemination of resistance determinants between pets and humans. However, further study is needed to elucidate this process.

In conclusion, this is the first report of CTX-M-64 from human patients in China, and dynamic transmission between humans and companion animals is indicated. Therefore, attention should be paid to the problem of antimicrobial resistance, and rigid management of antimicrobial agent administration is needed to prevent the emergence of novel chimeric CTX-M-type ESBLs.