Prevalence of Plasmid-Mediated AmpC β-Lactamases in a Chinese University Hospital from 2003 to 2005: First Report of CMY-2-Type AmpC β-Lactamase Resistance in China

Abstract

The aim of this study was to investigate the prevalences of plasmid-mediated AmpC β-lactamases (PABLs) in isolates of Escherichia coli and Klebsiella spp. from a university hospital in China. A total of 1,935 consecutive nonrepeat clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca were collected between January 2003 and July 2005. The isolates with cefoxitin zone diameters less than 18 mm (screen positive) were selected for PCR of the bla_AmpC genes and sequencing. Fifty-four (2.79%) isolates harbored PABLs, as demonstrated by PCR and isoelectric focusing. Sequence analysis revealed the presence of bla_DHA-1 and bla_CMY-2 genes. The Southern blot hybridization studies confirmed that bla_CMY-2 and bla_DHA-1 were located on plasmids. Based on species, PABLs were detected in 4.29% (29 isolates of DHA-1 and 1 isolate of CMY-2) of K. pneumoniae, 1.91% (11 isolates of DHA-1 and 12 isolates of CMY-2) of E. coli, and 3.03% (1 isolate of DHA-1) of K. oxytoca isolates. In contrast to our anticipation, the occurrence rate of DHA-1-producing K. pneumonia significantly decreased (P < 0.01), from 7.54% in 2003 to 2.72% in 2004. The results of random amplified polymorphic DNA analysis indicate that the prevalences of DHA-1-producing K. pneumoniae and CMY-2-producing E. coli strains were not due to epidemic strains. In conclusion, DHA-1 was the most prevalent acquired AmpC beta-lactamase in this collection of isolates from a medical center in China, and DHA-1-producing K. pneumoniae was the most prevalent bacterium harboring a PABL. To the best of our knowledge, this is the first report of CMY-2-type AmpC β-lactamases in the Chinese mainland.

Plasmid-mediated AmpC β-lactamases (PABLs) are derived from chromosomal ampC genes of the family Enterobacteriaceae, such as those of Citrobacter freundii, Enterobacter cloacae, and Aeromonas species.

PABLs have been reported to occur in the United States, Korea, Japan, etc. (4, 14, 24), but there are seldom data indicating PABLs in the Chinese mainland (28, 29). The aims of this study were to establish the prevalence rate of this resistance mechanism in isolates of Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli from a university hospital in Shanghai, China. The first identification of the CMY-2 AmpC β-lactamase in the Chinese mainland is also described.

MATERIALS AND METHODS

Bacterial isolates.

A total of 1,935 nonduplicate clinical isolates of Escherichia coli (n = 1,203), Klebsiella pneumoniae (n = 699), and Klebsiella oxytoca (n = 33) were consecutively selected from the Sixth Affiliate Hospital of Shanghai Jiao Tong University between January 2003 and July 2005. Positive controls were used, including Klebsiella pneumoniae (strain MISC 304, containing MIR-1) and clinical isolates of C. freundii, E. cloacae, Hafinia alvei, and Morganella morganii. Identification was performed by the GNI card of the Vitek system (BioMèrieux, MO) or Walk Away 40 (Dade-Bering, Sacramento, CA).

Antimicrobial susceptibility testing and screening of β-lactamases.

Isolates were tested for susceptibility by the standard disk diffusion method, and the results were interpreted according to the guidelines of the CLSI (formerly NCCLS). Isolates with cefoxitin zone diameters less than 18 mm (4) were considered positive for the AmpC β-lactamase screening test and were selected for MIC, isoelectric focusing (IEF), PCR, and sequencing analyses. The MICs and the presence of extended-spectrum β-lactamases (ESBLs) were determined by using the ESBL confirmation panel (Dade-Behring, Sacramento, CA). The presence of ESBLs was also investigated by the CLSI-recommended disk screening and confirmatory tests.

PCR and sequencing.

Multiplex PCR was performed to amplify six PABL group genes (16, 17). A series of specific primers were used for the detection of DHA-1, CMY-2, MOX, ACC, EBC, FOX, TEM, SHV, and CTX-M (7, 16, 23, 27) (Table 1). The PCR products were purified with a QIAquick PCR purification kit (Qiagen, Hilden, Germany) and cloned into DH5α. Plasmid DNA was prepared by using Qiagen columns and was sequenced on an ABI PRISM 377 automated sequencer (Applied Biosystems, Foster City, CA).

TABLE 1.

