Giancarlo Ripabelli

Occurrence of Vibrio and other pathogenic bacteria in Mytilus galloprovincialis (mussels) harvested from Adriatic Sea, Italy

Sixty-two samples of Mytilus galloprovincialis (mussels) harvested from approved shellfish waters in the Adriatic Sea were examined for the presence of Vibrio, Salmonella, Campylobacter, and verocytotoxin producing Escherichia coli. Vibrio spp. were isolated from 48.4% of samples; the species most frequently found were V. alginolyticus (32.2%) and V. vulnificus (17.7%), followed by V. cincinnatiensis (3.2%), V. parahaemolyticus (1.6%), V. fluvialis (1.6%) and V. cholerae non-O1 (1.6%). V. parahaemolyticus resulted negative to Kanagawa-phenomenon and to PCR amplification of tdh gene. V. cholerae resulted negative to PCR amplification of sto gene. No Salmonella, Campylobacter, or E. coli verocytotoxin-producing strains were isolated. The results of this study suggest the potential risk of ingesting raw or undercooked mussels due to the frequent presence of potentially pathogenic Vibrio species.

Prevalence of Salmonellae, Listeriae, and Yersiniae in the Slaughterhouse Environment and on Work Surfaces, Equipment, and Workers

In 1995 and 1996 a nine-month study was carried out in 11 pig abattoirs located in the Molise region (Italy) to evaluate the degree of contamination of- the slaughterhouse environment, work surfaces, equipment, and personnel by Salmonella spp., Listeria spp., and Yersinia spp. A total of 219 samples were taken over three replications including slaughtering floor and wall, hooks, work-tables, chopping blocks, knives, cleavers, dehairing devices, hands of personnel, clothing, hand-wash basins, and cold room handles, floor, wall, and hooks. Overall, six abattoirs (54.5%) had one or more positive sites, while only 14 of the 219 sites (6.4%) tested were positive for any of considered microorganisms. Salmonella spp. were isolated from 1 of 9 cleavers (11.1 %), 1 of 16 worktables (6.25%), and 1 of 18 slaughtering floors (5.6%). Yersinia enterocolitica was found on 3 slaughtering floors (16.7%) and on 2 worktables (12.5%). Yersinia kristensenii was detected on 2 slaughtering floor swabs (11.1 %). Listeria monocytogenes was isolated from 2 of 20 cold room floor swabs (13.3%) and from 1 of 14 hand-wash basins (7.1%). Other species of Listeria were detected on slaughtering wall and floor swabs and on chopping blocks. Our study indicates that slaughtering floors, cold room floors, and worktables are important sites in abattoirs that may possibly harbor pathogens like Salmonella spp., Yersinia enterocolitica , and Listeria monocytogenes , and that cleaning and sanitizing of the slaughterhouse environment and equipment need a greater emphasis.

Prevalence of Helicobacter-like organisms in porcine gastric mucosa: A study of swine slaughtered in Italy

Recent reports described some cases of gastritis in man caused by an uncultured gram-negative spiral bacterium morphologically identical to organisms observed in the stomachs of mammalians (e.g. cats, dogs, pigs). The aim of the present study is to confirm the presence of these bacteria in Italian swine. Tightly spiralled organisms (Gastrospirillum suis) were found in the stomach of eight (9.4%) out of 85 pigs examined. The bacteria were always associated with macroscopic lesions indicative of gastritis. Attempts to culture H. pylori or Helicobacter-like organisms were unsuccessful. The possibility that Gastrospirillum may be a zoonotic pathogen, with transmission occurring from pigs to humans, is discussed.

Prevalence of virulence-associated genes and cytolethal distending toxin production in Campylobacter spp. isolated in Italy.

The prevalence of virulence and cytolethal distending toxin (CDT) genes and the cytotoxic activity in Vero and HEp-2 cells was estimated in 29 Campylobacter jejuni and 36 Campylobacter coli from foods, animals and humans isolates. All C. jejuni showed flaA, cadF, cdtA, cdtB, cdtC and cdt cluster genes fragments, except for ceuE (86.2%) and cdt genes (93.1%). Amongst C. coli strains, a lower prevalence of ceuE gene (83.3%) was detected than that for cdtA, cdtB, cdtC genes (97.2%), cdt gene cluster (94.4%) and cdt genes (86.1%); whereas flaA and cadF genes were amplified in all isolates. Despite the high prevalence of CDT genes only 8 (27.6%) C. jejuni and 1 (2.8%) C. coli showed evidence for cytotoxin production in HEp-2 cells. However, how CDT positive and CDT negative strains differ in their biological properties remains unknown, but the relative higher prevalence of cytotoxicity in C. jejuni could be consistent with its predominant epidemiological role in human infections.

Immunomagnetic separation and conventional culture procedure for detection of naturally occurring Salmonella in raw pork sausages and chicken meat

The aim of the study was to compare immunomagnetic separation (IMS) and conventional selective enrichment procedures using selenite cystine broth (SC) and Rappaport–Vassiliadis broth (RV) in 137 naturally contaminated food samples (69 raw pork sausages and 68 chicken meat). The utilization of SC or IMS appeared to be the most appropriate enrichment procedure: 15 out of 18 Salmonella‐positive samples (83·3%) were detected by SC and 12 (66·7%) by IMS; RV yielded only seven positive isolations (38·9%). However, RV yielded the highest count of Salmonella colonies per plate and the lowest interference by competing organisms. IMS could become a reliable alternative to standard enrichment procedures and a combined IMS and selective enrichment broth could increase the chance of Salmonella recovery.

