ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2002, p. 1269–1272
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 5
Characterization of Plasmids Carrying CMY-2 from
Expanded-Spectrum Cephalosporin-Resistant Salmonella Strains
Isolated in the United States between 1996 and 1998
A. Carattoli,1F. Tosini,2W. P. Giles,3M. E. Rupp,3S. H. Hinrichs,4F. J. Angulo,5
T. J. Barrett,5and P. D. Fey3,4*
Laboratory of Bacteriology and Mycology1and Laboratory of Cellular Biology,2Istituto Superiore di Sanità, 00161 Rome, Italy;
Department of Internal Medicine3and the Nebraska Public Health Laboratory, Department of Pathology and Microbiology,4
University of Nebraska Medical Center, Omaha, Nebraska; and Foodborne and Diarrheal Diseases Branch,
Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia5
Received 11 May 2001/Returned for modification 24 July 2001/Accepted 24 January 2002
Sequencing of DNA from 15 expanded-spectrum cephalosporin (e.g., ceftriaxone)-resistant Salmonella iso-
lates obtained in the United States revealed that resistance to ceftriaxone in all isolates was mediated by cmy-2.
Hybridization patterns revealed three plasmid structures containing cmy-2 in these 15 isolates. These data
suggest that the spread of cmy-2 among Salmonella strains is occurring through mobilization of the cmy-2 gene
into different plasmid backbones and consequent horizontal transfer by conjugation.
Salmonellosis is primarily a food-borne disease that affects
an estimated 1.4 million people each year in the United States
(14). Expanded-spectrum cephalosporins (e.g., ceftriaxone and
cefotaxime) are the antimicrobial agents of choice in the treat-
ment of pediatric patients with invasive Salmonella infections
(9). Until recently, resistance to expanded-spectrum cephalo-
sporins was rarely reported among Salmonella strains (8). Re-
view of 1996 data from the National Antimicrobial Resistance
Monitoring System (NARMS) in the United States identified
only 1 (0.1%) ceftriaxone-resistant Salmonella isolate among
1,272 human Salmonella isolates. However, by 1999 almost 2%
review of 1999 NARMS data (6). Comparisons of these ceftri-
axone-resistant isolates found divergent strains, indicating
multiple probable sources. The isolates either were different
serotypes or, among patients infected with Salmonella enterica
serotype Typhimurium, were distinguishable by their pulsed-
field gel electrophoresis patterns, thus demonstrating that
these ceftriaxone-resistant human isolates did not represent
the epidemic spread of a clonal strain (6). This study was
undertaken to confirm the identity of the ?-lactamase confer-
ring resistance to expanded-spectrum cephalosporins and char-
acterize the associated plasmids from the apparently sporadic
human Salmonella isolates collected through NARMS from
1996 to 1998.
MATERIALS AND METHODS
The 15 bacterial strains used in the study are listed in Table 1. Thirteen of the
isolates were obtained by the Centers for Disease Control and Prevention
through NARMS. These 13 isolates represented 87% of the total expanded-
spectrum cephalosporin-resistant Salmonella isolates (n ? 15) obtained by the
Centers for Disease Control and Prevention from 1996 to 1998 (6). Isolate SS034
was isolated in Nebraska, whereas isolate 922 was isolated in Ohio. Susceptibility
testing of the Salmonella isolates and the Escherichia coli transconjugants and
transformants was performed by the disk diffusion methodology according to
NCCLS standards (16). The MIC for the pACYC184 construct containing cmy-2
was tested by the E-test (AB Biodisk, Solna, Sweden) methodology. The MICs of
ceftiofur (kindly provided by Pharmacia/Upjohn) were determined by broth
microdilution (15, 17). Plasmid DNA was extracted either by the method of Kado
and Liu (10) or with the Concert Purification Midi kit (Life Technologies, Milan,
Italy) and digested with PstI (Roche, Indianapolis, Ind.). Conjugation and trans-
formation experiments were performed as described previously with E. coli
C600N (ampicillin susceptible, nalidixic acid resistant) and E. coli DH5? as hosts
(Gibco BRL, Bethesda, Md.) (2, 18, 19). Transformants were selected on Luria-
Bertani agar (Difco, Detroit, Mich.) containing 50 ?g of ampicillin (Sigma) per
ml. All ceftriaxone-resistant C600N and DH5? transconjugants and transfor-
mants were subsequently named C6 or DH followed by the appropriate wild-type
Salmonella strain designation (e.g., C6/SS034 and DH/4656).
