1Center for Research in Anti-infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178, USA; 2Department of Microbiology and Infectious Diseases, South Western Area Pathology Service, Liverpool, Sydney, Australia
Received 9 November 2001; returned 9 January 2002; revised 15 February 2002; accepted 8 March 2002.
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Abstract |
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Introduction |
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AmpC ß-lactamases are primarily cephalosporinases but are capable of hydrolysing all ß-lactams to some extent. Overexpression of AmpC ß-lactamases usually confers resistance to all the ß-lactam drugs except dipolar ionic methoxyiminocephalosporins, such as cefepime and cefpirome, and the carbapenems.1 Plasmid-mediated AmpC ß-lactamases are derived from the chromosomally encoded enzymes of organisms such as Enterobacter cloacae, M. morganii and others.2 These enzymes have been detected in E. coli, Klebsiella species, Proteus mirabilis, Salmonella species and Enterobacter aerogenes.1 The movement of the ampC gene on to plasmids and transmission to other organisms is of major concern.
OXA-30 is a rare ß-lactamase that was recently reported to be produced by Hong Kong and Shanghai isolates of Shigella flexneri.3 It is closely related to OXA-1, differing by one amino acid. Compared with ESBLs and AmpC ß-lactamases, OXA-30 and OXA-1 have significantly more restricted substrate profiles, being capable of efficiently hydrolysing penicillins and early cephalosporins (e.g. cefalothin) but not the expanded-spectrum cephalosporins and aztreonam.
In recent years, there have been increasing reports of Salmonella isolates that produce either an ESBL or a plasmid-mediated AmpC ß-lactamase.4,5 To our knowledge, there are no reports of Salmonella isolates that produce both types of ß-lactamase. In this report, we describe an isolate of Salmonella enterica serotype Typhimurium that produced the ESBL SHV-9, the AmpC ß-lactamase CMY-7 and the OXA-30 ß-lactamase.
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Materials and methods |
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The S. enterica isolate was cultured from a stool sample of a 14-month-old girl hospitalized for diarrhoea and fever after a 4 week round trip from Australia through Pakistan, Turkey and Saudi Arabia, in February and March 2000. Stool culture yielded Campylobacter spp., Shigella boydii, Shigella flexneri and the S. enterica isolate. The S. enterica isolate was serologically typed as S. enterica serotype Typhimurium (4, 12:i:1,2), and was untypable by phage typing. It appeared to be unusual in its expression of resistance to cefoxitin, cefotaxime, ceftazidime and aztreonam, with the aztreonam resistance being partially reversible by the addition of clavulanate.
Susceptibility testing and ß-lactamase investigations
Antibiotic susceptibilities and resistance phenotypes were investigated by microdilution MIC methodology, double disc potentiation, VITEK (card GNS-424; bioMérieux, Inc., St Louis, MO, USA), NCCLS disc diffusion and the three-dimensional test.6,7
Crude ß-lactamase preparations derived from sonicated bacterial cultures of the S. typhimurium isolate and strains producing reference ß-lactamases were assessed for ß-lactamase pIs and general inhibitor characteristics by isoelectric focusing.6
Polymerase chain reaction (PCR)
Template DNA preparation and PCR amplifications were carried out as previously described using the following modifications: the final volume was 50 µL using 2 µL of template DNA with 0.5 µM primer.8 The annealing temperature and MgCl2 concentration required for each primer set are indicated with the appropriate primer set. The following oligonucleotide primer sets were used to amplify the entire structural gene of the following ß-lactamase genes: blaOXA30, prOXA30F and prOXA30R; blaCMY-7, prCMY25F1 and prCMY2DR1; and blaSHV-9, prSHV1F and prSHVDR (Table 1). The annealing temperature for primer sets amplifying blaCMY7 and blaOXA30 was 50°C and for blaSHV, 55°C. The MgCl2 concentration was 2 mM for all PCRs. PCR products were generated at least twice and sequenced by automated PCR cycle-sequencing with dye-terminator chemistry using a DNA stretch sequencer from Applied Biosystems. The PCR products were sequenced directly except for the blaCMY-7 amplicon, which was gel-purified using 1.5% agarose in Tris-acetate/EDTA buffer. DNA was extracted from the agarose using the Qiagen gel extraction kit (Qiagen, Valencia, CA, USA). Additional primers used to obtain internal sequences are listed in Table 1.
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Results |
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PCR amplification suggested the presence of a blaSHV-like gene, a blaOXA-like gene and a blaCMY-like ampC gene. Sequence data for each amplicon identified the genes as blaSHV-9,9 blaOXA-303 and blaCMY-7 (GenBank accession number AJ011291).
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Discussion |
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There are many gaps in our understanding of the development of antibiotic resistance in Salmonella. For example, both the isolate in this study and United States isolates produced AmpC ß-lactamases of C. freundii origin, CMY-7 and CMY-2, respectively. It would be of interest to determine whether Salmonella has a propensity to acquire plasmid-mediated AmpC ß-lactamases of C. freundii origin more readily than other types of plasmid-mediated AmpC ß-lactamases. Do C. freundii-derived ß-lactamases have special biological significance? Are they associated with a specific antibiotic selection pressure, pathogenic fitness, gene mobility or other attributes? In view of the importance of this pathogen and its capacity for international transmission, focused worldwide surveillance studies are needed to determine and monitor the extent and spread of expanded-spectrum ß-lactam resistance and other types of resistance in Salmonella, with emphasis on the mechanisms involved. A better understanding of the biology and epidemiology of resistant Salmonella isolates is needed to combat their emergence and spread, and to determine appropriate empirical therapy of infections caused by these organisms.
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Acknowledgements |
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Footnotes |
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References |
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Siu, L. K., Lo, J. Y., Yuen, K. Y., Chau, P. Y., Ng, M. H. & Ho, P. L. (2000). Beta-lactamases in Shigella flexneri isolates from Hong Kong and Shanghai and a novel OXA-1-like beta-lactamase, OXA-30. Antimicrobial Agents and Chemotherapy 44, 20348.
4 . Bradford, P. A. (2001). Whats new in beta-lactamases? Current Infectious Disease Reports 3, 139.
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7 . Thomson, K. S. & Sanders, C. C. (1992). Detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three-dimensional tests. Antimicrobial Agents and Chemotherapy 36, 187782.[Abstract]
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Hanson, N. D., Thomson, K. S., Moland, E. S., Sanders, C. C., Berthold, G. & Penn, R. G. (1999). Molecular characterization of a multiply resistant Klebsiella pneumoniae encoding ESBLs and a plasmid-mediated AmpC. Journal of Antimicrobial Chemotherapy 44, 37780.
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