Plasmid-mediated TEM-3 extended-spectrum ß-lactamase production in Salmonella typhimurium in Casablanca

Rajaa AitMhanda, Abdelaziz Soukrib, Najat Moustaouia, Hamid Amarouchb, Naima ElMdaghria, Danielle Sirotc and Mohamed Benbachira,*

a IbnRochd University Hospital and b Faculté des Sciences I AinChock, Casablanca, Morocco; c Faculté de Médecine Clermont-Ferrand, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Isolates of extended-spectrum ß-lactamase (ESBL)-producing Salmonella typhimurium were recovered from children admitted to the IbnRochd University Hospital of Casablanca in 1994. These isolates produced TEM-3 as shown by PCR, isoelectric focusing and sequencing. Production of TEM-3 and resistance to gentamicin were encoded by a 10 kb plasmid that could be transferred by conjugation and transformation. This report extends the list of ESBLs produced by S. typhimurium and stresses the need for continuous surveillance of non-typhoidal Salmonella to adapt antibiotic treatment and preventive measures.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The spread of resistance to ß-lactam antibiotics by extended-spectrum ß-lactamase (ESBL) production among members of the Enterobacteriaceae is a serious concern. ESBLs are increasingly associated with the genus Salmonella.1 In parallel with the increasing number of Salmonella serotypes, an increase in the variety of ESBLs has been reported.1

The ESBLs described in Salmonella typhimurium comprise SHV-2,2 CTX-M2,3 PER-1,4 PER-2,5 CTX-M4,6 CTX-M5 and CTX-M6.7 We report on TEM-3 production by S. typhimurium isolates recovered from children in Casablanca.8


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial isolates

The isolates had low levels of resistance to cefotaxime (MIC 4–8 mg/L) and ceftazidime (4–8 mg/L) and were resistant to gentamicin and co-trimoxazole. They were fully susceptible to imipenem, cefoxitin, cefotetan, kanamycin, tobramycin, amikacin, tetracycline, chloramphenicol and ciprofloxacin. The isolates were closely related8 and only one representative was used in the following experiments.

Plasmid analysis and transfer of resistance determinants

Plasmid analysis was performed by the alkaline lysis method as described by Sambrook et al. 9 Extracts were run on 0.8% agarose gels at 7 V/cm and stained with ethidium bromide. The sizes of the plasmids were estimated by comparing their migration with molecular weight standards (Supercoiled DNA Ladder; Gibco BRL, Cergy Pontoise, France).

Conjugation experiments were carried out with Escherichia coli K12 J53-2. Individual colonies of the donor and recipient isolates were suspended in 2 mL of Luria–Bertani broth supplemented with 0.5% sucrose and incubated for 18 h. The transconjugants were selected on MacConkey agar supplemented with rifampin (100 mg/L) and ampicillin (100 mg/L).

Each plasmid of the clinical isolates was electroeluted and transformed into E. coli DH5{alpha} using the CaCl2 procedure.9 The transformants were selected on MacConkey agar supplemented with ampicillin (100 mg/L) and cefotaxime (1 mg/L). The transformants and the transconjugants were assessed for their plasmid content, their antibiotic susceptibilities and ESBL production by the double disc synergy test.

Plasmid curing was performed by inoculating 103–104 cells of S. typhimurium, transformants and transconjugants into a series of tubes containing acridine at varying concentrations (10–10 000 mg/L). After incubation the cultures were streaked on to MacConkey agar with ampicillin (100 mg/L) and cefotaxime (1 mg/L).

Isoelectric focusing

Analytical isoelectric focusing was carried out with E. coli transconjugants using the method described by Matthew et al.10 Cells were harvested from overnight Luria–Bertani broth culture by centrifugation and resuspended in sodium phosphate buffer 0.05 M pH 7.0. ß-Lactamase was released by sonication. Enzymes were identified by isoelectric focusing in polyacrylamide minigels (Phast Gels IEF, pH gradient 3–9; Pharmacia, Uppsala, Sweden) and subsequent staining with nitrocefin (Oxoid, Dardilly, France); ESBLs of known pI were used as markers.

DNA amplification by PCR

PCR amplification of the TEM or SHV genes from S. typhimurium and from the transconjugants were carried out on a DNA thermal cycler, Progene (Techne, Duxford, UK). The PCR mixture contained in a total volume of 50 µL: 10 pmol of each primer, 0.2 pmol of deoxynucleotide triphosphates, 1 U of Taq polymerase (Promega) and 10 µL of bacterial lysate obtained by heating bacterial colonies to 100°C for 10 min. The following oligonucleotide pri-mers specific for the SHV and TEM genes were obtained from Genosphere Biotechnologie (Paris, France): for SHV genes: 5'-GCCCGGGTTATTCTTATTTGTCGC-3' and 5'-TCTTTCCGATGCCGCCGCCAGTCA-3'; and for TEM genes 5'-ATAAAATTCTTGAAGAC-3' and 5'-TTACCAATGCTTAATCA-3'. The PCR programme consisted of an initial denaturation at 94°C for 12 min, followed by 35 cycles of 30 s at 94°C, 30 s at 45°C (TEM) or 65°C (SHV), 90 s at 72°C. A final extension was performed at 72°C for 10 min. The PCR products were analysed on a 2% agarose gel stained with ethidium bromide and visualized by UV light. The specificities of the TEM and SHV primers for amplification of SHV and TEM genes, respectively, were tested using known controls.