Southern blot hybridizations were performed by standard methods (19) with a
cmy-2-specific DNA probe labeled with [?-32P]dCTP with an RTS RadPrimer
DNA Labeling kit (Life Technologies). DNA sequencing was performed with
primers derived from known sequences and an ABI Prism model 377 sequencer
(Perkin-Elmer Biosystems, Foster City, Calif.). The primers and DNA probes
used to detect potential class 1 integrons have been described previously (4).
Primers 92 (CCGTTTGTCAACACAGTAC [forward]) and 52 (TTGCAGCTT
TTCAAGAATGCGCC [reverse]) were used to amplify full-length blacmy.
Primer 92 was designed by using the sequence from the intercistronic region
between ampC and ampR in Citrobacter freundii (GenBank accession no.
X76636). Primer 52 was designed from the known cmy-2 sequence. Plasmid
vectors pCRII (Invitrogen, Carlsbad, Calif.) and pACYC184 (5) were used in
cloning experiments. Isoelectric focusing was performed at room temperature on
a mini isoelectric focusing gel system (model 111; Bio-Rad, Richmond, Calif.)
(13). The isoelectric points of unknown ?-lactamases were estimated by com-
parison with those of TEM-1, SHV-3, SHV-5, and CMY-2.
RESULTS AND DISCUSSION
The antibiotic resistance phenotypes of the 15 strains under
study are shown in Table 1. All isolates were resistant to am-
picillin, ceftriaxone, ceftiofur, and cefoxitin. After mating ex-
periments with C600N, 7 of 15 isolates were able to transfer
decreased susceptibilities to ceftriaxone to C600N (Table 1).
For those strains for which a transconjugant with decreased
* Corresponding author. Mailing address: Departments of Internal
Medicine and Pathology and Microbiology, University of Nebraska
Medical Center, 985400 Nebraska Medical Center, Omaha, NE 68198-
5400. Phone: (402) 559-2122. Fax: (402) 559-5581. E-mail: pfey@unmc
susceptibility to ceftriaxone was not isolated, plasmid DNA
was isolated and used to transform E. coli DH5?. From the
transformation experiments, an additional four transformants
with reduced susceptibilities to ceftriaxone were isolated. The
MIC of ceftriaxone was 8 to 32 ?g/ml for all E. coli C600N
transconjugants and DH5? transformants (hereafter these E.
coli transconjugants and transformants will be referred to as
ceftriaxone resistant). By isoelectric focusing, all ceftriaxone-
resistant E. coli transconjugants and transformants expressed a
?-lactamase (pI ?9.0) that comigrated alongside CMY-2 (data
not shown). In addition, primers specific for blacmyamplified
an appropriate 631-bp DNA product from all ceftriaxone-re-
sistant E. coli transconjugants and transformants and from the
four Salmonella strains for which a transformant or transcon-
jugant was not isolated (strains 2152, 2855, 4501, and 4528)
(data not shown). Other resistance factors cotransferred with
ceftriaxone resistance in 6 of 11 transconjugants or transfor-
mants (Table 1). Two strains (strains 922 and 2039) transferred
all resistance factors to their corresponding E. coli transconju-
gant. The remaining five transconjugants or transformants
were resistant only to ?-lactam antibiotics.
The sequence of the blacmygene obtained by PCR amplifi-
cation was determined. DNA sequencing revealed that all
strains encoded cmy-2, and no sequence divergence was de-
tected in any strain. The DNA sequence found in the U.S.
isolates was identical to the original cmy-2 sequence described
in Klebsiella pneumoniae (3), yet it was different from the
cmy-2-like sequence described in a ceftriaxone-resistant Sal-
monella serotype Senftenberg strain isolated in Algeria (11).
Compared with the U.S. isolates, the Algerian isolate had
three base pair changes within the first 50 bp, and two of these
changes resulted in amino acid changes, suggesting that the
cmy-2 gene disseminating throughout the United States is dis-
tinct from that in Algeria.
