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°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.

Transfer of Campylobacter from a Positive Batch to Broiler Carcasses of a Subsequently Slaughtered Negative Batch: A Quantitative Approach.

The present study was conducted to quantify Campylobacter cross-contamination from a positive batch of broiler chicken carcasses to a negative batch at selected processing steps and to evaluate the duration of this cross-contamination. During each of nine visits conducted in three broiler slaughterhouses, Campylobacter levels were determined on broiler carcasses originating from Campylobacter-negative batches processed immediately after Campylobacter-positive batches. Data were collected after four steps during the slaughter process (scalding, plucking, evisceration, and washing) at 1, 10, and 20 min after the start of the slaughter of the batches. Campylobacter levels in ceca of birds from Campylobacter-positive batches ranged from 5.62 to 9.82 log CFU/g. When the preceding positive batch was colonized at a low level, no (enumerable) carcass contamination was found in a subsequent negative batch. However, when Campylobacter levels were high in the positive batch, Campylobacter was found on carcasses of the subsequent negative batch but at levels significantly lower than those found on carcasses from the preceding positive batch. The scalding and the evisceration process contributed the least (< 1.5 log CFU/g) and the most (up to 4 log CFU/ g), respectively, to the Campylobacter transmission from a positive batch to a negative batch. Additionally, the number of Campylobacter cells transferred from positive to negative batches decreased over the first 20 min of sampling time. However, the reduction was slower than previously estimated in risk assessment studies, suggesting that pathogen transfer during crosscontamination is a complex process.

Identification of risk factors for Campylobacter contamination levels on broiler carcasses during the slaughter process.

Campylobacter carcass contamination was quantified across the slaughter line during processing of Campylobacter positive batches. These quantitative data were combined together with information describing slaughterhouse and batch related characteristics in order to identify risk factors for Campylobacter contamination levels on broiler carcasses. The results revealed that Campylobacter counts are influenced by the contamination of incoming birds (both the initial external carcass contamination and the colonization level of caeca) and the duration of transport and holding time that can be linked with feed withdrawal period. In addition, technical aspects of the slaughter process such as a dump based unloading system, electrical stunning, lower scalding temperature, incorrect setting of plucking, vent cutter and evisceration machines were identified as risk factors associated with increased Campylobacter counts on processed carcasses. As such the study indicates possible improvements of the slaughter process that can result in better control of Campylobacter numbers under routine processing of Campylobacter positive batches without use of chemical or physical decontamination. Moreover, all investigated factors were existing variations of the routine processing practises and therefore proposed interventions are practically and economically achievable.

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 µg/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.

Transmission electron microscopy sample preparation protocols for the ultrastructural study of cysts of free-living protozoa.

Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available. Transmission electron microscopy (TEM) is ideally suited for those analyses; however, conventional TEM protocols are not satisfactory for cysts of free-living protozoa. Here we report on the design and testing of four protocols for TEM sample preparation of cysts. Two protocols, one based on chemical fixation in coated well plates and one on high-pressure freezing, were selected as the most effective for TEM-based ultrastructural studies of cysts. Our protocols will enable improved analysis of cyst structure and a better understanding of bacterial survival mechanisms in cysts.

Comparison of sample types and analytical methods for the detection of highly campylobacter-colonized broiler flocks at different stages in the poultry meat production chain.

