Characterization of Listeria monocytogenes from encephalitis cases of small ruminants from different geographical regions, in Greece.

AIMS: The aim of this study was to evaluate the genetic diversity and resistance phenotypes of Listeria monocytogenes strains isolated from clinical encephalitis cases, and compare this population to isolates derived from tank milk of healthy animals. METHODS AND RESULTS: A total of 57 L. monocytogenes strains isolated from ruminant's listeriosis cases (n = 31) and from tank milk of healthy ruminants (n = 26) were characterized by species PCR, molecular serotyping, PCR detection of virulence genes, pulsed-field gel electrophoresis and antimicrobial susceptibility testing. All strains possessed inlA, inlC, inlJ, plcA, actA, hlyA and iap virulence-associated genes while serotyping analysis revealed that they were mainly assigned into IVb group. Genotyping revealed 50 pulsotypes among the 57 strains assigned into seven clusters while indistinguishable pulsotypes between clinical and milk strains were not identified. Resistance of L. monocytogenes isolates to 14-16 antimicrobial agents tested was observed and 23 antimicrobial resistance profiles (ARPs) were defined while no apparent predominant ARP type was observed among isolates. CONCLUSIONS: Small ruminants are exposed to a broad range of antimicrobial-resistant as well as genetically diverse strains of L. monocytogenes carrying virulence-associated genes but not all of them associated with the disease. Pulsed-field gel electrophoresis analysis suggests that pulsotypes associated with encephalitis are found in farms only in association with listeriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings are valuable in understanding the ecology of this important food-borne pathogen and creating awareness for the emerging antimicrobial resistance.

Culture phenotypes and molecular characterization of Mycobacterium avium subsp. paratuberculosis isolates from small ruminants.

In this study the suitability of different solid media was investigated for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) in order to identify the optimum single or combination of media to permit the isolation of all strain types from small ruminants. A subset of these Map strains was then further characterized by molecular typing methods to assess the genetic diversity of Map strains in the study area (Northern Greece). Map strains were isolated from tissues and faeces of infected goats (n=52) and sheep (n=8) and were analysed for polymorphisms in IS1311 to classify the strain type as Type C or S. The study found that M7H11 supplemented with mycobactin j, OADC and new born calf serum (M7H11+Mj) is the best single choice of medium for the primary isolation of Map of both Type C and S from small ruminants. The combination of M7H11+Mj and Herrolds egg yolk medium supplemented with mycobactin j and sodium pyruvate allowed the detection of all Map isolates in this study. Nineteen Map isolates were characterised by pulsed-field gel electrophoresis and the isolates demonstrated significant genetic diversity. Twelve different SnaBI and 16 distinct SpeI profiles were detected of which 25 have not been described previously and are new profiles. The combination of both enzyme profiles gave 13 different multiplex profiles. Ten different multiplex profiles were detected in goats and three in sheep. One ovine isolate gave the same multiplex profile as a caprine isolate and two different profiles were found within a single goat herd.

Prevalence, seasonal occurrence and antimicrobial resistance of Salmonella in poultry retail products in Greece.

AIMS: To detect the prevalence, the seasonal occurrence and distribution of Salmonella serotypes in poultry products and to determine the resistance profile of Salmonella isolates. METHOD AND RESULTS: A total of 96 skin-on chicken carcasses and 30 liver samples were analysed between May 2007 and May 2009 from twenty-two different commercial farm brands found in retail market countrywide. Salmonella was isolated from 38 (39·5%) of 96 chicken carcasses and from 10 (33·3%) of 30 liver samples. Higher isolation rate (60·4%) was observed in carcasses detected during summer (May to October), and lower isolation rate (18·7%) was observed in carcasses detected during winter (November to April); in liver samples, the positive rates were 53·4 and 13·2%, respectively. Twelve serotypes were detected with the serotypes Hadar, Enteritidis and Blockley being the most prevalent at 29·2, 22·9 and 12·5%, respectively. Nine of 11 Salm. Enteritidis isolates occurred during summer. Of 48 isolates, 38 (79%) were resistant to one or more of the antimicrobial agents used. The highest resistance rates were found to the following antimicrobials: streptomycin (64·5%), tetracycline (56·2%), nalidixic acid (39·5%), ampicillin and rifampicin (33·3%). CONCLUSIONS: The relatively high Salmonella spp. contamination rates of raw chicken meat and liver have been detected. Salm. Enteritidis isolates peaked in summer, increasing the risk to human health. Antibiotic resistance of Salmonella still remains a threat as resistance plasmids may be extensively shared between animal and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The study enabled us to improve the data on the seasonal occurrence of Salmonella and to determine the antimicrobial pattern profile and trends in Salmonella strains isolated from poultry retail products in Greece.

Diversity of vanA-type vancomycin-resistant Enterococcus faecium isolated from broilers, poultry slaughterers and hospitalized humans in Greece.

OBJECTIVES: This study investigated the prevalence of vancomycin-resistant enterococci (VRE) in the broiler production environment after the avoparcin ban and their epidemiological relationship with human clinical VRE from the same geographical regions in Greece. METHODS: Caecal contents from broilers (n = 500) from eight livestock farms and faecal samples from poultry slaughterers (n = 50), all collected in two slaughterhouses during 2005-08, were analysed for species and vancomycin resistance gene identification using multiplex PCR. Sixty-three human clinical vancomycin-resistant Enterococcus faecium (VREF) isolates, obtained during 2006-09, were also examined. Discriminant analysis (DA) was used to establish the relationship of antimicrobial resistance profiles (ARPs) among broiler, poultry slaughterer and human clinical VREF. PFGE was conducted to study the genetic relatedness among VREF from the different sources. RESULTS: A total of 120 VRE were recovered from 113 (22.6%) broiler samples. VREF carrying the vanA gene were predominant, being recovered from 72 (14.4%) samples from five (62.5%) broiler farms. Concerning poultry slaughterers, VREF were recovered from 10 (20%) samples. Susceptibility testing revealed that broiler VREF were consistently resistant to tetracycline, whereas 93.7% of clinical VREF were resistant to ampicillin. Furthermore, 92.1% of clinical VREF compared with 54.4% of broiler VREF were multiresistant (resistant to at least five antimicrobial classes). DA classified broiler and human clinical VREF into their corresponding source with high classification rates (100% and 85.7%, respectively), while the classification rate of poultry slaughterer VREF was relatively low (50%), with 40% of them classified closely to broiler VREF. PFGE patterns were clearly related to the source of the VREF, with broiler isolates being clustered distinctly from all human isolates. CONCLUSIONS: A remarkable persistence of VREF was observed in the broiler production environment even >10 years after the avoparcin ban. Human and broiler VREF belonged to clearly unrelated populations, strongly indicating no clonal spread of VREF among the different sources, even between broilers and poultry slaughterers, despite them sharing common ARPs, as also supported by DA.