DNA sequencing

The sequence was determined by direct sequencing of the specific amplified product obtained as described previously11 with a crude DNA preparation from S. typhimurium isolate as template. It was performed by the dideoxy chain termination procedure of Sanger et al.12 on an ABI 1377 automatic sequencer with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli-Taq DNA polymerase FS (Perkin-Elmer, Applied Biosystems Division, Foster City, CA, USA).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Isoelectric focusing showed that the S. typhimurium isolates produced a ß-lactamase with a pI of 6.3. PCR amplification of the ß-lactamase genes revealed that the ESBL belongs to the TEM group. Sequence analysis of the amplified PCR product confirmed the identification of the ESBL as TEM-3. To the best of our knowledge, this is the first report of TEM-3 ß-lactamase in S. typhimurium. A broad spectrum of ESBLs in non-typhoidal Salmonella has been reported,1 including molecular class A other than SHV and TEM and molecular class C enzymes. TEM-3 was first described in Klebsiella pneumoniae, and recent surveys of ESBLs in France revealed that this enzyme was frequently detected.13 This ß-lactamase has already been described in Salmonella panama,14 Salmonella kedougou15 and Salmonella enteritidis.16

The S. typhimurium isolates harboured three plasmids: P1 (10 kb), P2 (8 kb) and P3 (4.5 kb). Only the 10 kb plasmid was transferred by conjugation to E. coli K12 as determined by plasmid profile analysis. Transconjugants were ESBL producers, as determined by PCR and double disc synergy test, and were resistant to gentamicin and co-trimoxazole. Electroelution and transformation showed that plasmid P1 (10 kb) was responsible for the ESBL production and resistance to gentamicin but not to cotrimoxazole. Transformation with plasmids P2 and P3 did not confer resistance to ß-lactams. MICs for S. typhimurium, the two E. coli and the transconjugants and transformants are shown in the TableGo. In the curing experiments, no resistance was lost. Reports vary greatly as to resistance phenotypes, the number and sizes of the plasmids carried by ESBL-producing non-typhoidal Salmonella,1 probably reflecting different genetic events that occurred in distant geographical areas. ESBL production and resistance to gentamicin in the isolate reported here are encoded by a 10 kb plasmid whereas in S. enteritidis16 TEM-3 production was encoded by a 120 kb plasmid and was associated with resistance to amikacin, netilmicin, tobramycin, tetracycline and sulphonamides. In S. kedougou TEM-3 was due to the presence of an 85 kb plasmid and the isolates were resistant to all aminoglycosides except gentamicin and to sulphonamides and tetracycline.15


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Table. MICs for S. typhimurium and E. coli transconjugants and transformants
 
Resistance is easily transferred by the isolates and the list of antibiotics to which co-resistance is transferred is of great importance. Most isolates are co-resistant to at least one aminoglycoside,1 while some are resistant to chloramphenicol1 and/or sulphonamide15 and even to ciprofloxacin.1 A major risk would be the transmission of the resistance traits to Salmonella typhi via an ESBL-producing Enterobacteriaceae. This event could occur, since S. typhi is able to acquire antibiotic resistance in vivo17 and since in vivo acquisition of ESBL has been shown to occur in S. kedougou15 and S. enteritidis.16

This report extends the list of ESBLs produced by S. typhimurium, one of the two major serotypes of non-typhoidal Salmonella. The increasing number of Salmonella serotypes involved in ESBL production, the variety of the ESBLs, the high stability of the genetic determinants, and the co-transfer of resistance to antibiotics recommended for the treatment of systemic non-typhoidal Salmonella infection and typhoid fever are a serious problem especially in developing countries where these infections are still endemic.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
This work was partially supported by a grant from the Comité de Recherche of the IbnRochd University Hospital of Casablanca and from the Programme Thématique d'Appui à la Recherche Scientifique (PROTARS P1T2/04).