To further demonstrate that CMY-2 alone is responsible for
mediating expanded-spectrum cephalosporin resistance in
these isolates, CMY-2 was cloned by first amplifying cmy-2
from C6/SS034 with primers 92 and 52 and cloning it into
pACYC184. As shown in Table 2, all strains were resistant or
intermediate to ceftriaxone, ceftazidime, cefotaxime, ceftiofur,
and cefoxitin. Both SS034 and DH/pNF10 were resistant to
aztreonam; however, C6/SS034 was susceptible to aztreonam,
perhaps due to the lower plasmid copy number or genomic
background differences between C600N and DH5?. All strains
were susceptible to cefepime and imipenem. Both C6/SS034
and DH/pNF10 were susceptible to piperacillin-tazobactam, as
tazobactam is a known inhibitor of cmy-2 (3). The fact that
strain SS034 also produces TEM-1 may have contributed to its
resistance to piperacillin-tazobactam (7).
Plasmid DNA was isolated from the 11 E. coli transconju-
gants and transformants and the 4 wild-type Salmonella strains
that did not yield a ceftriaxone-resistant transconjugant or
TABLE 1. Ceftriaxone-resistant Salmonella strains used in the studya
State in which strain
Antibiotic resistance phenotype TraTransfer phenotypeIntegron
2152Typhim. California ACSSuTGmToKCroXnlFx NTA
aAbbreviations: A, ampicillin; C, chloramphenicol; S, streptomycin; Su, sulfisoxazole; T, tetracycline; Gm, gentamicin; To, tobramycin; K, kanamycin; Tp, trimethoprim;
Cro, ceftriaxone; Xnl, ceftiofur; Fx, Cefoxitin; Typhim, Typhimurium; Tra, self-transferred plasmids by conjugation; NT, no ceftriaxone-resistant transformants isolated;
ND, not done; Pos, positive; Neg, negative
TABLE 2. ?-Lactam MICs for CMY-2-containing strains
aAbbreviations: CRO, ceftriaxone; CAZ, ceftazidime; CTX, cefotaxime; XNL, ceftiofur; FEP, cefepime; TZP, piperacillin-tazobactam; IPM, imipenem; ATM,
aztreonam; FOX, cefoxitin.
1270CARATTOLI ET AL.ANTIMICROB. AGENTS CHEMOTHER.
transformant, and the plasmid DNA was probed with a cmy-
2-specific probe (amplified with primers 92 and 52). This anal-
ysis demonstrated that cmy-2 was encoded on large plasmids
(ca. 60 to 75 kb) in each strain (data not shown). The cmy-2-
containing plasmids isolated from the E. coli transconjugants
and transformants were additionally subjected to restriction
endonuclease digestion with PstI (Roche) since a single PstI
restriction site is present within cmy-2 (3), and the digests were
analyzed by Southern hybridization with cmy-2 as a probe.
Three PstI restriction fragment length polymorphism hybrid-
ization groups, referred to as types A, B, and C (Fig. 1), were
observed. The cmy-2 probe hybridized to bands of approxi-
mately 12 kb and 800 bp (type A) and 2.5 kb and 800 bp (type
B) for 8 and 5 of 15 cmy-2-containing plasmids, respectively
(Fig. 1A, type A, and Fig. 1B, type B). For strains 4528 (wild
type) and DH/4656, the cmy-2-specific probe hybridized to a
3.2-kb fragment and an 800-bp fragment (Fig. 1C, type C). PstI
digestion of type B plasmids, which encode resistance only to
?-lactam antibiotics, suggested that these plasmids were highly
related. Plasmids with the type A or C hybridization pattern
transferred resistance to at least four antibiotics (streptomycin,
chloramphenicol, tetracycline, and sulfonamides), in addition
to ceftriaxone (Table 1), but had different PstI restriction frag-
ment length polymorphism patterns (Fig. 1). The significance
of the conserved cmy-2 hybridization pattern in these plasmids
is not known.
