Antimicrobial susceptibility of Salmonella strains isolated from swine in Catalonia, Spain

E. M. Mateu, DVM, PhD, M. Martin, DVM, PhD, L. Darwich, DVM, W. Mejia, DVM, Departament de Sanitat i d'Anatomia Animals, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain N. Frias, DVM, F. J. Garcia Penia, DVM, PhD, Laboratorio de Sanidad Animal, Ministerio de Agricultura Pesca y Alimentaci6n, Carretera de Algete Km 5.400, 281 10 Algete, Spain ANTIMICROBIAL resistance is currently one of the key issues in public health debates. The World Health Organization (WHO 2000) recognises that if control measures are not taken, the 21st century could be the era of the socalled superbugs, namely, multidrug resistant bacteria that cannot be treated with the currently available antimicrobial agents. The source of this problem has been assumed to be the overconsumption of antibiotics, not only by humans but also by animals. Thus, apathogenic bacteria of animal origin could be a reservoir of resistant genes that could reach humans via the food chain. In other instances, multidrug resistant bacterial pathogens can infect humans through food consumption by other means. This is the case for Salmonella, particularly a phage type of Salmonella enterica subspecies enterica serovar Typhimurium called DT104. This phage type, which has emerged in a considerable number ofWestern countries, such as the UK (Threlfall and others 2000) and the USA (Akkina and others 1999), is characterised by a pentaresistance pattern called R-ACSSuT (accounting for ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline) which is encoded in the bacterial chromosome (Threlfall and others 1994). Some authors consider that this multidrug resistance confers an ecological advantage to the carrier strains which help them to survive in hostile environments (Low and others 1999). Recently, Molbak and others ( 1999) reported the first case of pig to human transmission of S Typhimurium DT104 via the food chain, which caused two deaths and severely affected several people. The strain involved in that outbreak also showed the multiple antibiotic resistance characteristic of the phage type but also a diminished susceptibility to fluoroquinolones, one of the first line antimicrobials in the therapy of Gram-negative sepsis in humans. This has also been reported by Threlfall and others (1998) who thought the use of these compounds in animals responsible for the emergence of such resistance. This short communication describes the antimicrobial susceptibility profiles of Salmonella strains isolated from healthy and diseased pigs in Catalonia, Spain. One hundred and fifty-five Salmonella strains isolated at the Veterinary Faculty of Barcelona were tested to determine their susceptibility patterns to common antimicrobial agents used in veterinary and human medicine. Isolates were obtained from faeces, mesenteric lymph nodes or other organs from either healthy pigs or swine suffering from diarrhoea or septicaemia. Most of the faecal samples or organs were submitted to the laboratory from several parts of Catalonia (north-east Spain), an area of high pig density (approximately 6,000,000 pigs in total). Once identified as belonging to the genus Salmonella, all strains were tested for susceptibility to 18 common antimicrobial agents used in human or veterinary medicine for the treatment of salmonellosis. This panel of antimicrobial agents included four penicillins or cephalosporins: ampicillin (A), amoxycillin and clavulanic acid (Amc), ceftiofur (Eft) and ceftriaxone (Cro); four quinolones: nalidixic acid (Nal), flumequine (Ar), enrofloxacin (Enr) and ciprofloxacin (Cip); four aminoglycosides and aminocylitoles: gentamicin (Gen), streptomycin (S), apramycin (Apr) and neomycin (Neo); two sulphonamides or combinations: sulphonamide (Su) and trimethoprim and sulphonamide (Tm); and four other miscellaneous antimicrobials: tetracycline (T), chloramphenicol (C), colistin (Col) and nitrofurantoin (Nit). Susceptibility tests were performed by the Kirby-Bauer method on Mueller-Hinton agar using as inoculum a fresh suspension made from an overnight culture in brain heart infustion (BHI) broth adjusted to fit the 0-5 McFarland standard with 0-85 per cent sterile saline. Measurement of growth inhibition diameters was performed with a calibrated scale and recorded. Interpretation of these diameters was done according to the National Committee for Clinical Laboratory Standards document for veterinary antimicrobial susceptibility tests (NCCLS 1999) or according to the microdisk manufacturer when such a standard was not available. Therefore, strains were classified as susceptible, intermediate or resistant. For the determination of multiple drug resistance patterns, intermediate results to a given antimicrobial were considered as a decreased susceptibility in order not to overestimate the rate of resistances. Epi Info version 6.01 was used to calculate descriptive statistics (Dean and others 1994). More than 50 per cent of the isolates belonged to two serovars: S Typhimurium accounted for 361 per cent of the isolates (56 of 155) and Salmonella serovar 4,5,12:i:(a monophasic variety of S Typhimurium) with 15-5 per cent of the total number of strains (24 of 155). The third most common serovar was Salmonella Anatum with 12-3 per cent of the isolates (19 of 155) followed by Salmonella Tilburg representing 7-7 per cent of the strains (12 of 155) and Salmonella Virchow with 4-5 per cent (seven of 155). Less common serovars were Salmonella Choleraesuis var kunzendorf (six strains, 3-9 per cent), Salmonella Brandenburg (five strains, 3-2 per cent), Salmonella Derby and Salmonella Enteritidis (four strains, 2-6 per cent each), Salmonella Bredeney and Salmonella Hadar (three strains, 1-9 per cent each) and Salmonella Kedougou (two isolates, 1-3 per cent). Another 10 serovars (Abony, Falkensee, Give, Goldcoast, Grumpensis, Kapemba, Manhattan, Ohio, Rissen, S enterica subspecies salamae) were represented only once, accounting in total for 6-5 per cent of the strains. Antimicrobial susceptibility tests showed that most strains were resistant to tetracycline (84.2 per cent), sulphonamides (81-6 per cent) or ampicillin (74-5 per cent), regardless of the serovar. Antimicrobials to which more than 50 per cent of the strains were found to be resistant were chloramphenicol (57-5 per cent), nitrofurantoin (53-0 per cent) and trimethoprim (52-7 per cent). In some serovars (S 4,5,12:i:-, Choleraesuis and Brandenburg) all strains were found to be resistant to chloramphenicol. In contrast, all S Derby, S Bredeney and six of seven isolates of S Virchow were susceptible to chloramphenicol. Regarding susceptibility to quinolones, most S Tilburg ( 1 of 12) and S Virchow (six of seven) isolates were resistant to nalidixic acid, though resistant strains were also found in serovars Typhimurium, S4,5,12:i:-, Choleraesuis, Enteritidis, Hadar and Brandenburg. Strains resistant to enrofloxacin were only detected in Typhimurium (four strains, one of them resistant to ciprofloxacin) and Tilburg (two isolates) serovars. With other antimicrobials, distribution of resistances were not clearly attributable to any serovar except for the combination of amoxycillin and clavulanic acid that was prevalent in the Typhimurium serovar (27 of 56 strains) and for gentamicin in serovars S 4,5,12:i:(16 of 24 strains) and Tilburg (10 of 12 strains). Table 1 summarises the results of susceptibility tests performed and breakpoints used. Different patterns of multiple antibiotic resistance (four or more antimicrobial agents) were observed comprising 77