Primers used for PCR of bla genes

Target	Primer	Sequencea (5′ to 3′ as synthesized)	Product size (bp)	Annealing temp (°C)	Reference
CIT	CIT-MF	TGG CCA GAA CTG ACA GGC AAA	462	64	17
	CIT-MR	TTT CTC CTG AAC GTG GCT GGC
	CMY25F1	CAA TGT GTG AGA AGC AGT C	1,432	50	7
	CMY2DR1	CGC ATG GGA TTT TCC TTG CTG
DHA	DHA-MF	AAC TTT CAC AGG TGT GCT GGG T	405	64	17
	DHA-MR	CCG TAC GCA TAC TGG CTT TGC
	DHA-1A	TGA TGG CAC AGC AGG ATA	998	52	27
	DHA-1B	GGC TTT GAC TCT TTC GGT A
MOX	MOXMF	GCT GCT CAA GGA GCA CAG GAT	520	64	17
	MOXMR	CAC ATT GAC ATA GGT GTG GTG C
ACC	ACCMF	AAC AGC CTC AGC AGC CGG TTA	346	64	17
	ACCMR	TTC GCC GCA ATC ATC CCT AGC
EBC	EBCMF	TCG GTA AAG CCG ATG TTG CGG	302	64	17
	EBCMR	CTT CCA CTG CGG CTG CCA GTT
FOX	FOXMF	AAC ATG GGG TAT CAG GGA GAT G	190	64	17
	FOXMR	CAA AGC GCG TAA CCG GAT TGG
TEM	TEM-F	TCA ACA TTT CCG TGT CG	860	42	13
	TEM-R	CTG ACA GTT ACC AAT GCT TA
SHV	SHV-F	ATG CGT TAT ATT CGC CTG TG	780	47	13
	SHV-R	AGA TAA ATC ACC ACA ATG CGC
CTX	CTXMU1	ATG TGC AGY ACC AGT AAR GT	593	50	16
	CTXMU2	TGG GTR AAR TAR GTS ACC AGA

^aR, A or G; M, A or C; Y, C or T; K, G or T; S, G or C; I, inosine.

IEF.

IEF was performed as described previously (9). Crude beta-lactamase extracts were subjected to analytical IEF on an ampholine polyacrylamide gel (pH 3.5 to 9.5; Pharmacia, Uppsala, Sweden). Preparations from standard strains known to harbor TEM-12 (pI 5.25), TEM-10 (pI 5.6), TEM-29 (pI 6.1), SHV-1 (pI 7.6), and ACT-1 (pI 9) plasmid-mediated β-lactamases were used as controls. Beta-lactamases were visualized with a 0.2-mg/ml nitrocefin solution (Oxoid Ltd., Basingstoke, England). A 1 mM solution of potassium clavulanate and a 0.3 mM solution of cloxacilin were used to visualize general inhibitor characteristics.

DNA fingerprinting.

Random amplified polymorphic DNA (RAPD) analysis was applied to type the PABL-producing isolates with the primer ERIC2. Amplified PCR products were separated using 1.5% agarose gels and visualized by UV transillumination. DNA fingerprints were compared by visual inspection. RAPD patterns were regarded as different if there were different bands on visual inspection (10, 12, 21).

Conjugation.

E. coli C600, with rifamycin resistance (Rif^r), was used as the recipient strain. Cultures of donor and recipient cells were grown to saturation, and 0.1 ml of each was added to the same medium (5 ml) and allowed to stand at 37°C for 2 h. The mixture was then incubated with shaking for 3 h, and 0.1-ml samples were streaked onto MacConkey agar containing 100 mg/liter rifampin and 10 mg/liter cefotaxime.

Southern hybridization.

Southern blot hybridizations were performed by standard methods (22) with a bla_CMY-2-specific and bla_DHA-1-specific digoxigenin (DIG)-labeled probe. Briefly, purified plasmid DNA was transferred onto a positively charged nylon membrane (Millipore) by capillary action. DIG-labeled bla_CMY-2-specific and bla_DHA-1-specific detection probes were generated according to the directions of the manufacturer (Roche Diagnostics). After prehybridization, hybridization of the membrane with the denatured, DIG-labeled probe (25 ng/ml of hybridization solution) was done overnight at 65°C. The hybridized probes were immunodetected with anti-DIG-alkaline phosphatase.

Statistical analysis.

All statistical tests were two-tailed χ² tests, and the results were considered statistically significant at a P value of <0.05. The data were stored and analyzed by using SPSS (version 12.0).

