Towards a typing strategy for Arcobacter species isolated from humans and animals and assessment of the in vitro genomic stability

Arcobacter species have a widespread distribution with a broad range of animal hosts and environmental reservoirs, and are increasingly associated with human illness. To elucidate the routes of infection, several characterization methods such as pulsed-field gel electrophoresis (PFGE), amplified fragment-length polymorphism, and enterobacterial repetitive intergenic consensus (ERIC)-PCR have already been applied, but without proper validation or comparison. At present, no criterion standard typing method or strategy has been proposed. Therefore, after the validation of PFGE, those commonly applied typing methods were compared for the characterization of six human- and animal-associated Arcobacter species. With a limited number of isolates to be characterized, PFGE with restriction by KpnI is proposed as the first method of choice. However, ERIC-PCR represents a more convenient genomic fingerprinting technique when a large number of isolates is involved. Therefore, a first clustering of similar patterns obtained after ERIC-PCR, with a subsequent typing of some representatives per ERIC cluster by PFGE, is recommended. As multiple genotypes are commonly isolated from the same host and food, genomic plasticity has been suggested. The in vitro genomic stability of Arcobacter butzleri and A. cryaerophilus was assessed under two temperatures and two oxygen concentrations. Variability in the genomic profile of A. cryaerophilus was observed after different passages for different strains at 37 degrees C under microaerobic conditions. The bias due to these genomic changes must be taken into account in the evaluation of the relationship of strains

Co-occurrence of free-living protozoa and foodborne pathogens on dishcloths: implications for food safety

&lt;p&gt;In the present study, the occurrence of free-living protozoa (FLP) and foodborne bacterial pathogens on dishcloths was investigated. Dishcloths form a potentially important source of cross-contamination with FLP and foodborne pathogens in food-related environments. First various protocols for recovering and quantifying FLP from dishcloths were assessed. The stomacher technique is recommended to recover flagellates and amoebae from dishcloths. Ciliates, however, were more efficiently recovered using centrifugation. For enumeration of free-living protozoa on dishcloths, the Most Probable Number method is a convenient method. Enrichment was used to assess FLP diversity on dishcloths (n=38). FLP were found on 89% of the examined dishcloths; 100% of these tested positive for amoebae, 71% for flagellates and 47% for ciliates. Diversity was dominated by amoebae: vahlkampfiids, vannellids, Acanthamoeba spp., Hyperamoeba sp. and Vermamoeba vermiformis were most common. The ciliate genus Colpoda was especially abundant on dishcloths while heterotrophic nanoflagellates mainly belonged to the genus Bodo, the glissomonads and cercomonads. The total number of FLP in used dishcloths ranged from 10 to 10(4) MPN/cm(2). Flagellates were the most abundant group, and ciliates the least abundant. Detergent use was identified as a prime determinant of FLP concentrations on used dishcloths. Bacterial load on dishcloths was high, with a mean total of aerobic bacteria of 7.47 log 10 cfu/cm(2). Escherichia coli was detected in 68% (26/38) of the used dishcloths, with concentrations up to 4 log 10 cfu/cm(2). Foodborne pathogens including Staphylococcus aureus (19/38), Arcobacter butzleri (5/38) and Salmonella enterica subsp. enterica ser. Halle (1/38) were also present. This study showed for the first time that FLP, including some opportunistic pathogens, are a common and diverse group on dishcloths. Moreover, important foodborne pathogens are also regularly recovered. This simultaneous occurrence makes dishcloths a potential risk factor for cross-contamination and a microbial niche for bacteria-FLP interactions.&lt;/p&gt;</p

Diversity and Habitat Specificity of Free-Living Protozoa in Commercial Poultry Houses

Despite stringent biosecurity measures, infections by bacterial food pathogens such as Campylobacter are a recurrent problem in industrial poultry houses. As the main transmission route remains unclear, persistence of these infections has been linked to bacterial survival and possibly multiplication within protozoan vectors. To date, however, virtually no information is available on the diversity and occurrence of free-living protozoa in these environments. Using a combination of microscopic analyses of enrichment cultures and molecular methods (denaturing gradient gel electrophoresis [DGGE]) on natural samples, we show that, despite strict hygiene management, free-living protozoa are common and widespread throughout a 6-week rearing period in both water and dry samples from commercial poultry houses. Protozoan communities were highly diverse (over 90 morphotaxa and 22 unique phylotypes from sequenced bands) and included several facultative pathogens and known bacterial vectors. Water samples were consistently more diverse than dry ones and harbored different communities, mainly dominated by flagellates. The morphology-based and molecular methods yielded markedly different results: amoebic and, to a lesser degree, ciliate diversity was seriously underestimated in the DGGE analyses, while some flagellate groups were not found in the microscopic analyses. Some recommendations for improving biosecurity measures in commercial poultry houses are suggested

Protozoan cysts act as a survival niche and protective shelter for foodborne pathogenic bacteria

The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 mu g/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain-and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed

Impact of Acanthamoeba cysts on stress resistance of Salmonella enterica serovar Typhimurium, Yersinia enterocolitica 4/O:3, Listeria monocytogenes 1/2a, and Escherichia coli O:26

The formation of robust resting cysts enables Acanthamoeba to resist harsh environmental conditions. This study investigated to what extent these cysts are resistant to physical and chemical stresses as applied in food industry cleaning and disinfection procedures. Moreover, it was assessed whether certain intracystic meat-borne bacterial pathogens are more stress resistant than free-living bacterial monocultures and if intracystic passage and subsequent association with trophozoites induces cross-tolerance toward other stressors. Several physical and chemical stressors (NaCl, H2O2, benzalkonium chloride, 55 degrees C, heating until boiling, ethanol, dishwashing detergent, and sodium hypochlorite) frequently used in domestic and industrial food-related environments were tested against (i) Acanthamoeba castellanii cysts, (ii) single strains of bacterial monocultures, (iii) intracystic bacteria, and (iv) bacteria after intracystic passage (cyst-primed bacteria). Only heating until boiling and hypochlorite treatment were cysticidal. After boiling, no viable trophozoites could be recovered from the cysts, and hypochlorite treatment caused a 1.34-to 4.72-log10 cells/ml reduction in cyst viability. All treatments were effective in reducing or even eliminating the tested bacterial monocultures, whereas bacteria residing inside cysts were more tolerant toward these stressors. All cyst-primed bacteria exhibited an increased tolerance toward subsequent H2O2 (>92% decrease in median log10 CFU/ml reduction) and 70% ethanol (>99% decrease) treatments. Moreover, intracystic passage significantly increased the survival of Yersinia enterocolitica (74% decrease in median log10 reduction), Escherichia coli (58%), and Salmonella enterica (48%) after NaCl treatment and of E. coli (96%), S. enterica (99%), and Listeria monocytogenes (99%) after sodium hypochlorite treatment compared with that of nonprimed bacteria. IMPORTANCE: The results from this study demonstrated that both viable and nonviable amoebal cysts can protect internalized bacteria against stressful conditions. Moreover, cyst passage can induce cross-tolerance in bacteria, increasing their survival when exposed to selected stressors. These findings underscore the potential importance of free-living amoebae in food-related environments and their impact on the persistence of meat-borne bacterial pathogens