Exclusion of broiler batches, highly colonized with Campylobacter (>7.5 log10 colony-forming units/g), from the fresh poultry meat market might decrease the risk of human campylobacteriosis. The objective of this study was to compare different sample types (both at the farm and the slaughterhouse) and methods (direct culture, quantitative real-time polymerase chain reaction [qPCR], propidium monoazide [PMA]-qPCR) applied for the quantification of the Campylobacter colonization level. In addition, the applicability of the lateral flow-based immunoassay, Singlepath(®) Direct Campy Poultry test (Singlepath(®) test), was evaluated as a rapid method for the qualitative detection of Campylobacter in highly colonized broiler batches. Campylobacter counts differed significantly between sample types collected at farm level (cecal droppings, feces, boot swabs) and at slaughterhouse level (cecal content, fecal material from crates). Furthermore, comparison of Campylobacter counts obtained by different methods (direct culture, qPCR, PMA-qPCR) in cecal droppings revealed significant differences, although this was not observed for cecal-content samples. Evaluation of the Singlepath(®) test on cecal droppings and cecal-content samples revealed an acceptable level of sensitivity and specificity. In conclusion, cecal droppings and cecal content are proposed as the most representative sample types for quantification of Campylobacter colonization level of broilers at farm and slaughterhouse, respectively. Direct culture and qPCR are equally sensitive for quantification of Campylobacter in fresh cecal-content samples. PMA treatment before qPCR inhibits the signal from dead Campylobacter cells. Consequently, when samples are extensively stored and/or transported, qPCR is preferred to direct culture and PMA-qPCR. Furthermore, the Singlepath(®) test offers a convenient alternative method for rapid detection of Campylobacter in highly colonized broiler batches.

Campylobacter carcass contamination throughout the slaughter process of Campylobacter-positive broiler batches.

Campylobacter contamination on broiler carcasses of Campylobacter colonized flocks was quantified at seven sampling sites throughout the slaughter process. For this purpose, in four slaughterhouses samples were collected from twelve Campylobacter positive batches. Broilers from all visits carried high numbers of campylobacters in their caeca (≥7.9log10cfu/g). Campylobacter counts on feathers (up to 6.8log10cfu/g), positively associated with the breast skin contamination of incoming birds and carcasses after plucking, were identified as an additional source of carcass contamination. A high variability in Campylobacter carcass contamination on breast skin samples within batches and between batches in the same slaughterhouse and between slaughterhouses was observed. In slaughterhouses A, B, C and D Campylobacter counts exceeded a limit of 1000cfu/g on 50%, 56%, 78% and 11% of carcasses after chilling, respectively. This finding indicates that certain slaughterhouses are able to better control Campylobacter contamination than others. Overall, the present study focuses on the descriptive analysis of Campylobacter counts in different slaughterhouses, different batches within a slaughterhouse and within a batch at several sampling locations.

Evaluation of a new chromogenic medium for direct enumeration of Campylobacter in poultry meat samples.

The present study was conducted to compare Campylobacter counts obtained by three selective media: modified charcoal cefoperazonedeoxycholate agar (mCCDA), Campy Food agar (CFA), and a novel agar RAPID' Campylobacter agar. Analysis of 12 artificially and 36 naturally contaminated samples indicated no significant differences in Campylobacter counts obtained with all three selective media. Lin's concordance correlation coefficient (CCC) and the Bland-Altman plot revealed a high level of agreement between Campylobacter counts when evaluating RAPID versus mCCDA and CFA plates. RAPID agar was the only medium tested that could effectively suppress the growth of the background microflora with naturally contaminated samples. Results of this study clearly indicated that RAPID agar is highly selective without loss of sensitivity for recovering Campylobacter. Results obtained are in agreement with those for other commonly used media; therefore, RAPID medium is suitable for Campylobacter enumeration in poultry meat samples.

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°C under microaerobic conditions. The bias due to these genomic changes must be taken into account in the evaluation of the relationship of strains.

Interaction of Aspergillus fumigatus conidia with Acanthamoeba castellanii parallels macrophage-fungus interactions.

Aspergillus fumigatus and free-living amoebae are common inhabitants of soil. Mechanisms of A. fumigatus to circumvent the amoeba's digestion may facilitate overcoming the vertebrate macrophage defence mechanisms. We performed co-culture experiments using A. fumigatus conidia and the amoeba Acanthamoeba castellanii. Approximately 25% of the amoebae ingested A. fumigatus conidia after 1 h of contact. During intra-amoebal passage, part of the ingested conidia was able to escape the food vacuole and to germinate inside the cytoplasm of A. castellanii. Fungal release into the extra-protozoan environment by exocytosis of conidia or by germination was observed with light and transmission electron microscopy. These processes resulted in structural changes in A. castellanii, leading to amoebal permeabilization without cell lysis. In conclusion, A. castellanii internalizes A. fumigatus conidia, resulting in fungal intracellular germination and subsequent amoebal death. As such, this interaction highly resembles that of A. fumigatus with mammalian and avian macrophages. This suggests that A. fumigatus virulence mechanisms to evade macrophage killing may be acquired by co-evolutionary interactions among A. fumigatus and environmental amoebae.

