Emergence of a New Delhi Metallo-β-Lactamase (NDM-1)-Encoding Gene in Clinical Escherichia coli Isolates Recovered from Companion Animals in the United States

LETTER

The rapid spread of extended-spectrum β-lactamases (ESBLs) and quinolone resistance in Escherichia coli in companion animals (1, 2) has increased concern among veterinarians to find an alternative therapy to treat clinical infections caused by these resistant organisms. Up until now, the prevalence of carbapenemases appeared to be restricted mainly to humans (3–5). More recently, studies have reported the presence of E. coli producing VIM-1 carbapenemase, isolated on a pig farm in Germany (6), and Acinetobacter baumannii expressing OXA-23, isolated from cattle in France (7). In the United States, carbapenems are not licensed for treatment of companion animals, and we are unaware of carbapenemase producers from companion animals. In this study, we describe the first report of detection of a New Delhi metallo-β-lactamase (NDM)-encoding gene (NDM-1) in multidrug-resistant strains of E. coli recovered from companion animals in the United States.

Susceptibility testing was performed on 944 canine and feline clinical E. coli isolates collected from clinical veterinary laboratories between May 2008 and May 2009 using custom microdilution susceptibility plates (2). E. coli strains were isolated from urine, wound, ear, genital tract, anal sac, nasal structure, and soft tissue samples. Unfortunately, information regarding the previous antimicrobial treatments these animals received is not available and a history of travel of owners with their pets was not collected by the veterinary clinics at the time of the medical visit. The MIC results were interpreted according to the CLSI interpretive standards (8). Of the 944 strains, isolates (n = 100) which exhibited reduced susceptibility to ceftazidime (MIC ≥ 16 μg/ml), cefotaxime (MIC ≥ 16 μg/ml), or meropenem (MIC ≥ 1 μg/ml) were analyzed. The presence of genes encoding carbapenemases (bla_KPC, bla_NDM, bla_AIM, bla_GIM, bla_SIM, bla_DIM, bla_IMP, bla_VIM, bla_SPM, and bla_OXA-48) was investigated by PCR as described previously (9). Of the 100 isolates tested, PCR and sequencing identified the presence of the bla_NDM gene in six E. coli isolates. Other carbapenemase-encoding genes were not detected. The meropenem MIC values for NDM-producing isolates ranged from 0.5 to 16 μg/ml (Table 1).

Table 1.

MICs (μg/ml) of cephalosporins tested for bla_NDM-positive Escherichia coli isolates

Demographic or antimicrobial	Demographic information or MIC (μg/ml) for each isolatea
	NCTR-1028	NCTR-1032	NCTR-1033	NCTR-1035	NCTR-1037	NCTR-929 (donor strain)	E. coli J53 (recipient strain)	Trans-929 (transconjugant Strain)
Demographics
Species	Canine	Canine	Canine	Canine	Feline	Canine
Source	Wound	Nasal	Urine	Urine	Urine	Urine
State	Illinois	Massachusetts	North Carolina	Illinois	North Carolina	California
Antimicrobials
Ampicillin	256	256	512	512	512	512	4	>32
Amoxicillin-clavulanic acid	64	64	64	64	32	64	8	>32
Cefazolin	NT	NT	NT	NT	NT	>16	≤8	>16
Cephalothin	2,048	2,048	2,048	2,048	2,048	2,048	≤8	>16
Cefoxitin	128	128	64	512	128	1,024	≤4	64
Cefotaxime	32	8	8	128	16	128	≤0.25	8
Cefpodoxime	512	512	512	512	256	256	1	>32
Ceftazidime	32	32	64	256	64	256	≤0.25	32
Meropenem	1	0.5	0.5	0.5	4	16	≤1	4
Chloramphenicol	8	8	8	16	4	8	8	8
Doxycycline	2	1	4	16	32	2	NT	NT
Enrofloxacin	0.03	0.03	0.03	0.06	32	1	NT	NT
Gentamicin	1	0.5	1	32	64	1	1	1
Trimethoprim-sulfamethoxazole	0.06	0.06	0.06	0.25	64	0.06	≤0.12	≤0.12
Cefotaxime-clavulanic acid	16	32	8	32	16	32	≤0.12	8
Ceftazidime-clavulanic acid	32	32	32	64	16	64	≤0.12	16

^aNT, not tested.

In order to determine the presence of different NDM variants, amplification of the entire open reading frame of bla_NDM was performed using the second set of primers (NDM-Full F, 5′-ATGGAATTGCCCAATATTATGCAC; NDM-Full R, 5′-TCAGCGCAGCTTGTCGGC) (10). The complete sequence of the bla_NDM gene was detected for isolates NCTR-1028, -1033, -1035, and -1037 in our study and showed no amino acid substitution relative to the NDM-1 peptide sequences available in GenBank (accession number AB604953) (data not shown). The entire bla_NDM region was successfully amplified in all but one NDM-positive isolate (i.e., NCTR-929). Although isolate NCTR-929 was successfully amplified using the first set of primers that amplified a 621-bp region of NDM, a negative reaction with the second set of primers may be attributed to the presence of a mutation(s) in the promoter region of NDM.

Conjugation experiments, which were performed on all NDM-positive isolates (n = 6) as donors and the recipient, an azide-resistant E. coli J53 strain, showed that only one isolate, NCTR-929, was successfully transferred to E. coli J53 under ampicillin selection (32 μg/ml). The unsuccessful conjugative attempts in the other five NDM-positive isolates could be attributed to either the location of the bla_NDM-1 gene on the bacterial chromosome instead of a plasmid or the presence of a bla_NDM-1-containing transposon structure, such as ISAba125, that may be integrated into the chromosome (11). The MICs for the transconjugant (Trans-929) are shown in Table 1. Isolate NCTR-929 exhibited the following MICs (μg/ml) for the donor, recipient, and transconjugant, respectively: for cefazolin, >16, ≤8, and >16; for ceftiofur, >8, 0.5, and >8; and for ceftriaxone, >64, ≤0.25, and 16. No genetic relationship was observed among the NDM-producing isolates by pulsed-field gel electrophoresis (PFGE) (Fig. 1).

Fig 1.

Dendrogram showing the genetic relatedness of bla_NDM-positive Escherichia coli isolates based on pulsed-field gel electrophoresis (PFGE) patterns with XbaI.

The emergence of carbapenem resistance may limit the treatment of E. coli infections in companion animals. This, in turn, may limit the treatment options in humans who may contract E. coli infections from their pets.