I. Van Damme

Contamination of freshly slaughtered pig carcasses with enteropathogenic Yersinia spp.: Distribution, quantification and identification of risk factors

A cross-sectional survey was undertaken to determine the overall prevalence of enteropathogenic Yersinia spp. in the tonsils, feces and on carcasses of pigs at slaughter. Moreover, factors associated with Yersinia contamination of freshly eviscerated pig carcasses were studied. Yersinia enterocolitica serotype O:3 was isolated from the tonsils and feces of 55.3% and 25.6% of pigs, and Y. pseudotuberculosis from 1.4% and 0.6%, respectively. The pathogens were also recovered from 39.7% of carcass surfaces post-evisceration. The highest prevalence was found at the mandibular region (28.9%), followed by the sternal region (16.4%), pelvic duct (7.8%), and split surface near the sacral vertebrae (6.9%). Regarding the quantification of the pathogen, the median concentration of pathogenic Y. enterocolitica was 4.14l og10 CFU/g in tonsils with countable numbers (n=143) and 2.80 log10 CFU/g for fecal samples with countable numbers (n=26). The quantitative load on the carcass surface was generally low as the majority of the carcass samples (97.0%) had Yersinia concentrations below the detection limit of enumeration (<1.30 log10 CFU/100 cm(2)). The initial presence of Y. enterocolitica in the tonsils and/or feces was significantly associated with carcass contamination at all sampled areas. Other risk factors for carcass contamination are the splitting of the head together with the carcass, and incision of the tonsils during removal of the pluck. Small adaptations in slaughter practices and the training of slaughterhouse personnel to respect basic hygienic instructions may diminish carcass contamination with enteropathogenic Yersinia.

Influence of isolation methods on the occurrence of plasmid-carrying Yersinia enterocolitica serotype O:3 in slaughter pig tonsils, faeces and carcass surface swabs

Yersinia enterocolitica is an important foodborne pathogen that is primarily transmitted to humans through the consumption of contaminated pork. Different matrices of pigs at slaughter were tested for the presence of human pathogenic types of Y. enterocolitica using direct plating, selective enrichment, and cold enrichment. Y. enterocolitica serotype O:3 was isolated from the tonsils and faeces of 55.3% and 25.6% of pigs, respectively. The pathogen was also recovered from 15.0% of swab samples taken from the carcass surface post-evisceration. Tonsils positive by direct plating revealed an average concentration of 3.99 log10 Y. enterocolitica per gram, whereas the majority of positive faecal and carcass samples were contaminated below the detection limit of enumeration. The relative sensitivity of the methods to recover pathogenic Y. enterocolitica varied among the different matrices. Nevertheless, cold enrichment was significantly more efficient than direct plating and selective enrichment for all three sample matrices. From the 2082 recovered Y. enterocolitica isolates, 1742 (83.7%) harboured the virulence plasmid. Isolates obtained from faeces were more likely to contain the virulence plasmid than isolates from tonsils and carcass swabs. To obtain reliable results regarding the presence of plasmid-carrying Y. enterocolitica isolates, sensitive isolation methods should be combined with testing of a sufficient number of isolates.

Behavior of Yersinia enterocolitica in the Presence of the Bacterivorous Acanthamoeba castellanii

ABSTRACT Free-living protozoa play an important role in the ecology and epidemiology of human-pathogenic bacteria. In the present study, the interaction between Yersinia enterocolitica, an important food-borne pathogen, and the free-living amoeba Acanthamoeba castellanii was studied. Several cocultivation assays were set up to assess the resistance of Y. enterocolitica to A. castellanii predation and the impact of environmental factors and bacterial strain-specific characteristics. Results showed that all Y. enterocolitica strains persist in association with A. castellanii for at least 14 days, and associations with A. castellanii enhanced survival of Yersinia under nutrient-rich conditions at 25°C and under nutrient-poor conditions at 37°C. Amoebae cultivated in the supernatant of one Yersinia strain showed temperature- and time-dependent permeabilization. Intraprotozoan survival of Y. enterocolitica depended on nutrient availability and temperature, with up to 2.8 log CFU/ml bacteria displaying intracellular survival at 7°C for at least 4 days in nutrient-rich medium. Transmission electron microscopy was performed to locate the Yersinia cells inside the amoebae. As Yersinia and Acanthamoeba share similar ecological niches, this interaction identifies a role of free-living protozoa in the ecology and epidemiology of Y. enterocolitica.

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

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.