Occurrence of putative virulence genes in arcobacter species isolated from humans and animals.

Interest in arcobacters in veterinary and human public health has increased since the first report of the isolation of arcobacters from food of animal origin. Since then, studies worldwide have reported the occurrence of arcobacters on food and in food production animals and have highlighted possible transmission, especially of Arcobacter butzleri, to the human population. In humans, arcobacters are associated with enteritis and septicemia. To assess their clinical relevance for humans and animals, evaluation of potential virulence factors is required. However, up to now, little has been known about the mechanisms of pathogenicity. Because of their close phylogenetic affiliation to the food-borne pathogen Campylobacter and their similar clinical manifestations, the presence of nine putative Campylobacter virulence genes (cadF, ciaB, cj1349, hecA, hecB, irgA, mviN, pldA, and tlyA) previously identified in the recent Arcobacter butzleri ATCC 49616 genome sequence was determined in a large set of human and animal Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii strains after the development of rapid and accurate PCR assays and confirmed by sequencing and dot blot hybridization.

Occurrence and diversity of free-living protozoa on butterhead lettuce.

The occurrence and diversity of free-living protozoa (FLP) on butterhead lettuce (Lactuca sativa L.) was investigated using four different sampling techniques (washing, swabbing, homogenization, and excising). FLP were recovered from all leaf samples (n=64), and cultures were FLP-positive after 1 week. Identification of FLP was performed by light microscopy and sequencing of denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments. Bodo saltans, Spumella (-like) spp. and Cercozoa were the most common heterotrophic nanoflagellates. Amoebae belonged mainly to the Vannellida and Tubulinida. Colpoda steinii and Cyclidium glaucoma were the most common ciliates. The total number of FLP on middle leaves estimated by the Most Probable Number method ranged from 9.3 × 10(2)MPN/g to 2.4 × 10(5)MPN/g leaf, with flagellates (92 MPN/g to 2.4 ×10(5)MPN/g) being more abundant than amoebae (<3 MPN/g to 9.3 × 10(3)MPN/g) and ciliates (<3 MPN/g to 9.3 × 10(2)MPN/g). Washing or rinsing leaves followed by spin-drying in a household salad spinner reduced the protozoan number with maximum one log unit. Our survey shows that FLP on lettuce leaves are a common and diverse but largely unexplored group of microorganisms.

Persistence of free-living protozoan communities across rearing cycles in commercial poultry houses.

The introduction and survival of zoonotic bacterial pathogens in poultry farming have been linked to bacterial association with free-living protozoa. To date, however, no information is available on the persistence of protozoan communities in these environments across consecutive rearing cycles and how it is affected by farm- and habitat-specific characteristics and management strategies. We therefore investigated the spatial and temporal dynamics of free-living protozoa in three habitats (pipeline, water, and miscellaneous samples) in three commercial poultry houses across three rearing cycles by using the molecular fingerprinting technique denaturing gradient gel electrophoresis (DGGE). Our study provides strong evidence for the long-term (ca. 6-month) persistence of protozoa in broiler houses across consecutive rearing cycles. Various free-living protozoa (flagellates, ciliates, and amoebae), including known vectors of bacterial pathogens, were observed during the down periods in between rearing cycles. In addition, multivariate analysis and variation partitioning showed that the protozoan community structure in the broiler houses showed almost no change across rearing cycles and remained highly habitat and farm specific. Unlike in natural environments, protozoan communities inside broiler houses are therefore not seasonal. Our results imply that currently used biosecurity measures (cleaning and disinfection) applied during the down periods are not effective against many protozoans and therefore cannot prevent potential cross-contamination of bacterial pathogens via free-living protozoa between rearing cycles.

