Journal of Antimicrobial Chemotherapy (2002) 49, 1011-1014
© 2002 The British Society for Antimicrobial Chemotherapy
Unusual Salmonella enterica serotype Typhimurium isolate producing CMY-7, SHV-9 and OXA-30 ß-lactamases
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.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
ß-Lactam resistance in Salmonella isolates is increasing. This paper describes the combination of three different ß-lactamases, OXA-30, SHV-9 and CMY-7, expressed by an isolate of Salmonella enterica serotype Typhimurium. This is the first report of an isolate of Salmonella having both an extended-spectrum ß-lactamase and an AmpC ß-lactamase.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Extended spectrum ß-lactamases (ESBLs) are most commonly produced by members of the Enterobacteriaceae, especially Klebsiella pneumoniae, and to a lesser extent, Escherichia coli.1 However, other ESBL-producing organisms, such as Morganella morganii, Serratia marcescens, Shigella dysenteriae, several species of Enterobacter, Salmonella, Proteus, Citrobacter, Pseudomonas aeruginosa, Burkholderia cepacia and Capnocytophaga ochracea, have been reported.1 There are two major concerns with pathogens producing ESBLs: their capacity to cause therapeutic failures with cephalosporins and aztreonam when the isolate is susceptible in vitro, and their capacity for undetected, widespread dissemination.1
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.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Strain and patient history
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.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The isolate was multiply antibiotic resistant, being susceptible to only imipenem, meropenem, ciprofloxacin and levofloxacin in MIC tests (Table 2). The MIC of the investigational agent, faropenem, was 2 mg/L. In the double disc potentiation test, it was intermediate to cefepime, but this agent was not included in the MIC tests. The isolate was resistant to other cephalosporins, penicillins, ß-lactam drug/ß-lactamase inhibitor combinations, aminoglycosides, chloramphenicol and co-trimoxazole. Phenotypic, inhibitor-based ESBL tests were positive, demonstrating potentiation of cefepime, cefpirome and aztreonam by clavulanate. The three-dimensional test indicated ß-lactamase-mediated hydrolysis of cefoxitin, a characteristic of AmpC ß-lactamases.
|
Isoelectric focusing revealed three ß-lactamases with pI values of 7.4, 8.2 and >9.0. These values were consistent with OXA-1 or OXA-30 (pI 7.4), SHV-9 (also known as SHV-5a) (pI 8.2) and AmpC (pI > 9.0). The ß-lactamases with pI values 7.4 and 8.2 were inhibited by clavulanate, but not cloxacillin, and the pI > 9.0 ß-lactamase was inhibited by cloxacillin, but not clavulanate.
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).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
This report describes a paediatric Salmonella isolate from an Australian patient that expressed an unusual combination of three ß-lactamases: a broad-spectrum ß-lactamase OXA-30, an ESBL, SHV-9, and an AmpC ß-lactamase, CMY-7. S. enterica serotype Typhimurium is an important pathogen that has demonstrated considerable versatility in its ability to acquire novel ß-lactamases, and in its complex epidemiology, which involves human, veterinary and food-borne transmission. Resistance to expanded-spectrum ß-lactam antibiotics, such as ceftriaxone, is rare but increasing in Salmonella isolates throughout the world.1,5 Outside the United States, this type of resistance has mostly been associated with ESBL production. In the United States, the incidence of human Salmonella isolates resistant to ceftriaxone increased from 0.1% in 1996 to 1.9% in 19995 and was associated with the production of the plasmid-mediated AmpC ß-lactamase of Citrobacter freundii origin, CMY-2. The S. enterica serotype Typhimurium isolate in this study produced both an ESBL and the AmpC ß-lactamase CMY-7. The acquisition of both an ESBL and an AmpC ß-lactamase is a significant and ominous event.
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.
|
Acknowledgements |
|---|
We thank Jennifer Black for expert technical support.
|
Footnotes |
|---|
* Corresponding author. Tel: +1-402-280-5837; Fax: +1-402-280-1875; E-mail: ndhanson{at}creighton.edu
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 . Thomson, K. S. & Smith Moland, E. (2000). Version 2000: The new beta-lactamases of Gram-negative bacteria at the dawn of the new millennium. Microbes and Infection 2, 1225–35.[Web of Science][Medline]
2 . Hanson, N. D. & Sanders, C. C. (1999). Regulation of inducible ampC beta-lactamase expression among Enterobacteriaceae. Current Pharmaceutical Design 5, 881–94.[Web of Science][Medline]
3
.