These data suggest that cmy-2 is being transferred among Sal-
monella strains by plasmid transfer to different genomic back-
bones as well as by independent acquisition of cmy-2 by dif-
ferent plasmid backbones, most of which carry multiple
antibiotic resistance determinants. The mechanism of transfer
and acquisition of cmy-2 is unknown; however, it appears that
cmy-2 is not encoded within a cassette that inserts into a class
1 integron. Experiments for the detection of class 1 integrons
were performed by both PCR amplification (12) and Southern
hybridization by using the integrase gene as a probe (data not
shown) (4). Class 1 integrons were detected in eight strains
(Table 1); however, the cmy-2 gene was not included as an
integron-borne gene cassette. Isolates 2039, SS034, 2152, 4204,
3977, 4501, and 2668 all contained an integron (In-t6) that
carries the aadA2 gene cassette, which confers resistance to
streptomycin and spectinomycin. Isolates 2039 and 2152 also
carried an additional integron (In-t4) that encodes the cmlA
and aadB gene cassettes, which confer resistance to chloram-
phenicol and kanamycin, respectively. One isolate, isolate
2855, contained a larger integron (In-t5) that carries the dfrA1
and aadA2 gene cassettes, which encode trimethoprim and
streptomycin-spectinomycin resistance, respectively. Integrons
were located on cmy-2-carrying plasmids only in isolates SS034
The results of this study demonstrate the emergence and
spread of a CMY-2 ?-lactamase in Salmonella strains isolated
from humans in the United States. The ceftriaxone resistance
reported in porcine, bovine, and human Salmonella isolates in
Iowa and Nebraska was also mediated by cmy-2 (20, 7). The
emergence of ceftriaxone resistance among Salmonella strains
isolated from food animals supports the transfer of ceftriax-
one-resistant Salmonella strains from food animals to humans
(21, 1). In this study, we demonstrated that CMY-2 alone can
mediate resistance to expanded-spectrum cephalosporins, in-
cluding ceftiofur, by cloning the cmy-2 gene into pACYC184.
Although the reasons for the emergence of resistance to ex-
panded-spectrum cephalosporins in humans remain uncertain,
the emergence of resistance in food animals may play a role.
The increased prevalence of ceftriaxone-resistant Salmonella
strains in food animals may in turn be related to the veterinary
FIG. 1. Restriction analysis (left panels in the pairs of panels) and cmy-2 Southern hybridization (right panels in the pairs of panels).
PstI-digested plasmids were extracted from E. coli C6/SS034, DH/4204, DH/3977, and C6/2039 (type A hybridization pattern) (A); E. coli C6/4255,
C6/3430, and C6/1358 (type B hybridization pattern) (B); and E. coli DH/4656 (type C hybridization pattern C) (C). A 1-kb marker (KiloBase DNA
marker; Pharmacia Biotech, Milan, Italy) (A and B) and a 12-kb ladder (Gibco BRL) (C) were used as standards.
VOL. 46, 2002CEFTRIAXONE-RESISTANT SALMONELLA1271
use of ceftiofur, an expanded-spectrum cephalosporin used Download full-text
only in veterinary medicine. Further studies are warranted to
determine the risk factors for dissemination of cmy-2-mediated
resistance and to determine whether limiting the use of ceft-
iofur in food animals, along with improvements in food pro-
cessing methods, might reduce the potential for dissemination
of ceftriaxone resistance.
We thank Emma Filetici, Susanna Mariotti, and Susan Greenwood
for technical assistance. We also thank the Ohio Public Health Labo-
ratory for Salmonella serotype Typhimurium isolate 922.
This research was partially supported by grants from the Italian
Ministry of Health’s “Progetto Antibiotico Resistenza” to A.C. and a
University of Nebraska Medical Center grant to P.D.F.
1. Angulo, F. J., K. R. Johnson, R. V. Tauxe, and M. L. Cohen. 2000. Origins
and consequences of antimicrobial-resistant nontyphoidal Salmonella: im-
plications for the use of fluoroquinolones in food animals. Microb. Drug
2. Bachman, B. 1987. Derivations and genotypes of some mutant derivatives of
Escherichia coli K-12, p. 1190–1219. In F. C. Niedhardt, J. L. Ingraham, K. B.
Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia
coli and Salmonella typhimurium: cellular and molecular biology, vol. 1.
American Society for Microbiology, Washington, D.C.
3. Bauernfeind, A., I. Stemplinger, R. Jungwirth, and H. Giamarellou. 1996.
Characterization of the plasmidic ?-lactamase CMY-2, which is responsible
for cephamycin resistance. Antimicrob. Agents Chemother. 40:221–224.
4. Carattoli, A., L. Villa, C. Pezzella, E. Bordi, and P. Visca. 2001. Expanding
drug resistance through integron acquisition by Inc plasmids of Salmonella
enterica Typhimurium. Emerg. Infect. Dis. 7:444–447.
5. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of
amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic
miniplasmid. J. Bacteriol. 134:1141–1156.
6. Dunne, E. F., P. D. Fey, P. Kludt, R. Reporter, F. Mostashari, P. Shillam, J.
Wicklund, C. Miller, B. Holland, K. Stamey, T. J. Barret, J. K. Rasheed,
F. C. Tenover, E. M. Ribot, and F. J. Angulo. 2000. Emergence of domesti-
cally acquired ceftriaxone-resistant Salmonella infections associated with
AmpC beta-lactamase. JAMA 284:3151–3156.
7. Fey, P. D., T. J. Safranek, M. E. Rupp, E. F. Dunne, E. Ribot, P. C. Iwen,
P. A. Bradford, F. J. Angulo, and S. H. Hinrichs. 2000. Ceftriaxone-resistant
Salmonella infections acquired by a child from cattle. N. Engl. J. Med.
8. Herikstad, H., P. S. Hayes, J. Hogan, P. Floyd, L. Snyder, and F. J. Angulo.
1997. Ceftriaxone-resistant Salmonella in the United States. Pediatr. Infect.
Dis. J. 9:904–905.
9. Hohmann, E. L. 2001. Nontyphoidal salmonellosis. Clin. Infect. Dis. 32:263–
10. Kado, C. I., and S. Liu. 1981. Rapid procedure for detection and isolation or
large and small plasmids. J. Bacteriol. 145:1365–1373.
11. Koeck, J.-L., G. Arlet, A. Philippon, S. Basmaciogullari, H. V. Thien, Y.
Buisson, and J. D. Cavallo. 1997. A plasmid-mediated CMY-2 ?-lactamase
from an Algerian clinical isolate of Salmonella senftenberg. FEMS Microbiol.
12. Levesque, C., L. Piche, C. Larose, and P. Roy. 1995. PCR mapping of
integrons reveals several novel combinations of resistance genes. Antimi-
crob. Agents Chemother. 39:185–191.
13. Mathew, A., A. M. Harris, M. J. Marshall, and G. W. Ross. 1975. The use of
analytical isoelectric focusing for detection and identification of ?-lactama-
ses. J. Gen. Microbiol. 88:169–178.
14. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro,
P. M. Griffen, and R. V. Tauxe. 1999. Food-related illness and death in the
United States. Emerg. Infect. Dis. 5:607–625.
15. National Committee for Clinical Laboratory Standards. 2000. Methods for
dilution antimicrobial susceptibility tests for bacteria that grow aerobically;
approved standard M7-A5. National Committee for Clinical Laboratory
Standards, Wayne, Pa.
16. National Committee for Clinical Laboratory Standards. 2001. Performance
standards for antimicrobial disk susceptibility tests; approved standard M2-
A7. National Committee for Clinical Laboratory Standards, Wayne, Pa.
17. National Committee for Clinical Laboratory Standards. 2001. Performance
standards for antimicrobial disk dilution susceptibility tests for bacteria iso-
lated from animals; approved standard M31-A. National Committee for
Clinical Laboratory Standards, Wayne, Pa.
18. Pitout, J., K. Thomson, N. Hanson, A. Ehrhardt, E. Moland, and C. C.
Sanders. 1998. Beta-lactamases responsible for resistance to expanded-spec-
trum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus
mirabilis isolates recovered in South Africa. Antmicrob. Agents Chemother.
19. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Har-
20. Winokur, P. L., A. Brueggemann, D. L. DeSalvo, L. Hoffmann, M. D. Apley,
E. K. Uhlenhopp, M. A. Pfaller, and G. V. Doern. 2000. Animal and human
multidrug-resistant, cephalosporin-resistant Salmonella isolates expressing a
plasmid-mediated CMY-2 AmpC beta-lactamase. Antimicrob. Agents Che-
21. Winokur, P. L., D. L. Vonstein, L. J. Hoffman, E. K. Uhlenhopp, and G. V.
Doern. 2001. Evidence for transfer of CMY-2 AmpC ?-lactamase plasmids
between Escherichia coli and Salmonella isolates from food animals and
humans. Antimicrob. Agents Chemother. 45:2716–2722.
1272CARATTOLI ET AL.ANTIMICROB. AGENTS CHEMOTHER.