JAC Advance Access originally published online on March 18, 2008
Journal of Antimicrobial Chemotherapy 2008 61(6):1389-1390; doi:10.1093/jac/dkn116
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Research letters |
Evidence for chromosomal and plasmid location of CMY-2 cephalosporinase gene in Salmonella serotype Typhimurium
1 Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens, Greece 2 Division of Foodborne, Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA 3 Department of Microbiology, Medical School, University of Athens, Greece
* Corresponding author. Tel: +30-210-6478810; Fax: +30-210-6425038; E-mail: miriagou{at}pasteur.gr
Keywords: β-lactamase , cephalosporin , resistance
Resistance to extended-spectrum cephalosporins has emerged among non-typhoid salmonellae (NTS) worldwide due to acquisition of plasmid-mediated β-lactamases including CMY-2.1 The isolation frequency of CMY-2-producing NTS in the USA has gradually increased since 1996 reflecting the widespread transmission of CMY-2-positive NTS among food animal reservoirs. The blaCMY-2 gene has been disseminated by plasmids to various Salmonella serotypes including Typhimurium.2–4 We report here on a human isolate of Salmonella serotype Typhimurium harbouring a CMY-2-encoding plasmid as well as a chromosomally located copy of blaCMY-2.
Salmonella serovar Typhimurium AM19083 was submitted to the National Antimicrobial Resistance Monitoring System in 2003 from Massachusetts. Antimicrobial susceptibility was assessed by broth microdilution (Sensititre®, Westlake, OH, USA). The isolate met the phenotypic criteria indicative of class C cephalosporinase production (i.e. resistance or decreased susceptibility to cefoxitin, ceftiofur and ceftriaxone). It was also resistant to ampicillin, amoxicillin/clavulanate and cefalotin, but susceptible to non-β-lactam drugs including streptomycin, sulphonamides, chloramphenicol, tetracycline, nalidixic acid and ciprofloxacin.
AM19083 was positive in PCR assays with blaCMY-specific primers,3 and the sequence of the amplicon (369 bp) was homologous to an internal fragment of blaCMY-2 (from nt 271 to nt 639, in GenBank accession no. X91840 [GenBank] ). Isoelectric focusing of sonicated cell extracts in polyacrylamide gels containing ampholines showed production of a β-lactamase species with an isoelectric point of >8.4 corresponding to CMY-2.
β-Lactam resistance was readily transferred by conjugation in broth cultures to a susceptible Escherichia coli host. The plasmid content analysis indicated transfer of an ~90 kb plasmid (designated p19083) that was positive in hybridization with a blaCMY-2 probe prepared as described previously.5 Plasmid p19083 belonged to the incompatibility Group I1 (IncI1), as found by a PCR-based Inc/rep typing method.6 Furthermore, the PstI restriction profile of p19083 resembled restriction type B of the IncI1, CMY-2-encoding plasmids previously described in Salmonella Typhimurium and Salmonella Thompson isolates from the USA.5,6
A plasmid DNA preparation from AM19083 was electrophoresed through agarose gels (0.8%). DNA was then transferred to Hybond-N nylon membranes (Amersham Biosciences, Buckingham, UK) and hybridized with a digoxigenin-labelled blaCMY-2 probe.5 These experiments showed not only the expected hybridization with plasmid p19083, but also a strong signal with the chromosomal material. This was not observed with preparations from two control strains of serotype Typhimurium, one of which (AM18447) also carried two CMY-2-encoding plasmids, p18447-S and p18447-L (~140 and 300 kb, respectively). To clarify this issue, an I-CeuI genomic analysis of isolates AM19083 and AM18447 was performed. The digested fragments were separated by PFGE (6.0 V/cm for 8 h/16 h with a pulsing time linearly ramped from 5 to 20 s/20 to 70 s) (Figure 1a). The blaCMY-2 probe was hybridized with a 16SrRNA-positive I-CeuI fragment of ~500 kb from isolate AM19083. Positive signals probably corresponding to the blaCMY-2-carrying plasmids were also seen (Figure 1b). Subsequent hybridization with Inc/rep probes6 showed that p19083 and p18447-S belonged to IncI1, whereas the blaCMY-2-carrying putative plasmid p18447-L and the 500 kb fragment from AM 19083 were not reactive (Figure 1c). In an attempt to rule out the existence of blaCMY-2-positive plasmids of high molecular weight in AM19083 and, also, to corroborate the plasmidic nature of p18447-L, the plasmid contents of AM19083 and AM18447 were analysed by PFGE after treatment of total DNA with S1 nuclease. The respective electrophoretic profiles indicated the absence of blaCMY-2-carrying ‘megaplasmids’ in AM19083, whereas a band of ~300 kb likely representing the linear form of p18447-L was observed in the preparations from AM18447 (data not shown).