Detection, seroprevalence and antimicrobial resistance of Yersinia enterocolitica and Yersinia pseudotuberculosis in pig tonsils in Northern Italy

Yersiniosis is the third most common reported zoonoses in Europe, with Y. enterocolitica and Y. pseudotuberculosis responsible for 98.66% and 0.94% of the confirmed human cases in 2013. From June 2013 to October 2014, 201 pigs at slaughter belonging to 67 batches were tested for Y. enterocolitica and Y. pseudotuberculosis in tonsils. Diaphragm muscle samples were tested for antibodies against Yersinia by a commercially available ELISA test. Y. enterocolitica 4/O:3 was detected in 55/201 pig tonsils (27.4%; 95% CI 23.1-37.1). The positive pigs came from 38/67 batches (56.7%) and were reared in 36/61 farms (59.0%). There was no statistical difference between farrow-to-finish and finishing farms. The mean count of Y. enterocolitica was 3.56±0.85log10CFU/g with a minimum of 2.0log10CFU/g and a maximum of 4.78log10CFU/g. Y. pseudotuberculosis was isolated from 4/201 pig tonsils (2.0%; 95% CI 0.0-4.5). Three isolates belonged to serotype O:3 and one to serotype O:1. The positive pigs belonged to 4/67 batches (6.0%) and came from finishing farms only. Y. pseudotuberculosis could be enumerated in one sample only (4.27log10CFU/g). The ELISA test demonstrated that 56.1% of the meat juice samples were positive for Yersinia antibodies. Serological positivity was found in 67.9% (36/53) of the Y. enterocolitica- and 75.0% (3/4) of the Y. pseudotuberculosis positive pigs. A significant association was found between serological results and the presence of Y. enterocolitica in tonsils (OR=1.97, p=0.044). All the Y. enterocolitica 4/O:3 isolates were susceptible to amoxicillin-clavulanic acid, gentamicin, ceftazidime, ertapenem and meropenem, 94.5% to cefotaxime, 89.1% to kanamycin and 78.2% to tetracycline. The highest resistance rates were observed for ampicillin (100%), sulphonamides (98.2%) and streptomycin (78.2%). Y. pseudotuberculosis strains were sensitive to all the antimicrobials tested, i.e. amoxicillin, amoxicillin/clavulanic acid, azithromycin, cephalothin, cefoxitin, ceftriaxone, ciprofloxacin, nalidixic acid, sulphonamide, tetracycline and ticarcillin. The study shows that Italian fattening pigs are frequently infected with human pathogenic Y. enterocolitica 4/O:3. Although the isolation rate is slightly lower than in other European countries, the serological test demonstrates that the infection is widespread among pig population. In fact, seroprevalence is similar to other EU countries. The detection of Y. pseudotuberculosis serotypes O:1 and O:3 in pig tonsils is of concern. Since tonsils may represent a contamination source for pig meat at slaughter, further studies regarding human infections by both microbial species are strongly recommended.

Abundance, diversity and community composition of free-living protozoa on vegetable sprouts.

Interactions with free-living protozoa (FLP) have been implicated in the persistence of pathogenic bacteria on food products. In order to assess the potential involvement of FLP in this contamination, detailed knowledge on their occurrence, abundance and diversity on food products is required. In the present study, enrichment and cultivation methods were used to inventory and quantify FLP on eight types of commercial vegetable sprouts (alfalfa, beetroot, cress, green pea, leek, mung bean, red cabbage and rosabi). In parallel, total aerobic bacteria and Escherichia coli counts were performed. The vegetable sprouts harbored diverse communities of FLP, with Tetrahymena (ciliate), Bodo saltans and cercomonads (flagellates), and Acanthamoeba and Vannella (amoebae) as the dominant taxa. Protozoan community composition and abundance significantly differed between the sprout types. Beetroot harbored the most abundant and diverse FLP communities, with many unique species such as Korotnevella sp., Vannella sp., Chilodonella sp., Podophrya sp. and Sphaerophrya sp. In contrast, mung bean sprouts were species-poor and had low FLP numbers. Sampling month and company had no significant influence, suggesting that seasonal and local factors are of minor importance. Likewise, no significant relationship between protozoan community composition and bacterial load was observed.