RESULTS

Three hundred twenty-seven (16.9%) of the 1,935 clinical isolates yielded cefoxitin zone diameters less than 18 mm (screen positive), 54 of which were demonstrated to harbor PABLs by multiplex PCR; 41 isolates carried bla genes of the DHA group, and 13 isolates carried genes belonging to the CIT group.

Two types of PABLs were detected by PCR with specific primers and confirmed as DHA-1 (41 isolates) and CMY-2 (13 isolates) by sequencing analysis. Each isolate produced one to four β-lactamases, with pI values of 5.4, 7.6, 8.2, 7.8, and 9.0. The pI 5.4 β-lactamase corresponds to the TEM-like enzyme (inhibited by clavulanate), and the pI 7.6 and 8.2 β-lactamases correspond to SHV-like and CTX-like enzymes (inhibited by clavulanate), respectively. The β-lactamases with pI values of 7.8 and 9.0 were inhibited by cloxacilin but not by clavulanate and were consistent with DHA-1 and CMY-2 enzymes, respectively.

Based on species, PABLs were detected in 30 (4.29%) of 699 K. pneumoniae, 23 (1.91%) of 1,203 E. coli, and 1 (3.03%) of 33 K. oxytoca isolates. The genotypes of PCR amplification showed that DHA-1 and CMY-2 were harbored by 29 and 1 of 30 PABL-producing K. pneumoniae isolates and 11 and 12 of the 23 E. coli isolates, respectively. The prevalence rate of DHA-1-producing K. pneumoniae was 7.54% in 2003, and this proportion significantly decreased, to 2.72%, in 2004 (P < 0.01) but was not significant between 2003 and 2005 (2.96%) (Table 2). ERIC2 PCR was performed to investigate whether the prevalence of DHA-1-producing K. pneumoniae was due to nosocomial outbreaks of infections caused by epidemic strains. The 15 isolates of DHA-1-producing K. pneumoniae from 2003 had seven different patterns (Fig. 1). Two of the DHA-1-producing K. pneumoniae isolates from 2004 displayed the same RAPD pattern. Only 5 of 13 DHA-1-producing E. coli strains showed one pattern (data not shown). Two CMY-2-producing E. coli isolates from 2003 and six from 2005 indicated very similar RAPD patterns, while four isolates from 2004 presented different types.

TABLE 2.

Prevalence of plasmid-mediated AmpC-producing clinical isolates in a Chinese university hospital from 2003 to 2005

Organism and yr	No. of isolates
	Total	Nonsusceptible to cefoxitin	Multiplex PCR positive	Producing indicated AmpC β-lactamase
				DHA-1	CMY-2
E. coli
2003	400	75	7	5	2
2004	513	88	7	3	4
2005	290	79	9	3	6
Total	1,203	242	23	11	12
K. pneumoniae
2003	199	30	15	15	0
2004	331	31	10	9	1
2005	169	23	5	5	0
Total	699	84	30	29	1
K. oxytoca
2003	16	1	1	1	0
2004	11	0	0	0	0
2005	6	0	0	0	0

FIG. 1.

RAPD fingerprinting of isolates investigated in this work, carried out with primer ERIC2. Lanes 1 to 15, the 15 isolates of DHA-1-producing K. pneumoniae from 2003; lane 16, ATCC 25922; M, 250-bp ladder.

Twenty-seven DHA-1-producing isolates and 10 CMY-2-producing isolates were positive for ESBL production according to phenotypic ESBL confirmatory tests (Table 3). Based on species, 78.3% (18/23) of E. coli strains and 60% (18/30) of K. pneumoniae strains produced PABL and ESBL simultaneously. In order to determine the ESBL gene relatively exactly, we analyzed the beta-lactamases of these isolates by PCR experiments with a series of primers specific for bla_TEM, bla_SHV, and bla_CTX-M. All 54 PABL-producing isolates harbored bla_TEM-like genes. bla_SHV-like and bla_CTX-M-like genes were found in 37 and 29 of the 54 isolates, respectively. Thirty-seven of 41 DHA-1-producing isolates harbored bla_SHV-like genes, and 20 of these 37 isolates harbored bla_CTX-M-like genes simultaneously. Nine of 13 CMY-2-producing isolates harbored bla_CTX-M-like genes. Four of 13 CMY-2-producing isolates transferred bla_CMY-2 to transconjugants, 3 had bla_CMY-2 alone, and 1 had bla_CMY-2 and bla_CTX-M. The Southern blot analysis further revealed that bla_DHA-1 (data not shown) and bla_CMY-2 were located on plasmids (Fig. 2).

