Identification and Expression of Cephamycinase bla_CMY Genes in Escherichia coli and Salmonella Isolates from Food Animals and Ground Meat

Abstract

Twenty-one Salmonella and 54 Escherichia coli isolates, recovered from food animals and retail ground meats, that exhibited decreased susceptibilities to ceftiofur and ceftriaxone were shown to possess a bla_CMY gene. The bla_CMY-4 gene was identified in an E. coli isolate recovered from retail chicken and was further shown to be responsible for resistance to cephalothin, ampicillin, and amoxicillin-clavulanic acid and elevated MICs of ceftriaxone, cefoxitin, and ceftiofur.

Resistance to broad-spectrum cephalosporins has increased among Salmonella species over the past 2 decades (17, 20). The majority of cephalosporin-resistant Salmonella strains express an extended-spectrum β-lactamase such as TEM and SHV, that is able to hydrolyze oxyimino cephalosporins and monobactams but not cephamycins (2). However, recent reports indicate that several species of Enterobacteriaceae have acquired plasmids encoding AmpC-like β-lactamases that hydrolyze cephalosporins, as well as cephamycins such as cefoxitin and ceftriaxone (14, 20). Resistance to ceftriaxone is most commonly mediated by a cephalomycinase (CMY) encoded by the bla_CMY gene (2), and 11 bla_CMY sequence variants have been deposited in the GenBank database to date. Plasmid-mediated AmpC-like β-lactamases have been identified in clinical isolates of Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Enterobacter aerogenes in Europe and the United States and other countries (8–10, 18, 22). They have also been recently described among Salmonella isolates recovered from food animals and humans in the United States (4, 20). The source of ceftriaxone-resistant Salmonella is often not identified in human cases, but it may have been acquired through the consumption of contaminated food, particularly mishandled or undercooked foods of animal origin (5).

Ceftiofur is the only cephalosporin approved for systemic use in food-producing animals in the United States. This drug was first approved in 1988 as an injectable therapeutic agent for the treatment of acute bovine respiratory disease and has been subsequently approved for use in other food animal species, including pigs, sheep, chickens, and turkeys (4). Because ceftiofur-resistant organisms also exhibit decreased susceptibility to cephamycins and extended-spectrum cephalosporins, the use of this antimicrobial in food animals has come under increasing scrutiny as a selective agent responsible for the emergence and dissemination of ceftriaxone-resistant enteric pathogens such as Salmonella (6, 8, 20, 21). The objectives of this study were to determine the presence of cephamycinase bla_CMY genes and their contribution to the decreased ceftiofur-ceftriaxone susceptibility phenotypes observed among Salmonella and E. coli isolates recovered from food animals and retail ground meats.

Eighty-one (60 E. coli and 21 Salmonella) isolates from cattle, poultry, swine, and retail meats that displayed decreased susceptibility to ceftiofur (MIC, ≥8 μg/ml) and ceftriaxone (MIC, ≥16μg/ml) were selected for study. All bacterial isolates were recovered from 1998 to 2000 in five states (Table 1). The majority of the Salmonella and E. coli isolates recovered from animals did not have a history of cephamycin or cephalosporin exposure. Primers cmy-F 5′-GACAGCCTCTTTCTCCACA-3′ and cmy-R 5′-TGGAACGAAGGCTACGTA-3′ were used in the PCR assay based on the bla_CMY-2 gene sequence of K. pneumoniae (GenBank accession no. Y16784). Amplifications were carried out as described previously with 50°C as the annealing temperature (23). For each set of PCRs, cephalosporin-susceptible Salmonella enterica serotype Typhimurium CVM785 and E. coli CVM4168 were included as negative controls. Fifty-four (90%) of the E. coli isolates and all 21 of the Salmonella isolates tested possessed a bla_CMY gene. The PCR-generated DNA fragments were purified and sequenced. Sequence comparisons were made using the National Center for Biotechnology Information BLAST program (1). DNA sequence analysis of 1.0-kb PCR amplicons obtained from five Salmonella and four E. coli isolates confirmed that the PCR products were bla_CMY genes demonstrating 95 to 99% homology to previously reported bla_CMY-2 genes in K. pneumonia and S. enterica serotype Senftenberg.