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PFGE, New England Biolabs) are in the first lane. Arrow in (c) indicates the 500 kb chromosomal fragment from AM19083, which was positive for blaCMY-2. Hybridization signals in (d) represent the blaCMY-2-positive, IncI1 plasmids carried by both isolates.Finally, to further strengthen our hypothesis for the chromosomal acquisition of blaCMY-2 in AM19083, the relevant I-CeuI fragment was purified by electroelution. The resulting preparation contained blaCMY-2 as confirmed by PCR and sequencing. It was also negative in PCR assays designed for replicon typing of plasmids including IncI1 and IncA/C. Nevertheless, the possibility that plasmid segments including rep could have been removed during I-CeuI treatment cannot be excluded.
Production of CMY-2 has been described in a wide variety of NTS serotypes in the USA. The plasmidic location of blaCMY-2 is critical for spread of this resistant determinant. The present findings confirm the prominent role of IncI1 plasmids in this process. Additionally, the likely chromosomal carriage of blaCMY-2 underscores the operation of efficient mobilization mechanisms commonly involving ISEcp1 and/or IS26.4,7
Multiple copies of a resistance gene may increase the antimicrobial resistance levels and provide more of a selective advantage. Expression of the chromosomal CMY-2 and its possible contribution to β-lactam resistance levels were not studied. β-Lactam MICs for Salmonella Typhimurium AM19083, however, were comparable with those observed for other Salmonella strains carrying only plasmidic blaCMY-2, such as AM18447, but, for many of the drugs, exceeded the range of dilutions tested (ampicillin, amoxicillin/clavulanate, ceftiofur, cefoxitin and cephalothin). Additionally, a comparison of total cephalosporinase activities by UV spectrophotometry using cell extracts and cefalotin as a reporter substrate did not indicate any significant influence of the extra chromosomal copy (data not shown). Therefore, the role of the chromosomal blaCMY-2 in determining resistance levels in this isolate is unclear. Nevertheless, Salmonella strains with chromosomally located blaCMY-2 may act as reservoirs for de novo acquisition of resistance by completely different plasmid backbones and other mobile structures. To the best of our knowledge, this is the first study providing indications as to the simultaneous chromosomal and plasmid location of blaCMY-2 in Salmonella.
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This work was supported by the Hellenic Pasteur Institute. NARMS is funded through an interagency agreement between the United States Food and Drug Administration Center for Veterinary Medicine and the Centers for Disease Control and Prevention.
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None to declare.
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1 Miriagou V, Tassios PT, Legakis NJ, et al. Expanded-spectrum cephalosporin resistance in non-typhoid Salmonella. Int J Antimicrob Agents (2004) 23:547–55.[CrossRef][Web of Science][Medline]
2
Winokur PL, Vonstein DL, Hoffman LJ, et al. Evidence for transfer of CMY-2 AmpC β-lactamase plasmids between Escherichia coli and Salmonella isolates from food animals and humans. Antimicrob Agents Chemother (2001) 45:2716–22.
3 Whichard JM, Joyce K, Fey PD, et al. β-Lactam resistance and Enterobacteriaceae, United States. Emerg Infect Dis (2005) 11:1464–6.[Web of Science][Medline]
4
Giles WP, Benson AK, Olson ME, et al. DNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbones. Antimicrob Agents Chemother (2004) 48:2845–52.
5
Carattoli A, Tosini F, Giles WP, et al. Characterization of plasmids carrying CMY-2 from expanded-spectrum cephalosporin-resistant Salmonella strains isolated in the United States between 1996 and 1998. Antimicrob Agents Chemother (2002) 46:1269–72.
6 Carattoli A, Miriagou V, Bertini A, et al. Replicon typing of plasmids encoding resistance to newer β-lactams. Emerg Infect Dis (2006) 12:1145–8.[Web of Science][Medline]
7
Kang M-S, Besser TE, Call DR. Variability in the region downstream of the blaCMY-2 β-lactamase gene in Escherichia coli and Salmonella enterica plasmids. Antimicrob Agents Chemother (2006) 50:1590–3.
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