    Notes
 
* Correspondence address. Faculté de Médecine, Laboratoire de Microbiologie, BP 9154 Casablanca, Morocco. Tel/Fax: +212-22269057; E-mail: benbachir{at}casanet.net.ma Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Revathi, G., Shannon, K. P., Stapleton, P. D., Jain, B. K. & French, G. L. (1998). An outbreak of extended-spectrum ß-lactamase-producing Salmonella seftenberg in a burns ward. Journal of Hospital Infection 40, 295–302. [ISI][Medline]

2 . Benredjeb, S., BenYaghlane, H., Boujnah, A., Philippon, A. & Labia, R. (1988). Synergy between clavulanic acid and newer ß-lactams on nine clinical isolates of Klebsiella pneumoniae, Escherichia coli and Salmonella typhimurium resistant to thirdgeneration cephalosporins. Journal of Antimicrobial Chemotherapy 21, 263–6. [ISI][Medline]

3 . Bauernfeind, A., Casellas, J. M., Goldberg, M., Holley, M., Jungwirth, R., Mangold, P. et al. (1992).A new plasmidic cefotaximase from patients infected with Salmonella typhimurium. Infection 20, 158–63. [ISI][Medline]

4 . Vahaboglou, H., Dodanli, S., Eroglu, C., Ozturk, R., Soleytir, G., Yildirim, I. et al. (1996). Characterization of multiple-antibioticresistant Salmonella typhimurium strains: molecular epidemiology of PER-1-producing isolates and evidence for nosocomial plasmid exchange by a clone. Journal of Clinical Microbiology 34, 2942–6. [Abstract]

5 . Bauernfeind, A., Stemlinger, I., Jungwirth, R., Mangold, P., Amann, S., Akalin, E. et al. (1996). Characterization of ß-lactamase gene blaPER-2, which encodes an extended-spectrum class A ß-lactamase. Antimicrobial Agents and Chemotherapy 40, 616–20. [Abstract]

6 . Gazouli, M., Tzelepi, E., Sidorenko, S. V. & Tzouvelkis, L. S. (1998). Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A ß-lactamase CTX-M-4: involvement of serine 237 in cephalosporin hydrolysis. Antimicrobial Agents and Chemotherapy 42, 1259–62. [Abstract/Free Full Text]

7 . Gazouli, M., Tzelepi, E., Markogiannakis, A., Legakis, N. J. & Tzouvelkis, L. S. (1998). Two novel plasmid-mediated cefotaxime-hydrolyzing ß-lactamases (CTX-M-5 and CTX-M-6) from Salmonella typhimurium. FEMS Microbiology Letters 165, 289–93. [ISI][Medline]

8 . AitMhand, R., Brahimi, N., Moustaoui, N., Elmdaghri, N., Amarouch, H., Grimont, F. et al. (1999). Characterization of extended-spectrum ß-lactamase producing Salmonella typhimurium by phenotypic and genotypic methods. Journal of Clinical Microbiology 37, 3769–73. [Abstract/Free Full Text]

9 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (Eds) (1989). Plasmid vectors. In Molecular Cloning: A Laboratory Manual, 2nd edn, pp. 1.25, 1.74. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

10 . Matthew, M., Harris, A. M., Maeshall, M. J. & Ross, G. W. (1975). The use of analytical isoelectric focusing for detection and identification of ß-lactamases. Journal of General Microbiology 88, 169–78. [ISI][Medline]

11 . Mabilat, C., Goussard, S., Sougakoff, W., Spencer, R. C. & Courvalin, P. (1990). Direct sequencing of the amplified structural gene and promoter for the extended broad-spectrum ß-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae. Plasmid 23, 27–34. [ISI][Medline]

12 . Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, USA 74, 5463–7. [Abstract]

13 . Chanal, C., Sirot, D., Romaszko, J. P., Bret, L. & Sirot, J. (1996). Survey of prevalence of extended spectrum ß-lactamase among Enterobacteriaceae. Journal of Antimicrobial Chemotherapy 38, 127–32. [Abstract]

14 . Cabie, A., Jouanelle, J. & Saintaime, C. (1989 ). ß-lactamase à spectre élargi CTX-chez Salmonella panama à Fort-de-France (Martinique). Médecine et Maladies Infectieuses 19, 418–20. [ISI]

15 . Archambaud, M., Gerbaud, G., Labau, E., Marty, N. & Courvalin, P. (1991). Possible in-vitro transfer of ß-lactamase TEM-3 from Klebsiella pneumoniae to Salmonella kedougou. Journal of Antimicrobial Chemotherapy 27, 427–36. [Abstract]

16 . Barguellil, F., Burucoa, C., Amor, A., Fauchère, J. L. & Fendri, C. (1995). In vivo acquisition of extended-spectrum ß-lactamase in Salmonella enteritidis during antimicrobial therapy. European Journal of Clinical Microbiology and Infectious Diseases 14, 703–6. [ISI][Medline]

17 . Datta, N., Richards, H. & Datta, C. (1981). Salmonella typhi in vivo acquires resistance to both chloramphenicol and cotrimoxazole. Lancet i, 1181–3.

Received 13 November 2000; returned 11 April 2001; revised 31 July 2001; accepted 31 August 2001