TABLE 1.

Ceftiofur-resistant bacteria used in this study and their carriage of the bla_CMY gene

Isolate(s)	No. of isolates	No. isolates with blaCMY	Source	State(s)
E. coli	15	12	Avian	Georgia
E. coli	34	31	Bovine	Michigan, North Dakota
E. coli	1	1	Swine	Oklahoma
E. coli	1	1	Ground beef	Maryland
E. coli	9	9	Ground chicken	Maryland
All E. coli	60	54
Salmonella	3	3	Bovine	Michigan
Salmonella	6	6	Ground beef	Maryland
Salmonella	2	2	Ground chicken	Maryland
Salmonella	10	10	Ground turkey	Maryland
All Salmonella	21	21
Total	81	75

Conjugation studies were conducted to determine whether ceftriaxone-ceftiofur resistance is transferable between bacterial strains. S. enterica serotype Agona CVM1132 (from ground beef), E. coli CVM1320 (from ground chicken), and E. coli CVM1897 (from cattle) were used as donor strains, and E. coli O157:H7 strains CVM 990 and JM263 were used as recipient strains. Ceftiofur (16 μg/ml) and ceftriaxone (64 μg/ml) were used as counterselective agents for donor strains, and kanamycin (64 μg/ml) and nalidixic acid (100 μg/ml) were used as selective agents for recipient strains CVM 990 and JM263, respectively. Transconjugants were confirmed as E. coli O157:H7 by a latex agglutination test (Unipath, Oxoid Division, Ogdensburg, N.Y.). Antimicrobial MICs were determined by using the Sensititre automated antimicrobial susceptibility system (Trek Diagnostic Systems, Westlake, Ohio) and interpreted in accordance with the NCCLS guidelines for broth microdilution methods using recommended quality control organisms (12, 13).

E. coli CVM1320 transferred the ceftriaxone-ceftiofur resistance phenotype to two E. coli O157:H7 strains, CVM990 and JM263. However, S. enterica serotype Agona CVM1132 and E. coli CVM1897 were not able to transfer the resistance phenotype to recipient strains. Transconjugant 1320/990 acquired resistance to ceftriaxone and ceftiofur and other β-lactams, including ampicillin, amoxicillin-clavulanic acid, cephalothin, and cefoxitin (Table 2). Transconjugant 1320/263 exhibited resistance to ceftriaxone, ceftiofur, ampicillin, and amoxicillin. Resistance to cefoxitin and cephalothin could not be measured because JM263 was resistant to these two antimicrobials prior to the conjugation experiments. Additionally, for both transconjugants, the MICs of ceftriaxone and ceftiofur were increased compared to those for the wild-type donor strains. Transfer of the bla_CMY gene to the transconjugants was confirmed by PCR analysis (Fig. 1). Interestingly, the MICs of ceftiofur and ceftriaxone for the transconjugants were two- to fourfold higher than those for the donor strain. This may be due to synergism with resident β-lactamases present in the recipient strains, as chromosome-encoded β-lactamases are present in most gram-negative bacteria and these enzymes are often expressed at low levels (22). On two occasions, conjugal transfer of cephalosporin resistance could not be achieved with E. coli CVM1897 and S. enterica serotype Agona CVM1132 as donors. This may be due to integration of the bla_CMY genes in the chromosome or their inclusion on nonconjugative plasmids.

TABLE 2.

