Maria Fredriksson-Ahomaa

Low Occurrence of Pathogenic Yersinia enterocolitica in Clinical, Food, and Environmental Samples: a Methodological Problem

SUMMARY While Yersinia enterocolitica is an important pathogen, which can cause yersiniosis in humans and animals, its epidemiology remains obscure. The pig is the major reservoir of pathogenic Y. enterocolitica of bioserotype 4/O:3, the most common type found in humans. Y. enterocolitica is thought to be a significant food-borne pathogen, although pathogenic isolates have seldom been recovered from foods. The low isolation rate of this pathogenic bacterium in natural samples, including clinical, food, and environmental samples, may be due to the limited sensitivity of culture methods. During the last decade, numerous DNA-based methods, such as PCR and colony hybridization assays, have been designed to detect pathogenic Y. enterocolitica in natural samples more rapidly and with better sensitivity than can be achieved by culture methods. In addition, the occurrence of pathogenic Y. enterocolitica in natural samples is clearly higher with PCR than with culture methods. The methods available for detection of pathogenic Y. enterocolitica in natural samples are reviewed in this article.

Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry.

Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level.

Correspondence of genotypes of sporadic Yersinia enterocolitica bioserotype 4/O:3 strains from human and porcine sources.

The sources and transmission routes of sporadic Yersinia enterocolitica bioserotype 4/O:3 infections in Finland were studied. A total of 212 human strains were compared with 334 non-human strains, including 163 strains from pig slaughterhouses, 164 strains from retail outlets and 7 strains from pet animals. All strains were characterized using pulsed-field gel electrophoresis (PFGE) with NotI enzyme. When the 194 human and 287 non-human strains of 22 identical NotI profiles were further characterized with ApaI and XhoI enzymes, 126 genotypes (DI = 094) were distinguished. Of all 212 human strains, 80% were genetically indistinguishable from the strains found in samples of pig origin when characterized with the three enzymes. A major contamination source of sporadic Y. enterocolitica 4/O:3 infections was revealed to be edible pig offal: 71% of the human strains were indistinguishable from the strains isolated from tongues, livers, kidneys and hearts of pigs. These results reveal that in Finland contaminated pig offal is an important vehiclein the transmission of Y. enterocolitica bioserotype 4/O:3 from slaughterhouses to humans.

High Prevalence of Yersinia enterocolitica 4:O3 on Pig Offal in Southern Germany: A Slaughtering Technique Problem

Prevalence and contamination routes of pathogenic Yersinia enterocolitica were studied in Southern Germany. Tonsil and faeces samples of 50 fattening pigs, 140 offal samples and 120 minced meat samples were examined. Pig and offal samples were collected from a slaughterhouse approved by the European Union, and minced meat samples from two large meat factories. Yersinia enterocolitica was isolated using direct plating, overnight enrichment and selective enrichment in MRB and ITC broth. The isolates were bio- and serotyped, and pathogenicity was studied using two plasmid-encoded virulence markers: calcium dependence and Congo red absorption. The genotypes were studied with pulsed-field gel electrophoresis using NotI enzyme. Prevalence of pathogenic Y. enterocolitica 4:O3 was 60% and 10% in tonsils and faeces of fattening pigs, respectively. Besides tonsils, prevalence of pathogenic Y. enterocolitica 4:O3 was also high in other pluck set samples, including tongues, lungs, hearts, diaphragms and livers. However, the highest isolation rate was obtained from the tonsils. Kidneys, which were not attached to the pluck set and did not hang together with tonsils on the rack, had the lowest isolation rate. Yersinia enterocolitica 4:O3 was isolated from 12% of minced meat samples. A total of 25 NotI profiles were obtained from porcine samples. The most common genotype, NBI, found in tonsils was also the most common type recovered from offal and minced meat samples. The high contamination rate of tonsils, and the indistinguishable NotI profiles obtained from tonsils and offal indicate that the tonsils contaminate offal when they are removed and hung on the rack together. When the head, with the tonsils and tongue, is not removed prior to evisceration and is not handled and inspected separately, it is difficult to control the spread of Y. enterocolitica 4:O3 from tonsils to the carcass, and subsequently, to meat.