High abundance and diversity of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faeces and tonsils of pigs at slaughter

This cross-sectional study investigates the abundance of cefotaxime-resistant Escherichia coli (CREC) in the faeces and tonsils of 96 pigs during slaughter. Moreover, different isolates from a selected number of pigs were tested to study the diversity of blaESBL genes within E. coli isolates from one pig. Cefotaxime-resistant bacteria (based on enumeration results on MacConkey agar supplemented with 1mg/L cefotaxime) were found in the faeces of 77 pigs (80%; 95% CI: 70-87%) and the tonsils of 91 pigs (95%; 95% CI: 88%-98%). Cefotaxime-resistant E. coli (based on enumeration results on Tryptone Bile X-glucuronide agar supplemented with 1mg/L cefotaxime) were detected in 72 faecal samples (75%; 95% CI: 64-83%) and 45 tonsil samples (47%; 95% CI: 35-59%), in numbers up to 5.5 and 5.6log10 CFU/g, respectively. On average, around 1/10,000 E. coli in both faeces and tonsils were cefotaxime-resistant, though large variations were observed between pigs. Within one sample, CREC isolates with up to five different combinations of ESBL genes were observed. In three out of 16 faecal samples and six out of 14 tonsil samples, only one ESBL gene profile was found. The high numbers of CREC that are occasionally found in the faeces and tonsils of pigs during slaughter may represent an important source of contamination of carcasses and subsequently pork.

Free-living protozoa in the gastrointestinal tract and feces of pigs: Exploration of an unknown world and towards a protocol for the recovery of free-living protozoa.

Associations with free-living protozoa (FLP) have been implicated in the persistence of foodborne pathogenic bacteria in food-related environments. To date however no information is available on the presence of FLP in the gastrointestinal tract (GIT) of pigs, which represents an important reservoir for zoonotic foodborne bacteria and hence a potential location for associations with FLP. This is at least partly due to the lack of adequate protocols to recover FLP from intestinal content and feces. In the present study different protocols to recover FLP from the porcine GIT and feces were tested. The most effective protocols were then applied to explore the presence of live FLP in the pig GIT and feces. A filtration based protocol was identified as the most suitable method to recover viable FLP from the porcine GIT and feces. Cultivable FLP were recovered from different parts of the GIT, suggesting at least a transient presence of FLP in this habitat. Free-living amoebae species (Acanthamoeba spp., Hyperamoeba sp., Vannella sp., Vermamoeba vermiformis, hartmannellids and vahlkampfiids) but also ciliates (Colpoda sp. and Tetrahymena/Glaucoma lookalike) and flagellates (cercomonads, bodonids and glissomonads) were recovered and cultured from pig intestinal content. Acanthamoeba hatchetti and Filamoeba sinensis were isolated for the first time from pig intestinal content. Despite high gastric acidity, non-cyst forming amoeba species were also detected which suggests survival of their trophozoites in the animal GIT.

Quantification, distribution and diversity of ESBL/AmpC-producing Escherichia coli on freshly slaughtered pig carcasses

This study quantified cefotaxime-resistant E. coli (CREC) on nine different carcass areas of 104 freshly slaughtered pig carcasses. In 49% [95% confidence interval (95% CI): 29-69%] of the carcasses CREC could be isolated and enumerated (using Tryptone Bile Agar with X-Glucuronide supplemented with 1 mg/L cefotaxime). Proportions of positive samples varied between carcass areas from 1% [95% CI: 0-10%] (loin) to 23% [95% CI: 10-44%] (head). Maximum concentrations on positive samples ranged between -0.6 log10 CFU/cm2 (loin, elbow before evisceration) and 1.7 log10 CFU/cm2 (head). The head was significantly more frequently contaminated than the loin (p = 0.027) and ham (3% [95% CI: 1-15%]). The foreleg was significantly more frequently contaminated (20% [95% CI: 13-30%]) than the ham. Combination disk diffusion assays revealed that 81% of the CREC isolates were extended-spectrum beta-lactamases (ESBL) producers, 13% were AmpC cephalosporinases (AmpC) producers and 2% ESBL and AmpC co-producers. Genotyping denoted blaCTX-M-gr1 (63%) and blaTEM (40%) as most present antibiotic resistance genes. Multiple gene combinations in one isolate and multiple combinations of genotypes and phenotypes among isolates of one sample were observed. These quantitative data can be used for intervention strategies to lower human exposure to CREC.

Reduced contamination of pig carcasses using an alternative pluck set removal procedure during slaughter

This study compared the current pig slaughter procedure where the pluck set is completely removed with a procedure where the pluck set is partially removed, leaving the highly contaminated oral cavity, tonsils and tongue untouched. The effect on carcass contamination was investigated by enumerating hygiene indicator bacteria (total aerobic count, Enterobacteriaceae and E. coli) and cefotaxime-resistant E. coli (CREC) as well as assessing Salmonella and Yersinia enterocolitica presence on the sternum, elbow and throat of pig carcasses. Using the alternative pluck set removal, significantly lower mean numbers of hygiene indicator bacteria on throat samples and E. coli on elbow samples were found. Less pig carcasses were highly contaminated and a lower presence and level of CREC was observed. No difference in Salmonella or Yersinia enterocolitica presence was seen. The data in this study can help to assess the effect of this alternative procedure on the safety of pork and subsequently public health.