Antimicrobial susceptibility profiles of E. coli strains used in conjugation and expression experiments^a

Strain	Designation	MIC (μg/ml)
		Amo	Amp	Cef	Cet	Cep	Fox	Cml	Kan	Nal	Str	Sul	Tet
CVM1320	Donor	>32	>32	8	16	>32	>32	≤4	≤16	≤4	128	≤128	>32
CVM990	Recipient	4	4	≤0.5	≤0.25	8	≤4	≤4	>64	≤4	>256	>512	>32
1320/990	Transconjugant	>32	>32	>16	64	>32	>32	8	>64	≤4	>256	>512	>32
JM263	Recipient	8	8	1	≤0.25	32	32	32	≤16	>256	≤32	≤128	8
1320/263	Transconjugant	>32	>32	>16	64	>32	32	≤16	>256	≤32	≤128	8
BL21(DE3)pLysS	Host cell	2	≤2	≤0.5	≤0.25	2	≤4	>32	≤16	≤4	≤32	≤128	≤4
BL21(DE3)pLysS + pET34b	Host cell + vector	2	≤2	≤0.5	≤0.25	2	≤4	>32	>64	≤4	≤32	≤128	≤4
BL21 (DE3)pLysS + pET34b-cmy	Transformant	>32	>32	8	4	>32	16	>32	>64	≤4	≤32	≤128	≤4

^aAmo, amoxicillin-clavulanic acid; Amp, ampicillin; Cef, ceftiofur; Cet, ceftriaxone; Cep, cephalothin; Fox, cefoxitin; Cml, chloramphenicol; Kan, kanamycin; Nal, nalidixic acid; Str, streptomycin; Sul, sulfamethoxazole; Tet, tetracycline. All isolates were susceptible to amikacin, apramycin, ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole. β-Lactam antimicrobials are bolded and italicized. MICs were determined via broth microdilution methods in accordance with NCCLS guidelines (12, 13). Resistance is indicated by boldface values. 

FIG. 1.

PCR amplifications of the bla_CMY gene in donor E. coli CVM1320 (lane 1), recipients E. coli O157: H7 CVM990 and JM263 (lanes 2 and 3), transconjugants 1320/990 and 1320/263 (lanes 4 and 5), and negative control S. enterica serotype Typhimurium CVM785 (lane 6), which were used in conjugation experiments. Lane MW contained molecular size standards.

Conjugation experiments showed that two transconjugants (1320/990 and 1320/263) acquired resistance to all six of the β-lactam antimicrobials tested. Cloning and expression experiments were then performed in order to determine if the bla_CMY gene was solely responsible for the resistance phenotype observed in the transconjugants. The entire 1,146-bp bla_CMY gene from CVM1320 was amplified with primers cmy-F-exp (5′-KpnI- BamHI-GGATCCTAGGATCCATGATGAAAAAATCGT-3′) and cmy-R-exp, (5′-SacI-HindIII-EcoRI-GAGCTCAAGCTTGAATTCTTATTGCAGCTTTTC-3′) by PCR. The underlined sequences indicate restriction sites. The PCR product was digested with BamHI and EcoRI, purified, and cloned into expression vector pET34b+ (Novagen, Inc., Madison, Wis.). Recombinants were then introduced into E. coli BL21(DE3)pLysS (Novagen), and transformants were selected on tryptic soy agar supplemented with kanamycin (30 μg/ml) and either ceftriaxone (16 μg/ml) or ceftiofur (8 μg/ml).

Transformants from the expression experiments displayed resistance or decreased susceptibility to the six β-lactams tested compared to the host strain, BL21(DE3)pLysS, or the host strain possessing expression vector pET34b+ alone (Table 2). For transformants, the MICs of cefoxitin increased 4-fold and those of ceftriaxone, ceftiofur, amoxicillin-clavulanic acid, ampicillin, and cephalothin increased up to 16-fold. The MICs of amoxicillin-clavulanic acid, ampicillin, ceftiofur, and cephalothin for the transformants were similar to those for wild-type donor strain CVM1320; however, the MICs of cefoxitin and ceftriaxone were two- to fourfold lower (Table 2). This interesting observation, which has also been reported by other investigators (3), may be due, in part, to the dissimilar genetic backgrounds of the recipient and cloning host strains, resulting in decreased β-lactamase expression and activity relative to that of the wild-type strain.