TABLE 3.

Prevalence of AmpC and ESBL genes and correlation with phenotypes from ESBL tests

Species and β-lactamase	No. of isolatesa
	Harboring genes in indicated group				Positive for ESBL production	Cefr
	AmpC	TEM	CTX-M	SHV
E. coli
DHA-1	11	11	5	8	8	8
CMY-2	12	12	9	0	10	6
K. pneumoniae
DHA-1	29	29	15	28	18	11
CMY-2	1	1	0	0	0	0
K. oxytoca
DHA-1	1	1	0	1	1	0
Total	54	54	29	37	37	25

^aCef^r represents resistance to cefepime.

FIG. 2.

Plasmid profiles and Southern blotting. (A) plasmid DNA prepared from CMY-2-producing isolates and their transconjugants. Lane M, molecular size marker. (B) Southern blotting of the plasmid DNAs in panel A with the bla_CMY-2 probe. The two photographs were aligned so that the size marker can apply to both.

The susceptibilities of the 54 isolates were as follows: piperacillin, 0%; cefazolin, 0%; cefotaxime, 13%; ceftazidime, 25.9%; cefepime, 53.7%; imipenem, 98%; and meropenem, 98%. The MICs and pI values of the isolates harboring CMY-2-type PABLs are given in Table 4.

TABLE 4.

Results for IEF and susceptibility testing of clinical isolates carrying the CMY-2-type bla_AmpC gene

Strain	pI values	Organismb	MIC (μg/ml)a for:
			IMP	CTN	MER	CPE	CAZ	CFT	CFX	PI	AZT	CPD	CAX
8488	5.4, 8.2, 9.0	ECO	≤0.5	32	≤0.5	>32	32	>128	>32	>64	64	>64	>64
8562	5.4, 9.0	ECO	≤0.5	32	≤0.5	2	32	8	>32	>64	16	>64	16
9882	5.4, 9.0	KPN	≤0.5	32	≤0.5	<1	16	4	>32	>64	16	>64	16
9894	5.4, 7.6, 9.0	ECO	≤0.5	>32	≤0.5	>32	>128	>128	>32	>64	>64	>64	>64
10908	5.4, 9.0	ECO	≤0.5	4	≤0.5	<1	8	4	32	>64	4	>64	4
11174	5.4, 8.2, 9.0	ECO	≤0.5	>32	≤0.5	>32	64	>128	>32	>64	32	>64	>64
11245	5.4, 8.2, 9.0	ECO	≤0.5	32	≤0.5	>32	128	>128	>32	>64	64	>64	>64
12232	5.4, 8.2, 9.0	ECO	≤0.5	32	≤0.5	>32	128	>128	>32	>64	>64	>64	>64
12404	5.4, 8.2, 9.0	ECO	16	>32	16	>32	>128	>128	>32	>64	>64	>64	>64
12488	5.4, 8.2, 9.0	ECO	≤0.5	32	≤0.5	>32	128	>128	>32	>64	64	>64	>64
12535	5.4, 9.0	ECO	≤0.5	32	≤0.5	>32	128	>128	>32	>64	64	>64	>64
12575	5.4, 8.2, 9.0	ECO	≤0.5	>32	≤0.5	>32	64	>128	>32	>64	>64	>64	>64
13317	5.4, 8.2, 9.0	ECO	≤0.5	32	≤0.5	<1	32	8	>32	>64	8	>64	16

^aIMP, imipenem; CTN, cefotetan; MER, meropenem; CPE, cefepime; CAZ, ceftazidime; CFT, cefotaxime; CFX, cefoxitin; PI, piperacillin; AZT, aztreonam; CPD, cefpodoxime; CAX, ceftriaxone.

^bECO, E. coli; KPN, K. pneumoniae.

DISCUSSION

Plasmid-mediated class C β-lactamases have been discovered most frequently in naturally AmpC-negative species, such as K. pneumoniae, K. oxytoca, Salmonella spp., and Proteus mirabilis, and also in E. coli, which normally weakly expresses the chromosomal AmpC enzyme by gene duplication or mutation in the ampC promoter or attenuator, with consequent enhanced gene expression. Since CMY-1 was first reported (isolated from South Korea in 1989), over 40 types of PABLs have been reported worldwide. The prevalence of PABLs among clinical isolates differs depending on the countries and institutions. In China, though PABL (ACT-1 type) was first reported in 2002 (3), the true rate of occurrence of PABLs remains unknown. In this study, the prevalence rate (2.79%) of PABLs in Escherichia coli and Klebsiella spp. is similar to that in Korea (3.1%) but higher than that in the United States (1.2%) (4, 25). The inducible PABL DHA-1 is the most prevalent PABL in China. DHA-1 is also the most prevalent PABL in other areas of Asia (25, 30).