Influence of temperature, oxygen and bacterial strain identity on the association of Campylobacter jejuni with Acanthamoeba castellanii.

Campylobacteriosis is the most frequently reported foodborne disease in the industrialized world, mainly through consumption of contaminated chicken meat. To date, no information is available on the primary infection sources of poultry. In this study, the ability of five Campylobacter jejuni strains with different invasion potential towards Caco-2 cells to survive and replicate in the protozoan Acanthamoeba castellanii was tested under simulated in situ conditions (i.e. chicken broiler houses). Results indicate that environmental conditions play a crucial role in C. jejuni-A. castellanii interactions. Co-culture in general did not result in an increase of either bacteria or amoebae. However, co-culture with Acanthamoeba did result in a delayed decline and an increased long-term survival of Campylobacter. Bacterial strain-specific effects were observed, with higher survival rates for low-invasive strains. The presence of C. jejuni in general did not affect A. castellanii viability, except at 37 °C under microaerobic conditions, where the presence of the reference and low-invasive Campylobacter strains resulted in a significant decline in amoebal viability. Confocal laser scanning microscopy revealed that intra-amoebal campylobacters were not always colocated with acidic organelles, suggesting potential bacterial interference with digestive processes. As Acanthamoeba enhances the persistence of C. jejuni, the presence of the amoeba in broiler house environments may have important implications for the ecology and epidemiology of this food pathogen.

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.

Microscopic and molecular studies of the diversity of free-living protozoa in meat-cutting plants.

The diversity of free-living protozoa in five meat-cutting plants was determined. Light microscopy after enrichment culturing was combined with sequencing of PCR-amplified, denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments, which was used as a fast screening method. The general results of the survey showed that a protozoan community of amoebae, ciliates, and flagellates was present in all of the plants. Protozoa were detected mainly in floor drains, in standing water on the floor, on soiled bars of cutting tables, on plastic pallets, and in out-of-use hot water knife sanitizers, but they were also detected on surfaces which come into direct contact with meat, such as conveyer belts, working surfaces of cutting tables, and needles of a meat tenderizer. After 7 days of incubation at refrigerator temperature, protozoa were detected in about one-half of the enrichment cultures. Based on microscopic observations, 61 morphospecies were found, and Bodo saltans, Bodo spp., Epistylis spp., Glaucoma scintillans, Petalomonas spp., Prodiscophrya collini, and Vannella sp. were the most frequently encountered identified organisms. Sequencing of DGGE bands resulted in identification of a total of 49 phylotypes, including representatives of the Amoebozoa, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. Sequences of small heterotrophic flagellates were affiliated mainly with the Alveolata (Apicomplexa), Stramenopiles (Chrysophyceae), and Rhizaria (Cercozoa). This survey showed that there is high protozoan species richness in meat-cutting plants and that the species included species related to known hosts of food-borne pathogens.

Dogs as carriers of the emerging pathogen Arcobacter.

Dogs and cats living in a household have previously been identified as a risk factor for human infection with Campylobacter and Helicobacter. In this study, carried out between July 2006 to September 2007, feces and oral swabs from 267 dogs and 61 cats were examined for the presence of the emerging pathogen Arcobacter. Isolates, obtained by an Arcobacter selective isolation procedure, were identified with an Arcobacter species-specific multiplex-PCR and characterized by modified enterobacterial repetitive intergenic concensus PCR. No arcobacters were isolated from cats. Five dogs excreted arcobacters in the feces and two other dogs carried arcobacters in the mouth. In the follow-up, one dog excreted the same Arcobacter butzleri strain for at least 1 week. Six dogs carried each an unique A. cryaerophilus strain although three of them lived in the same family. Therefore, beside the consumption of food and water, contact with dogs is another potential source of Arcobacter infection.