Wide variety of bioserotypes of enteropathogenic Yersinia in tonsils of English pigs at slaughter

The tonsils of 630 pigs from 45 English farms using three different rearing methods (Assured British Pigs, Open Management and Organic) were examined between 2003 and 2005 in order to investigate if the low incidence of human yersiniosis could be attributed to a low prevalence of enteropathogenic Yersinia among English pigs. In addition, different isolation methods were compared, possible differences in prevalence among pigs were studied, as well as the prevalence of different bioserotypes of enteropathogenic Yersinia. A high prevalence and a wide diversity of bioserotypes of enteropathogenic Yersinia compared to other European countries were observed. The prevalence of pathogenic Yersinia enterocolitica was 44% and of Yersinia pseudotuberculosis 18%. Overall, 60% of pigs carried enteropathogenic Yersinia. Y. pseudotuberculosis was detected on 78% of farms and Y. enterocolitica on 69%. The most common bioserotypes of Y. enterocolitica were 2/O:9 (33%) and 2/O:5 (26%), and of Y. pseudotuberculosis 2/O:3 (34%), 1/O:1 (26%) and 1/O:4 (24%). Cold enrichment gave the highest isolation rate for both species. Y. enterocolitica was more prevalent (P<0.001) and Y. pseudotuberculosis less prevalent (P<0.05) in winter than in summer in Eastern England. Y. enterocolitica was more common in Eastern England and in assured British pigs, whereas Y. pseudotuberculosis was more common in Western England and in organic pigs. Y. pseudotuberculosis 1/O:1 was predominant (P<0.05) in Western England. Types 1/O:4 (P<0.05) and 2/O:3 (P<0.001) predominated in Eastern England. The high prevalence of Y. enterocolitica bioserotypes 2/O:9 and 2/O:5 found in this study suggests that English pigs are an important reservoir of these bioserotypes whereas in other European countries bioserotype 4/O:3 predominates.

Prevalence of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis in wild boars in Switzerland

Between October 2007 and March 2008, 153 wild boars shot in the Canton of Geneva in Switzerland were sampled. Fifty-one percent of the animals were males and 49% were females. The age of most (81%) animals varied between 6months and 2years. Prevalence of enteropathogenic Yersinia in tonsils and faeces was studied using culture and PCR methods and in tissue fluid of tonsils using an ELISA system. Prevalence of anti-Yersinia antibodies in tissue fluid was 65%. Detection rate of enteropathogenic Yersinia in tonsils of 153 wild boars by real-time PCR was 44%. Ail-positive Yersiniaenterocolitica and inv-positive Yersiniapseudotuberculosis were detected in 35 and 20% of the animals, respectively. Both species were detected in 10% of the animals. Isolation rate of enteropathogenic Yersinia was low; ail-positive Y. enterocolitica and inv-positive Y. pseudotuberculosis were found in 9 and 3% of the animals, respectively. Prevalence was shown to be significantly higher in tonsils than in faeces. Furthermore, females were more commonly positive than males. This study shows that the prevalence of enteropathogenic Yersinia is high and both enteropathogenic Y. enterocolitica and Y. pseudotuberculosis are common findings in tonsils of wild boars in Switzerland.

Pathogenic Yersinia enterocolitica 2/O:9 and Yersinia pseudotuberculosis 1/O:1 strains isolated from human and non-human sources in the Plateau State of Nigeria

Foodborne yersiniosis, caused by enteropathogenic Yersinia, especially Yersinia enterocolitica, is an important cause of diarrhea in developed countries, especially in temperate zones. Since studies concerning the presence of enteropathogenic Yersinia in humans and foods are rare in developing countries and tropical areas, human and non-human samples were studied in Plateau state of Nigeria to obtain information on the epidemiology of Y. enterocolitica and Yersinia pseudotuberculosis. Surprisingly, ail-positive Y. enterocolitica and inv-positive Y. pseudotuberculosis were isolated in Plateau state of Nigeria from several samples of human and non-human origin. Bioserotype 1/O:1 was the only Y. pseudotuberculosis type found. Y. enterocolitica belonging to bioserotype 2/O:9 was the dominating type found in most samples. Bioserotype 4/O:3 was isolated only from one pig and one sheep. Using PFGE, 5 genotypes were obtained among 45 Y. enterocolitica 2/O:9 strains with NotI, ApaI and XhoI enzymes and 3 among 20 Y. pseudotuberculosis 1/O:1 strains with NotI and SpeI enzymes. All human Y. pseudotuberculosis 1/O:1 strains were indistinguishable from pig, sheep or food strains. The dominating genotype of Y. enterocolitica 2/O:9 strains among humans was also found among strains isolated from pig, fermented cow milk and traditional intestine pepper soap samples.