Based on species, 1.9% of E. coli and 4.3% of K. pneumoniae isolates harbored PABLs in this study. In Korea, the occurrence rates for these species are 1.5% and 5.4% (or 2.9%), respectively (24, 25). But in the United States, they decreased to 1.6% (4) and 3.3% (13), respectively. The prevalence rate of DHA-1-producing K. pneumonia significantly decreased, from 7.54% to 2.72%, between 2003 and 2004 in this study. In contrast, it significantly increased in Korea, from 0.6% in 2002 to 2.8% in 2003 and to 4.3% in 2004 (24). The results of RAPD analysis indicate that the prevalences of DHA-1-producing K. pneumoniae (Fig. 1) and CMY-2-producing E. coli strains were due to a combination of clonal spread and horizontal transmission of plasmids (Fig. 3).

FIG. 3.

RAPD fingerprinting of isolates investigated in this work, carried out with primer ERIC2. Lanes 1 to 12, the 12 isolates of CMY-2-producing E. coli from 2003 to 2005. M, 250-bp ladder.

Because PABLs provided a broader spectrum of resistance than ESBLs and were not inhibited by commercially available inhibitors, the antimicrobial agents that could be used are limited. Previous studies suggested that cefepime might be effective for the treatment of infections caused by an AmpC-producing organism. However, in this study, only 53.7% (29 of 54) PABL producers were susceptible to cefepime. Song et al. reported that the association of PABLs with ESBLs may cause failure of treatment, and there is also a report indicating the high inoculum effect of cefepime in PABL-producing isolates (11, 20, 26). Thirty-seven (68.5%) of the 54 PABL-producing isolates also produced an ESBL, which was confirmed by CLSI-recommended confirmatory tests. By the PCR method, 47 (87.0%) of the 54 isolates were detected to harbor bla_CTX-like or bla_SHV-like genes. Based on species, 93.3% (28/30) of K. pneumoniae and 78.3% (18/23) of E. coli isolates harbored beta-lactamase genes of AmpC and bla_CTX-like or bla_SHV-like genes simultaneously. This rate of K. pneumoniae is far higher than that in Korea (8.7%) but is lower than that in Japan (100%) (14, 24). In this study, one isolate (no. 12404), which harbored bla_CMY-2, bla_TEM-like, and bla_CTX-like genes, was simultaneously resistant to imipenem and meropenem. Poirel et al. reported that one E. coli strain with CMY-2 and without outer membrane proteins OmpF and OmpC was resistant to carbapenem and other broad-spectrum cephalosporins (19). The conjugation test of this isolate was not successful, and further research is in progress.

Southern blot hybridization revealed that the CMY-2 gene is located on plasmid. The different bands that hybridized to the CMY-2 probe, though the size of each band differed in each isolate, could presumably represent another gene that has homology to bla_CMY-2 or partial or complete duplications of CMY-2.

The CMY-2 β-lactamase was first described in 1990 (2). It is the ancestor of the other C. freundii ampC alleles that have been found on plasmids since then (1). CMY-2 was detected not only in E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis but also in Salmonella and Shigella (5, 8, 15), the last two having caused an outbreak. CMY-2 is the most prevalent and geographically the most widely distributed PABL (18). In China, the most prevalent type of PABL is DHA-1. The CMY-2 β-lactamase has not been previously reported to occur in China. In 2004, Guan et al. (6) reported a new CMY-type cephalosporinase which is 99% identical to the deduced amino acid sequences of CMY-2 and CMY-7. To the best of our knowledge, this is the first report of plasmid-mediated CMY-2-type AmpC β-lactamases in mainland China.

In conclusion, the prevalence rate of PABLs is 2.79% in Escherichia coli and Klebsiella spp. in this collection of isolates from a medical center in China. DHA-1 is the predominant PABL, and DHA-1-producing K. pneumoniae is the most prevalent bacterial species harboring PABLs. The results of this study reinforce the need for increasing concern for therapy for clinical infections caused by isolates that coproduce PABL and ESBL. This study also represents the first time that CMY-2 has been described to occur in China.