The bla_CMY gene insert of the expression experiments was sequenced by using flanking primers (pET34F [5′-ATCTGGGTACCGATGACGACGACAA-3] and pET34R [5′-ATCAATTAGTGGTGGTGGTGGTGGT-3′]) derived from the pET34b+ vector DNA sequence. DNA sequence analysis revealed that the 1,146-bp DNA insert was 100% homologous to the bla_CMY-4 gene described in E. coli (16). The sequence of the retail meat E. coli bla_CMY-4 gene has been assigned GenBank accession number AF420597. This gene has previously been identified in an E. coli strain from stool specimens of a leukemia patient in the United Kingdom (16) and a P. mirabilis strain from a urine sample collected from a woman in Tunisia (18). Using BLAST, it was determined that the cloned bla_CMY gene also demonstrated 99.9% similarity to the bla_CMY-2 gene identified in K. pneumoniae and S. enterica serotype Senftenberg and 99% similarity to the bla_CMY-3 gene identified in P. mirabilis (2, 18).

The extended-spectrum cephalosporin ceftiofur has been approved in the United States for therapeutic use in animals beginning with cattle in 1988. Since its approval, ceftiofur has commonly been used for treatment of respiratory tract infections, metritis, and foot rot (20). Our findings confirm those of other investigators that Salmonella and E. coli isolates of animal and food origin that possess bla_CMY genes display decreased susceptibility to ceftiofur, as well as to ceftriaxone (21). Such an association may have been a contributing factor in the development of ceftriaxone-resistant Salmonella phenotypes. Indeed, the isolation of extended-spectrum cephalosporin-resistant E. coli and Salmonella from retail ground meats presents compelling evidence for potential food-borne transmission of these resistant pathogens. Interestingly, resistance to extended-spectrum β-lactams was not associated with one particular chromosomal genotype and is most likely due to horizontal dissemination of the bla_CMY genes via large, broad-host-range plasmids and/or a mobile transposon(s) (11, 21). This suggests the possibility that ceftriaxone resistance in E. coli and Salmonella arose from intraspecies transfer of broad-host-range plasmids, which has been observed for other antimicrobial resistance phenotypes (15, 19). This is in contrast to a recent report attributing aminoglycoside resistance in S. enterica serotype Typhimurium DT104 to clonal dissemination of this pathogen (7). Additionally, since the isolates in our study displayed resistance to other β-lactams, including ampicillin, amoxicillin-clavulanic acid, cephalothin, and cefoxitin, the use of any of these antimicrobials may contribute to the overall selective pressures involved in maintaining these resistance genes within enteric pathogens that are present in the animal production environment.

In summary, the present study indicates that bla_CMY genes are commonly present in E. coli and Salmonella isolates of animal origin that display decreased susceptibility to ceftiofur and ceftriaxone. Additionally, bla_CMY genes were shown to be transferred between bacteria through conjugation. To our knowledge, this is the first report of bla_CMY-4 being detected in E. coli recovered from retail ground meats. This significant finding warrants continued surveillance for bla_CMY variants in bacterial pathogens isolated from animals and animal-derived foods. Of particular importance is the fact that many of these isolates possessing bla_CMY genes were recovered from animals that had no history of exposure to cephamycins and extended-spectrum cephalosporins. As the presence and expression of this gene in a bacterium result in decreased susceptibility to penicillins, cephalosporins, and cephamycin, its dissemination in E. coli and Salmonella may present future therapeutic challenges in animal and human health care.

Nucleotide sequence accession number. The sequence of the retail meat E. coli bla_CMA−4 gene has been assigned GenBank accession number AF420597.