Molecular characterization and antimicrobial susceptibility of Clostridium difficile isolated from rabbits raised for meat production.

Clostridium difficile is an important cause of enteric disease in humans and animals. Recent studies demonstrated a genetic overlap between C. difficile isolated from animals and humans suggesting animals as possible reservoir for human pathogenic strains. This study was a preliminary investigation on the occurrence of C. difficile in rabbits raised in industrial holdings for food production and aimed to characterise isolates and estimate their antimicrobial susceptibility. C. difficile isolates were characterized by toxin profiles, toxinotyping and PCR-ribotyping. The MICs of six antibiotics were determined using E-test. Between 2007 and 2013, 285 industrial holdings (representing 40% of the national census) submitted rabbits to our laboratory for diagnostic purposes, among these holdings, groups of three to five post-weaned rabbits were sampled once by convenience. 1279 samples of caecal content were collected. The overall isolation rate of C. difficile from the enteric specimen was 3% (38/1279), with no difference among animals affected or not by enteric disorders. Among isolates 66% (25/38) were toxigenic. Sixteen different PCR-ribotypes (RTs) were identified. Among the toxigenic strains RT-014/020, RT-078 and RT-012 were found in at least three rabbit holdings. According to the ECOFF threshold, 82% (31/38) C. difficile isolates displayed a reduced susceptibility to at least one and 18% (7/38) to three tested antimicrobials. Rabbits are colonized by heterogeneous C. difficile ribotypes many of which are commonly isolated in humans. One third of isolates displayed a reduced susceptibility to MTZ, the first choice antimicrobial for human CDI treatment. According to our findings rabbits are a potential source of C. difficile for humans.

Antibiotic resistance patterns and PCR-ribotyping of Clostridium difficile strains isolated from swine and dogs in Italy.

Recent studies suggest animals, in particular farm and companion animals, as possible reservoir for Clostridium difficile human pathogenic strains. The aim of this study was to give a first characterization of C. difficile isolates from Italian swine and dogs. In total, 10 different PCR-ribotypes were identified among porcine strains and six among canine strains. The predominant type found among porcine strains was 078 (50%), whereas the most frequently detected among canine strains was the non-toxinogenic 010 (64%). Considering the CLSI breakpoints, 60% of porcine isolates was resistant to ERY, 35% to MXF, 15% to CLI, 5% to RIF, and none to MTZ or VAN. Among dogs, 51% of strains was resistant to CLI, 46% to ERY, 21% to MTZ and 5% to MXF or RIF, and none to VAN. Five porcine strains (10%) and 9 canine isolates (41%) were MDR. Interestingly, 8 MDR canine strains were highly resistant to MTZ, with MICs ≥32 mg/L. Considering the EUCAST cut-off for MTZ (MIC >2 mg/L), 13 canine isolates and one porcine strain were found with reduced susceptibility to MTZ (MICs ranging from 3 to ≥256 mg/L). Swine and canine strains showing resistance or reduced susceptibility to MTZ belonged to PCR-ribotype 010 and 078. These PCR-ribotypes have been associated to reduced susceptibility to MTZ also in human, suggesting a potential risk for the emergence of C. difficile strains resistant to the current first-line antibiotic for CDI treatment. The agar incorporation method (AIM) was confirmed as the best method to detect C. difficile strains with this phenotype also after strains manipulations. The results obtained add further evidences about the possible role of animals as source of MDR C. difficile strains and reservoir of antibiotic resistance determinants.

First reporting of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in an industrial rabbit holding and in farm-related people.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been described in food-producing animals and farm or slaughterhouse workers involved in the primary industrial production of swine, bovine and poultry. This communication describes the first case of LA-MRSA (ST398, spa types t034 and t5210) occurring in rabbits raised intensively for meat production and involving farm workers or their family members. In 2012-2013, in a study involving 40 rabbit industrial holdings in Italy, one farm was found to have rabbits colonized or infected with MRSA. Four farm workers and one of their relatives were found to be carrying MRSA. In this case holding, rabbits, people and the holding environment were further investigated and followed up by a second sampling five months later. MRSA was found in 48% (11/23) and 25% (15/59) of the rabbits carrying S. aureus at first and second samplings, respectively. Five months after first detection, some farm workers or family members were still MRSA carriers. Surface samples (2/10) and air samples (2/3) were contaminated with MRSA. Air samples yielded MRSA counts of 5 and 15CFU/m(3). MRSA from rabbits and people collected at first sampling were spa types t034 and t5210 belonging to ST398. The MRSA isolates from rabbits and persons tested at second sampling were t034 and t5210, but spa types t1190 and t2970 were also detected in MRSA isolates from rabbits. Tracing the epidemiological pattern earlier may prevent further spread of LA-MRSA in these food producing animals.

Development of PCR protocols for specific identification of Clostridium spiroforme and detection of sas and sbs genes.

Rabbit diarrhoea caused by toxigenic Clostridium spiroforme is responsible for significant losses in commercial rabbitries but the accurate identification of this micro-organism is difficult due to the absence of both a commercial biochemical panel and biomolecular methods. The aim of this study was therefore to develop PCR protocols for specific detection of C. spiroforme and its binary toxin encoding genes. The C. spiroforme specie-specific primers were designed based on its 16S rDNA published sequences and the specificity of these primers was tested with DNA extracted from closely related Clostridium species. The sa/bs_F and sa/bs _R C. spiroforme binary toxin specific primers were designed to be complementary, respectively, to a sequence of 21 bases on the 3' and of sas gene and on the 5' of the sbs gene. The detection limits of in house developed PCR protocols were 25CFU/ml of bacterial suspension and 1.38x10(4)CFU/g of caecal content for specie-specific primers and 80CFU/ml of bacterial suspension and 2.8x10(4)CFU/g of caecal content in case of sa/bs primers. These results indicated that the described PCR assays enable specific identification of C. spiroforme and its binary toxin genes and can therefore be considered a rapid, reliable tool for the diagnosis of C. spiroforme-related enterotoxaemia.