CMY-29 and CMY-30, Two Novel Plasmid-Mediated AmpC β-Lactamases

Plasmid-mediated AmpC β-lactamases (PMACBLs) have been reported in Escherichia coli, Klebsiella, Salmonella, and Proteus. The genes for these β-lactamases originated from the chromosomally encoded AmpC β-lactamases found in other Enterobacteriaceae. Typically, PMACBLs confer resistance to all β-lactams, except the fourth-generation cephalosporins and carbapenems (3).

Here, we report two novel PMACBLs identified in cefoxitin-resistant E. coli isolates from New Zealand. Both E. coli strains (our laboratory numbers ARL05-909 and ARL06-39) were isolated in 2005 from the urine of elderly, hospitalized patients on antibiotic treatment.

The PMACBL genes were identified using the multiplex PCR developed by Pérez-Pérez and Hanson (4). Amplicons of 462 bp in size were obtained for both isolates, and sequencing of these amplicons indicated that both PMACBLs were CMY-2-like. The entire CMY gene was amplified with primers ampC1 and ampC2 as previously described (7) and sequenced using these primers and the CITMF and CITMR primers from the original multiplex PCR as internal primers. The amino acid sequences were predicted from the nucleotide sequences and compared to those in GenBank. Both sequences were novel. The CMY β-lactamase in ARL05-909 was designated CMY-29, and the CMY β-lactamase in ARL06-39 was designated CMY-30 by the Lahey Clinic.

CMY-29 is most closely related to CMY-7, from which it differs by an Ile141Phe substitution. CMY-30 is most closely related to CMY-2, from which it differs by a Val231Gly substitution. Comparison of the DNA sequences showed that the CMY-29 gene and CMY-30 gene differ by single-point mutations from CMY-7 (GenBank accession number AJ011291) and CMY-2 (GenBank accession number X91840), respectively.

The susceptibility of ARL05-909 and ARL06-39, and local isolates with CMY-2 and CMY-7, was determined according to CLSI agar and broth microdilution methods (1). The β-lactam susceptibility patterns were as expected for organisms with PMACBLs (Table 1).

TABLE 1.

β-Lactam MICs for isolates with CMY-29, CMY-30, and closely related β-lactamases

Isolate identification	CMY type	MIC (μg/ml)a
		FOX	CRO	CAZ	FEP	AMC	TIM	TZP	IPM	MEM
ARL05-909	29	≥256	128	≥128	1	64	≥256	64	0.5	≤0.12
ARL06-39	30	128	32	32	0.5	128	≥256	8	0.5	≤0.12
ARL04-413	2	≥256	32	64	0.25	64	64	16	0.25	≤0.12
ARL05-705	7	64	32	64	0.25	64	128	2	0.5	≤0.12

^aFOX, cefoxitin; CRO, ceftriaxone; CAZ, ceftazidime; FEP, cefepime; AMC, amoxicillin (amoxicilline)-clavulanic acid; TIM, ticarcillin-clavulanic acid; TZP, piperacillin-tazobactam; IPM, imipenem; MEM, meropenem.

Isoelectric focusing of crude cellular protein extracts of ARL05-909 and ARL06-39 was carried out as previously described (6). The extracts of both isolates contained only β-lactamase with a pI value of ∼9.0.

The location of the CMY-29 and CMY-30 genes was determined by Southern blot analysis (5). DNA probes were prepared using the CITMF and CITMR primers from the original multiplex PCR and the digoxigenin nucleic acid labeling and detection system (Roche). The results confirmed that both genes were on plasmids.

CMY-29 and CMY-30 are the first novel PMACBLs identified in New Zealand. They are most closely related to CMY-7 and CMY-2, respectively, which are the only other CMY-2-like PMACBLs that have been identified in New Zealand. CMY-2 appears to be the most prevalent PMACBL in this country and accounted for five of the six PMACBLs identified among the urinary E. coli isolates included in a 2006 national survey (2).

The sequences of the CMY-29 and CMY-30 genes are available under GenBank accession numbers EF685371 and EF685372, respectively.

Isolates ARL05-909 (with CMY-29) and ARL06-39 (with CMY-30) have been deposited in the New Zealand Reference Culture Collection, Medical Section, under accession numbers NZRM 4389 and NZRM 4390, respectively.