A Brisabois ¹, I Cazin^2,4 , J Breuil³ , E Collatz ²
1. Centre National d’Etudes Vétérinaires et Alimentaires (CNEVA), Paris, France
2. Laboratoire de Recherche Moléculaire sur les Antibiotiques, Paris, France
3. Collège de Bactériologie, Virologie et Hygiène des Hôpitaux de France
4. Hôpital Saint-Louis, Paris, France

Introduction

The World Health Organisation has recently pointed out an alarming increase in the incidence of antibiotic resistant strains of Salmonella, which are due to the use of antibiotics in intensive breeding.

In France, until recent years, no or few cases of antibiotic resistant Salmonellas were isolated in clinical practice and the antibiotic treatment (not given routinely) of salmonellosis was rarely a therapeutic issue.

For two decades, however, the serotyping centre of Centre National d'Etudes Vétérinaires et Alimentaires (CNEVA) in Paris has been surveying the antibiotic resistance of strains received. In parallel, CNEVA-Lyon has set up a surveillance network to monitor antimicrobial resistance among pathogenic bacteria that are commonly isolated from cattle and, in particular, salmonella strains.

A rapid increase in the incidence of antibiotic resistance and the emergence of multiresistant strains of Salmonella typhimurium isolated from animals (1,2) and humans (3,4) has recently been observed through these networks.

The Laboratoire de Recherche Moléculaire sur les d'Antibiotiques (LRMA) has collaborated with CNEVA-Paris to compare the phenotypes of antibiotic resistance and the distribution of genes encoding for beta-lactamase of S. typhimurium strains isolated either from humans or from animals, mainly cattle (5). Different types of beta-lactamase have already been identified in salmonella: TEM-1, TEM-2, OXA-1 types are the commonest, SHV-1 type seems to predominate in Africa, and PSE-1 and PSE-2 types are usually detected in Pseudomonas aeruginosa.

Materials and methods

1) Bacteria strains and antimicrobial sensitivity

One hundred and eighty-two ampicillin resistant strains of S. typhimurium isolated in 1994 have been compared; 82 from humans collected by hospitals of the “Collège de Bactériologie, Virologie et Hygiène des hôpitaux de France” and 100 from animals sent by the local veterinary laboratories and other public or private laboratories to the Salmonella Serotyping Centre of CNEVA-Paris. Strains were tested for resistance throughout France to the following antibiotics: ampicillin, piperacillin, cefalotine, cefoperazone, cefamandole, cefuroxime, combinations of amoxycillin with clavulanic acid and ticarcillin with clavulanic acid, ceftazidime, ceftriaxone, tetracycline, streptomycin, kanamycin, tobramycin, gentamicin, amikacin, chloramphenicol, sulphonamides, trimethoprim sulphamethoxazole, and nalidixic acid.

2) Preparation of probes and hybridation technique

The DNA specific probes were prepared using a polymerase chain reaction (PCR) from the following reference strains: Escherichia coli K12 (PBR 322) for TEM-1, E. coli K12 (p453) for SHV-1, P. aeruginosa PA 038 (RPL 11) for PSE-1; P. aeruginosa PA 038 (R159) for PSE-2, and E. coli K12 (R46) for OXA-2 and integrase.

The detection of DNA fragments homologous to these genes on the strains tested was achieved by hybridisation on nylon membrane with the same probes marked with fluorescein.

Results

Over 80% of strains from both human and animal sources showed resistance to tetracyclines, sulphonamides, streptomycin, and chloramphenicol (table 1). Ampicillin resistance was never found in isolation, and was associated with resistance to at least four antibiotics in 78% of human strains and 83% of animal strains. Resistance patterns were similar among strains from humans and animals: the commonest phenotype comprised resistance to ampicillin, sulphonamides, streptomycin, chloramphenicol, and tetracycline and was found in 76% of human and 73% of animal strains.

Ampicillin resistance was caused for the most part by two genes encoding for a beta-lactamase with a similar distribution in both groups. The TEM type was found in 20% of human and 22% of animal strains, the CARB type in 73% and 77% respectively. Both TEM and CARB types were found in five strains. Only one human strain had the gene encoding the beta-lactamase (table 2).

The gene encoding integrase was found in 62 human and 89 animal strains associated with CARB-type gene encoding -lactamase for 60 human strains and 77 animal strains; this gene was also found in 12 of the 22 animal strains with TEM-type beta-lactamase.

Discussion

The presence of the CARB-type gene in 78% of S. typhimurium from both human and animal origins seems surprising since it is usually found in pseudomonas. This gene is located on an integron, a new family of genetic components into which many resistance agents can fit, in a form of mobile sequences, by specific site recombination under the effect of integrase.

These results suggest that integrons carrying multiple resistance genes encoding CARB-type beta-lactamase have been acquired and spread in S. typhimurium of human and animal origin. These preliminary results strengthen the hypothesis that salmonella strains may acquire antibiotic resistance by recombination and transfer between them; some strains having all the genetic material required for transfer of resistance genes.

The reasons and the means of this apparently widespread acquisition of these structures by S. typhimurium are still to be explained. Animals are known to be the main reservoir of Salmonella and a common source of human contamination, however, ensuring their dissemination and persistence. A link between resistance observed among strains isolated from animals and in human medicine could be established, therefore, but a selective pressure also exists in hospital environments that strongly contributes to the increasing resistance of strains isolated in such environments.

All these observations are alarming and have mobilised public and scientific institutions. Surveillance and research programmes should be undertaken with the collaboration of veterinary, food hygiene, and public health institutions.

References

1. Brisabois A, Fremy S, Moury F, Oudard C, Piquet C, Pires Gomes C. Inventaire des Salmonella 1994-1995. Edition du CNEVA.

2. Martel JL, Chaslus-Dancla E, Coudert M, Lafont JP. Evolution de la sensibilité aux antibiotiques des salmonelles d'origine bovine en France. Med Mal Infect 1996; 26: 415-9

3. Breuil J, Berger N, Dublanchet A et le collège BVH. Sensibilité aux antibiotiques de 2800 souches de salmonelles et shigelles isolées en France en 1994. Med Mal Infect 1996; 26: 420-5

4. Casin I, Brisabois A, Berger N, Breuil J, Collatz E. Phenotypes et genotypes de résistance de 182 souches de Salmonella serotype typhimurium résistances à l'ampicilline d'origine humaine et animale. Med Mal Infect 1996; 26: 426-30.

5. Lee LA, Puhr ND, Malonet EK, Bean NH, Tauxe RV. Increase in antimicrobial-resistant Salmonella infections in the United States, 1989-1990. J Infect Dis 1994; 170: 128-34.