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, 2034–8.
4 . Bradford, P. A. (2001). What’s new in beta-lactamases? Current Infectious Disease Reports 3, 13–9.
5
.
Dunne, E. F., Fey, P. D., Kludt, P., Reporter, R., Mostashari, F., Shillam, P. et al. (2000). Emergence of domestically acquired ceftriaxone-resistant Salmonella infections associated with AmpC beta-lactamase. Journal of the American Medical Association 284, 3151–6.
6
.
Thomson, K. S., Sanders, C. C. & Washington, J. A. (1991). High-level resistance to cefotaxime and ceftazidime in Klebsiella pneumoniae isolates from Cleveland, Ohio. Antimicrobial Agents and Chemotherapy 35, 1001–3.
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, 1877–82.
8
.
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, 377–80.
9 . Prinarakis, E. E., Tzelepi, E., Gazouli, M., Mentis, A. F. & Tzouvelekis, L. S. (1996).Characterization of a novel SHV beta-lactamase variant that resembles the SHV-5 enzyme. FEMS Microbiological Letters 139, 229–34.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. D. Hanson, E. S. Moland, S. G. Hong, K. Propst, D. J. Novak, and S. J. Cavalieri Surveillance of Community-Based Reservoirs Reveals the Presence of CTX-M, Imported AmpC, and OXA-30 {beta}-Lactamases in Urine Isolates of Klebsiella pneumoniae and Escherichia coli in a U.S. Community Antimicrob. Agents Chemother., October 1, 2008; 52(10): 3814 - 3816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, Q. Li, Y. Du, X. Jiang, J. Tang, J. Wang, G. Li, and Y. Jiang Prevalence of Plasmid-Mediated AmpC {beta}-Lactamases in a Chinese University Hospital from 2003 to 2005: First Report of CMY-2-Type AmpC {beta}-Lactamase Resistance in China J. Clin. Microbiol., April 1, 2008; 46(4): 1317 - 1321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. D. Pitout, D. L. Church, D. B. Gregson, B. L. Chow, M. McCracken, M. R. Mulvey, and K. B. Laupland Molecular Epidemiology of CTX-M-Producing Escherichia coli in the Calgary Health Region: Emergence of CTX-M-15-Producing Isolates Antimicrob. Agents Chemother., April 1, 2007; 51(4): 1281 - 1286. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. E. Sidjabat, N. D. Hanson, E. Smith-Moland, J. M. Bell, J. S. Gibson, L. J. Filippich, and D. J. Trott Identification of plasmid-mediated extended-spectrum and AmpC {beta}-lactamases in Enterobacter spp. isolated from dogs J. Med. Microbiol., March 1, 2007; 56(3): 426 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Ahmed, K. Furuta, K. Shimomura, Y. Kasama, and T. Shimamoto Genetic characterization of multidrug resistance in Shigella spp. from Japan. J. Med. Microbiol., December 1, 2006; 55(Pt 12): 1685 - 1691. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. E. Sidjabat, K. M. Townsend, N. D. Hanson, J. M. Bell, H. W. Stokes, K. S. Gobius, S. M. Moss, and D. J. Trott Identification of blaCMY-7 and associated plasmid-mediated resistance genes in multidrug-resistant Escherichia coli isolated from dogs at a veterinary teaching hospital in Australia J. Antimicrob. Chemother., May 1, 2006; 57(5): 840 - 848. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Rankin, J. M. Whichard, K. Joyce, L. Stephens, K. O'Shea, H. Aceto, D. S. Munro, and C. E. Benson Detection of a blaSHV Extended-Spectrum {beta}-Lactamase in Salmonella enterica Serovar Newport MDR-AmpC J. Clin. Microbiol., November 1, 2005; 43(11): 5792 - 5793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Moubareck, F. Doucet-Populaire, M. Hamze, Z. Daoud, and F.-X. Weill First Extended-Spectrum-{beta}-Lactamase (CTX-M-15)-Producing Salmonella enterica Serotype Typhimurium Isolate Identified in Lebanon Antimicrob. Agents Chemother., February 1, 2005; 49(2): 864 - 865. [Full Text] [PDF]
|
|
|
|
