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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, December 2001, p. 3647-3650, Vol. 45, No. 12
Division of Animal and Food Microbiology,
Office of Research, Center for Veterinary Medicine, U.S. Food and
Drug Administration, Laurel, Maryland 207081;
Department of Nutrition and Food Science, University of
Maryland, College Park, Maryland 207422;
Department of Avian Medicine, University of Georgia,
Athens, Georgia, 306023; and Department
of Veterinary Diagnostic and Production Animal Medicine, Iowa State
University, Ames, Iowa 500114
Received 7 March 2001/Returned for modification 17 August
2001/Accepted 20 September 2001
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 blaCMY
gene. The blaCMY-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 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
blaCMY 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,
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3647-3650.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification and Expression of Cephamycinase
blaCMY Genes in Escherichia coli
and Salmonella Isolates from Food Animals and Ground
Meat
ABSTRACT
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-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 -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 blaCMY
gene (2), and 11 blaCMY
sequence variants have been deposited in the GenBank database to date. Plasmid-mediated AmpC-like -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).
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 blaCMY-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
blaCMY 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 blaCMY genes demonstrating 95 to 99%
homology to previously reported blaCMY-2 genes in K. pneumonia and S. enterica serotype Senftenberg.
TABLE 1.
Ceftiofur-resistant bacteria used in this study and
their carriage of the blaCMY gene
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 -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 blaCMY 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 -lactamases present in the recipient strains, as chromosome-encoded -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
blaCMY genes in the chromosome or their
inclusion on nonconjugative plasmids.
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Conjugation experiments showed that two transconjugants (1320/990 and
1320/263) acquired resistance to all six of the -lactam antimicrobials tested. Cloning and expression experiments were then performed in order to determine if the
blaCMY gene was solely responsible
for the resistance phenotype observed in the transconjugants. The
entire 1,146-bp blaCMY 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 -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 -lactamase expression and activity relative to that of the
wild-type strain.
The blaCMY 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 blaCMY-4 gene described in E. coli (16). The sequence of the retail meat E. coli blaCMY-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 blaCMY gene also demonstrated 99.9% similarity to the blaCMY-2 gene identified in K. pneumoniae and S. enterica serotype Senftenberg and 99% similarity to the blaCMY-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 blaCMY 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 -lactams
was not associated with one particular chromosomal genotype and is most
likely due to horizontal dissemination of the
blaCMY 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 -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 blaCMY genes are commonly present in E. coli and Salmonella isolates of animal origin that display decreased susceptibility to ceftiofur and ceftriaxone. Additionally, blaCMY genes were shown to be transferred between bacteria through conjugation. To our knowledge, this is the first report of blaCMY-4 being detected in E. coli recovered from retail ground meats. This significant finding warrants continued surveillance for blaCMY variants in bacterial pathogens isolated from animals and animal-derived foods. Of particular importance is the fact that many of these isolates possessing blaCMY 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 blaCMA
4 gene has been
assigned GenBank accession number AF420597.
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FOOTNOTES |
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* Corresponding author. Mailing address: Office of Research, U.S. FDA/CVM, 8401 Muirkirk Rd., Laurel, MD 20708. Phone: (301) 827-8139. Fax: (301) 827-8127. E-mail: szhao{at}cvm.fda.gov.
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