Detection, enumeration and characterization of Yersinia enterocolitica 4/O:3 in pig tonsils at slaughter in Northern Italy

Abstract

Tonsils from 150 pigs slaughtered at 270days or older were tested for Yersinia enterocolitica with different cultural methods. Samples were collected in three different abattoirs of Northern Italy between April and November 2012 and were analysed by direct plating on cefsulodin–irgasan–novobiocin (CIN) agar and by enrichment procedures following the ISO 10273:2003 reference method. Twenty-three (15.3%) samples were positive: 22 tonsils (14.7%) were positive for human pathogenic Y. enterocolitica bio-serotype 4/O:3 and one tonsil (0.7%) for Y. enterocolitica bio-serotype 1A/7,8-8,8,19. Seventeen samples out of 23 (73.9%) were positive by direct plating method. Among the enrichment procedures, the best recovery rate (8 positives out of 23; 34.8%) was obtained by the two-day enrichment in peptone–sorbitol–bile (PSB) broth followed by plating on CIN agar plates. The two-day enrichment in PSB followed by potassium hydroxide (KOH) treatment before plating onto CIN agar gave 7 positives out of 23 (30.4%), decreasing to 3 positives (13.0%) without KOH treatment. The worst results were obtained by prolonged (five days) enrichment in PSB, with or without KOH treatment, followed by plating on CIN agar: 4.3% (1 out of 23) and 0.0% recovery rates, respectively. The mean concentration was 1.9×104CFU/g, with a minimum of 1.0×102CFU/g and a maximum of 5.8×104CFU/g, thus demonstrating that tonsils may play an important role in contamination of pluck sets, carcasses, and slaughterhouse environment.Prevalence of virulence genes among the Y. enterocolitica 4/O:3 isolates was as follows: 12/22 (54.5%) for yadA, 21/22 (95.5%) for ail, 21/22 (95.5%) for inv and 22/22 (100%) for ystA.All Y. enterocolitica 4/O:3 isolates were sensitive to amoxicillin/clavulanic acid, ciprofloxacin and ceftazidime and resistant to ampicillin and cephalotin. High proportions of 4/O:3 isolates (95%) were sensitive to cefotaxime, gentamicin, kanamicin and nalidixic acid. High levels of resistance were observed to sulphonamide compounds (91%), streptomycin (64%) and chloramphenicol (55%). Multi-resistant isolates were very common; resistance to three or more antimicrobials was observed in 91% (20/22) of 4/O:3 isolates. High level of resistance to chloramphenicol was possibly due to coresistance to tiamphenicol, which was detected in 100% of the isolates.XbaI-PFGE detected four clusters among the 22 Y. enterocolitica 4/O:3 isolates. The most represented accounted for 77% (17/22) of the isolates, the second most common was found in 14% (3/22) of the isolates and the two other profiles were observed in single isolates. The comparison with a selection of human isolates supported the role of the pig as reservoir of 4/O:3 Y. enterocolitica.