|
|
T. Kruger, D. Szabo, K. H. Keddy, K. Deeley, J. W. Marsh, A. M. Hujer, R. A. Bonomo, and D. L. Paterson Infections with Nontyphoidal Salmonella Species Producing TEM-63 or a Novel TEM Enzyme, TEM-131, in South Africa Antimicrob. Agents Chemother., November 1, 2004; 48(11): 4263 - 4270. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Hossain, M. J. Ferraro, R. M. Pino, R. B. Dew III, E. S. Moland, T. J. Lockhart, K. S. Thomson, R. V. Goering, and N. D. Hanson Plasmid-Mediated Carbapenem-Hydrolyzing Enzyme KPC-2 in an Enterobacter sp. Antimicrob. Agents Chemother., November 1, 2004; 48(11): 4438 - 4440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Edelstein, M. Pimkin, T. Dmitrachenko, V. Semenov, N. Kozlova, D. Gladin, A. Baraniak, and L. Stratchounski Multiple Outbreaks of Nosocomial Salmonellosis in Russia and Belarus Caused by a Single Clone of Salmonella enterica Serovar Typhimurium Producing an Extended-Spectrum {beta}-Lactamase Antimicrob. Agents Chemother., August 1, 2004; 48(8): 2808 - 2815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Mulvey, D. A. Boyd, L. Baker, O. Mykytczuk, E. M. F. Reis, M. D. Asensi, D. P. Rodrigues, and L.-K. Ng Characterization of a Salmonella enterica serovar Agona strain harbouring a class 1 integron containing novel OXA-type {beta}-lactamase (blaOXA-53) and 6'-N-aminoglycoside acetyltransferase genes [aac(6')-I30] J. Antimicrob. Chemother., August 1, 2004; 54(2): 354 - 359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Antunes, J. Machado, J. C. Sousa, and L. Peixe Dissemination amongst humans and food products of animal origin of a Salmonella typhimurium clone expressing an integron-borne OXA-30 {beta}-lactamase J. Antimicrob. Chemother., August 1, 2004; 54(2): 429 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Hossain, M. D. Reisbig, and N. D. Hanson Plasmid-encoded functions compensate for the biological cost of AmpC overexpression in a clinical isolate of Salmonella typhimurium J. Antimicrob. Chemother., June 1, 2004; 53(6): 964 - 970. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Trott, L. J. Filippich, J. C. Bensink, M. T. Downs, S. E. McKenzie, K. M. Townsend, S. M. Moss, and J. J.-C. Chin Canine model for investigating the impact of oral enrofloxacin on commensal coliforms and colonization with multidrug-resistant Escherichia coli J. Med. Microbiol., May 1, 2004; 53(5): 439 - 443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. D. Pitout, M. D. Reisbig, M. Mulvey, L. Chui, M. Louie, L. Crowe, D. L. Church, S. Elsayed, D. Gregson, R. Ahmed, et al. Association between Handling of Pet Treats and Infection with Salmonella enterica Serotype Newport Expressing the AmpC {beta}-Lactamase, CMY-2 J. Clin. Microbiol., October 1, 2003; 41(10): 4578 - 4582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Smith Moland, N. D. Hanson, V. L. Herrera, J. A. Black, T. J. Lockhart, A. Hossain, J. A. Johnson, R. V. Goering, and K. S. Thomson Plasmid-mediated, carbapenem-hydrolysing {beta}-lactamase, KPC-2, in Klebsiella pneumoniae isolates J. Antimicrob. Chemother., March 1, 2003; 51(3): 711 - 714. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Mulvey, G. Soule, D. Boyd, W. Demczuk, and R. Ahmed Characterization of the First Extended-Spectrum Beta-Lactamase-Producing Salmonella Isolate Identified in Canada J. Clin. Microbiol., January 1, 2003; 41(1): 460 - 462. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. S. Moland, J. A. Black, J. Ourada, M. D. Reisbig, N. D. Hanson, and K. S. Thomson Occurrence of Newer {beta}-Lactamases in Klebsiella pneumoniae Isolates from 24 U.S. Hospitals Antimicrob. Agents Chemother., December 1, 2002; 46(12): 3837 - 3842. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||