Prevalence and biomolecular characterization of Campylobacter spp. isolated from retail meat.

We estimated the prevalence of Campylobacter spp. in retail meat (n = 352 samples; 104 chicken, 106 pork, and 142 beef) collected in Campobasso, Italy, comparing two microbiological methods. All the isolates were characterized by biomolecular techniques for epidemiological purposes. Campylobacter isolation was performed by selective culture and membrane filtration methods. Phenotypic and genotypic methods for genus and species identification were evaluated together with antimicrobial resistance and plasmid profiling. Sixty-nine (86.2%) samples were positive by selective culture, 49 (61.2%) by membrane filtration, and 38 (47.5%) by both methods. Only 74 of 80 strains were confirmed as Campylobacter spp. by PCR, and two Campylobacter coli were identified as Campylobacter jejuni. Chicken meat was more frequently contaminated than other meats. Selective culture was more sensitive than membrane filtration (85 versus 66%), and specificity of the methods was 98 and 100%, respectively. Among Campylobacter isolates from chicken meat, 86.5% were multidrug resistant. Resistance to ciprofloxacin (51.3%) and enrofloxacin (52.7%) was lower than to nalidixic acid (71.6%). C. coli strains showed the highest cross-resistance for quinolones (82.6%) and fluoroquinolones (60.9%) as well as a high resistance to tetracycline. Plasmids were isolated from six C. coli and two C. jejuni isolates, but no association was detected between antimicrobial resistance and plasmid DNA carriage. Selective culture is considered as the optimal method for Campylobacter isolation, although it was unable to detect all contaminated samples. Membrane filtration provided more specific results but with low sensitivity. A combination of both techniques may offer better results.

Typing of Listeria monocytogenes strains isolated in Italy by inlA gene characterization and evaluation of a new cost-effective approach to antisera selection for serotyping.

Aims:  In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost‐effective methodological approach based on preliminary PCRs screening was proposed.

Microbiological and toxicological quality of dried herbs

Aims:  The microbiological and toxicological quality of 51 samples of dried herbs (Melissa officinalis, Salvia officinalis, Malva sylvestris, Matricaria chamomilla, Alchemilla vulgaris and Centaurea cyanus) cultivated in family‐managed farms in Molise Region (Italy) was evaluated.

Evaluation of immunomagnetic separation and plating media for recovery of Salmonella from meat.

Immunomagnetic separation (IMS) was compared with selective enrichment in selenite cystine (SC) broth for isolation of Salmonella from 86 artificially contaminated ground beef samples. Both Rambach agar (RA) and Hektoen enteric (HE) agar were used as selective plating media. The highest count of Salmonella colonies per plate was obtained after enrichment in SC broth and plating on RA (mean value: 111.1+/-58.1 CFU per plate); the lowest count was obtained after IMS and plating on HE agar (mean value: 65.4+/-36.6 CFU per plate). Salmonella in preenrichment was concentrated 1.7-fold by IMS and represented 31% of the microbial population captured by the beads, but nonspecific binding was high. As a result of the large numbers of competing bacteria, isolations on both RA and HE agar following IMS were quite difficult (mean value for Salmonella colonies: 79.9+/-42.7 CFU per plate; mean value for non-Salmonella colonies: 144.1+/-75.7 CFU per plate; ratio of Salmonella to non-Salmonella colonies: 0.8). This study indicates that SC broth is superior to IMS in the isolation of Salmonella from raw ground meat.

Prevalence and genotype identification of human papillomavirus in women undergoing voluntary cervical cancer screening in Molise, Central Italy

We examined the prevalence of HR- and LR-HPV by Linear Array genotyping test in 299 women aged 18-63 years who consecutively visited Molise Region main hospitals for routine Pap smear between February and August 2008. Ninety women were positive for any HPV (30.1%), and 66 for any HR-HPV (22.1%). The most prevalent HR-HPV types were HPV 16 (22.2% of all women with HPV infection), HPV 53 (14.4%), and HPV 66 (14.4%). HPV infections increased from 15.8% in the 18-20 years group to 50.0% in the 21-23 years group and then decreased to 9.1% in those aged 50 years or more (p=0.008). Multiple HPV infections were observed in 15.7% of the study sample (52.2% of all HPV positive). There is a significantly higher prevalence of multiple infections in 18-32 years group women (24.5%) compared with females aged 33 years or more (6.8%) (p<0.005). Current smokers were at increased risk of HPV infection (44.2% of HPV infections compared with 23.5% in never smokers, and 25.3% of multiple HPV infections compared with 11.3%; p=0.001). HR-HPV infections were higher in women never been pregnant (27.1% compared with 7.7%; p=0.001). Oral contraceptive use was completely unrelated to infection. Among the 122 women who had both cytological examination and HPV results, multiple HR-HPV types were found in 36.8% of those with abnormal cervical findings, and in 13.6% of those with normal cervical findings (p=0.05). The results of the present investigation provide further evidence for the notion that cervical HPV infection is more widespread than previously suggested.