Yersinia enterocolitica and Yersinia pseudotuberculosis

Yersinia enterocolitica and Yersinia pseudotuberculosis are included in the genus Yersinia. These species were formerly included in the genus Pasteurella and later placed into the genus Yersinia, named in honor of the French bacteriologist A. J. E. Yersin, a discoverer of the plague bacillus (1). Y. pseudotuberculosis was the first species identified in this genus (2). This organism was described in 1889 as a disease in guinea pigs. However, Y. pseudotuberculosis has shown to be the ancestor of Y. pestis, which was the cause of pandemic plague already during 541–767 AD (3). The Y. enterocolitica was identified in 1939 and named by Frederiksen in 1964 (4). The genus Yersinia is presently composed of 11 species, three of which can cause disease in humans and animals: Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis (5, 6, 7, 8).

Different Enteropathogenic Yersinia Strains Found in Wild Boars and Domestic Pigs

Yersinia enterocolitica and Yersinia pseudotuberculosis strains isolated from wild boars and fattening pigs were characterized and compared with each other. In wild boars, ail-positive Y. enterocolitica strains belonged to bioserotypes 4/O:3 (36%, 5/14), 2/O:9 (29%, 4/14), and 2/O:5,27 (21%, 3/14). Additionally, two ail-positive strains were untypable. Among fattening pigs, the bioserotype 4/O:3 was dominating (91%, 71/78), and bioserotypes 2/O:5,27 (8%, 6/78) and 2/O:9 (1%, 1/78) were rare. inv-positive Y. pseudotuberculosis strains of serotypes O:1 and O:2 were isolated only from wild boars. Antimicrobial resistance patterns between wild boar and fattening pig strains differed. Most of the ail-positive Y. enterocolitica strains carried yst, hreP, and virF genes. Several genotypes of Y. enterocolitica strains were obtained by PFGE using NotI, ApaI, XhoI, and SpeI enzymes. All genotypes of wild boar strains differed from fattening pig strains. Especially strains of bioserotype 4/O:3 were clearly different with all four enzymes. These results show that wild boar strains differed from domestic pig strains. More wild boar strains should be isolated to show that wild boars and domestic pigs are reservoirs for different Y. enterocolitica and Y. pseudotuberculosis strains.

High bacterial contamination of pig tonsils at slaughter

Food-borne zoonoses have a major health impact in industrial countries. Campylobacter spp., Salmonella enterica, Yersinia enterocolitica and Listeria monocytogenes are high-risk food-borne zoonotic hazards in finishing pigs. The objectives of this work were (1) to study the isolation rate of pathogenic Y. enterocolitica, Salmonella spp., Campylobacter spp. and L. monocytogenes in the tonsils and feces and (2) to determine the number of mesophilic aerobic bacteria (MAB) and Escherichia coli in the tonsils of fattening pigs at slaughter. The samples, which were collected from one slaughterhouse on five occasions, originated from 50 pigs and 15 farms. The number of MAB varied from 6.40 to 7.82 log(10) CFU/g and E. coli from 4.38 to 6.53 log(10) CFU/g. Additionally, 31 (62%) of the tonsils were colonized with Y. enterocolitica and 16 (32%) with L. monocytogenes. Campylobacter spp. were more frequently excreted in feces and only 3 (6%) of the pigs carried Campylobacter spp. in the tonsils. No Salmonella spp. were isolated. The pig tonsils were shown to be colonized with a high number of bacteria including E. coli, which is the most important indicator for fecal contamination, and with Y. enterocolitica and L. monocytogenes, which are important food-borne pathogens. This study demonstrates that the tonsils are highly contaminated with micro-organisms and can be a very important source of contamination in the slaughterhouse.