Antibiotic-resistance patterns of faecal indicator organisms and occurrence of Salmonella spp. in wild boar (Sus scrofa scrofa) in Italy.

Antibiotics have become commonplace in our environment in consequence of their wide use in animal husbandry and medical therapy. Although data concerning antibiotic resistance in wild animals are scarce, some surveys have demonstrated the presence of multi-resistant indicator organisms in their faeces. The aim of this study was monitoring antibiotic-resistance in faecal indicator organisms isolated from wild boar (Sus scrofa scrofa) population living in two natural Regional Parks (RP) of Bologna province. Furthermore, occurrence and antibiotic resistance of Salmonella spp. were evaluated. From September 2002 to June 2003, faeces from 55 wild boars from Gessi Bolognesi RP (48.15 Km2) and 55 from Monte Sole RP (69.34 Km2) located in the Emilia-Romagna Region were collected. No contact between the different populations of the two RP had been recorded. The possibility of interactions and hybridisation between wild boars and domestic pigs was demonstrated in both parks, by capturing hybrid phenotype animals. Faecal samples were collected directly from the rectum, placed in BBL-Cary-Blair Transport Medium, stored at 4°C, transported to the laboratory and processed within 24 h after collection. In order to isolate E. coli, faeces were streaked directly onto BBL-MacConkey Agar and incubated at 37°C for 18-24 h. Strains identification was carried out by API20E. For the isolation of E. faecalis and E. faecium, 1 g of faeces was suspended in 4 ml of saline solution and serially 10-fold diluted until 10-4; 0,1 ml of each dilution was then inoculated on Bile Esculin Azide Agar and incubated at 37°C for 72 h. Isolates were identified at genus level on the basis of colony morphology, Gram staining, catalase production, esculin hydrolysis, growth in 6,5% NaCl, bile tolerance and L-pyrrolydonyl-β-naphtylamide hydrolysis. Identification of E. faecium and E. faecalis isolates was performed by PCR. In order to isolate Salmonella spp. 5 g of each sample were inoculated in Difco-Muller-Kauffmann Tetrathionate Broth and Selenite Broth and incubated at 42°C and 37°C for 24 h, respectively. Enrichment broth’s culture were then seeded onto two different media: Brilliant Green Agar and XLT4 Agar. Salmonella suspect colonies were identified by commercial system API 20E. A single isolate of E. coli, E. faecalis, E. faecium and Salmonella spp. from each sample was tested for antimicrobial susceptibility using the agar diffusion method recommended by CLSI.The following 16 antimicrobial agents were included in the study for E. coli and Salmonella spp. using BBL Sensi-Disc: amikacin, amoxicillin-clavulanic acid, ampicillin, cefazolin, cefotaxime, chloramphenicol, colistin, enrofloxacin, gentamicin, kanamycin, nalidixic acid, spectinomycin, streptomycin, sulfisoxazole, tetracycline and trimethoprim-sulfamethoxazole The antibiotic susceptibility of Enterococcus spp. was assessed using the following molecules: ampicillin, chloramphenicol, ciprofloxacin, erythromycin, penicillin, rifampin, tetracycline and teicoplanin. CLSI interpretative standards for Enterobacteriacae and Enterococcus were used and the intermediate category was considered, in this study, as resistant. Multiple resistance was defined when resistance to three or more unrelated antimicrobial agents was found. Vancomycin and the high level aminoglycoside resistances of E. faecalis and E. faecium were evaluated by the screening test described by CLSI. From the 110 wild boars tested a total of 110 E. coli, 48 E. faecium and 5 E. faecalis strains were isolated. Differences in isolation rate from the animals living in the two Regional parks were observed for Enterococcus spp.: E. faecium was isolated from 33 subjects from Gessi Bololgnesi RP and 15 animals from Monte Sole RP while E. faecalis was isolated only from 5 wild boars in Monte Sole RP; no antibiotic resistance was detected in these strains. In this study, E. coli and Salmonella strains showed a low antibiotic resistance level.