A bacteriophage cocktail can efficiently reduce five important Salmonella serotypes both on chicken skin and stainless steel.

Salmonella is one of the most important zoonotic pathogens and is mostly transmitted through food of animal origin. Application of bacteriophages is a promising tool to biocontrol Salmonella on both food and food contact surfaces. In this study, we evaluated the effectiveness of a six-phage cocktail for the reduction of Salmonella Enteritidis and a mixture of five major Salmonella serotypes (S. Enteritidis, Salmonella Typhimurium, Salmonella Infantis, Salmonella Paratyphi B, and Salmonella Indiana) on chicken skin and stainless steel. A phage cocktail with a final concentration of 107 PFU/cm2 was sprayed on these surfaces. After adding the phage cocktail, the samples were incubated at RT (~23°C) for different periods of time. The phage cocktail caused a significant reduction of S. Enteritidis and the mixed culture on chicken skin 30 min after phage addition, with 1.8 log10 and 1 log10 units, respectively. Reduction rates (1.2-1.7 log10 units) on stainless steel after 30 min were similar. Four hours after addition, the phage cocktail caused a significant reduction on both surfaces up to 3 log10 units on chicken skin and 2.4 log10 units on stainless steel. In a further experiment, bacteria added to stainless steel were not allowed to dry to simulate a fresh bacterial contamination. In this case, the bacterial count of S. Enteritidis was reduced below the detection limit after 2 h. The results demonstrate that this phage cocktail has potential to be used in post-harvest applications to control Salmonella contaminations.

The Novel Yersinia enterocolitica Telomere Phage vB_YenS_P840 Is Closely Related to PY54, but Reveals Some Striking Differences.

Telomere phages are a small group of temperate phages, whose prophages replicate as a linear plasmid with covalently closed ends. They have been isolated from some Enterobacteriaceae and from bacterial species living in aquatic environments. Phage PY54 was the first Yersinia (Y.) enterocolitica telomere phage isolated from a nonpathogenic O:5 strain, but recently a second telomeric Yersinia phage (vB_YenS_P840) was isolated from a tonsil of a wild boar in Germany. Both PY54 and vB_YenS_P840 (P840) have a siphoviridal morphology and a similar genome organization including the primary immunity region immB and telomere resolution site telRL. However, whereas PY54 only possesses one prophage repressor for the lysogenic cycle, vB_YenS_P840 encodes two. The telRL region of this phage was shown to be processed by the PY54 protelomerase under in vivo conditions, but unlike with PY54, a flanking inverted repeat was not required for processing. A further substantial difference between the phages is their host specificity. While PY54 infects Y. enterocolitica strains belonging to the serotypes O:5 and O:5,27, vB_YenS_P840 exclusively lyses O:3 strains. As the tail fiber and tail fiber assembly proteins of the phages differ significantly, we introduced the corresponding genes of vB_YenS_P840 by transposon mutagenesis into the PY54 genome and isolated several mutants that were able to infect both serotypes, O:5,27 and O:3.

A Bacteriophage Cocktail Reduces Five Relevant Salmonella Serotypes at Low Multiplicities of Infection and Low Temperatures.

Salmonella are important pathogenic bacteria and, following Campylobacter, they are the second most common cause of bacterial foodborne infections worldwide. To reduce the presence of bacteria along the food chain, the application of bacteriophages (phages) may be a promising tool. In this study, the lytic properties of six phages against five relevant Salmonella serotypes (S. Enteritidis, S. Typhimurium, S. Infantis, S. Paratyphi B and S. Indiana) were analyzed. Three phages were able to lyse all five serotypes. We determined the lytic potential of each phage on indicator strains in vitro at room temperature (RT) and at 37 °C using low multiplicities of infection (MOIs). Most phages reduced their host more efficiently at RT than at 37 °C, even at the lowest MOI of 0.001. Following this, the lytic activity of a cocktail comprising five phages (MOI = 0.1) was examined with each of the five serotypes and a mix of them at RT, 15, 12, 10, 8 and 6 °C. All cultures of single serotypes as well as the mixture of strains were significantly reduced at temperatures as low as 8 °C. For single serotypes, reductions of up to 5 log10 units and up to 2.3 log10 units were determined after 6 h (RT) and 40 h (8 °C), respectively. The mixture of strains was reduced by 1.7 log10 units at 8 °C. The data clearly suggest that these phages are suitable candidates for biocontrol of various Salmonella serotypes under food manufacturing conditions.

Ophiodimyces ophiodiicola, Etiologic Agent of Snake Fungal Disease, in Europe since Late 1950s.

The fungus Ophiodimyces ophiodiicola is the etiologic agent of snake fungal disease. Recent findings date US occurrence at least as far back as 1945. We analyzed 22 free-ranging snakes with gross lesions consistent with snake fungal disease from museum collections from Europe. We found 5 positive samples, the oldest collected in 1959.

Phage vB_YenS_P400, a Novel Virulent Siphovirus of Yersinia enterocolitica Isolated from Deer.

Phage vB_YenS_P400 isolated from deer, is a virulent siphovirus of Y. enterocolitica, whose circularly permutated genome (46,585 bp) is not substantially related to any other phage deposited in public nucleotide databases. vB_YenS_P400 showed a very narrow host range and exclusively lysed two Y. enterocolitica B4/O:3 strains. Moreover, lytic activity by this phage was only discernible at room temperature. Together with the finding that vB_YenS_P400 revealed a long latent period (90 to 100 min) and low burst size (five to ten), it is not suitable for applications but provides insight into the diversity of Yersinia phages.

Phylogeographic inference of Sumatran ranids bearing gastromyzophorous tadpoles with regard to the Pleistocene drainage systems of Sundaland.

Rivers are known to act as biogeographic barriers in several strictly terrestrial taxa, while possibly serving as conduits of dispersal for freshwater-tolerant or -dependent species. However, the influence of river systems on genetic diversity depends on taxa-specific life history traits as well as other geographic factors. In amphibians, several studies have demonstrated that river systems have only minor influence on their divergence. Here, we assess the role of the paleodrainage systems of the Sunda region (with a focus on the island of Sumatra) in shaping the evolutionary history of two genera of frogs (Sumaterana and Wijayarana) whose tadpoles are highly dependent on cascading stream habitats. Our phylogenetic results show no clear association between the genetic diversification patterns of both anurans genera and the existence of paleodrainage systems. Time-calibrated phylogenies and biogeographical models suggest that these frogs colonized Sumatra and diversified on the island before the occurrence of the Pleistocene drainage systems. Both genera demonstrate phylogenetic structuring along a north-south geographic axis, the temporal dynamics of which coincide with the geological chronology of proto Sumatran and -Javan volcanic islands. Our results also highlight the chronic underestimation of Sumatran biodiversity and call for more intense sampling efforts on the island.

Host Range, Morphology and Sequence Analysis of Ten Temperate Phages Isolated from Pathogenic Yersinia enterocolitica Strains.

Yersinia enterocolitica is a heterogeneous species comprising highly pathogenic, weakly pathogenic and non-pathogenic strains. Previous data suggest that gene exchange may occur in Yersinia. Only scarce information exists about temperate phages of Y. enterocolitica, even though many prophage sequences are present in this species. We have examined 102 pathogenic Y. enterocolitica strains for the presence of inducible prophages by mitomycin C treatment. Ten phages were isolated from nine strains belonging to the bio (B)/serotypes (O) B2/O:5,27, B2/O:9 and 1B/O:8. All phages are myoviruses showing lytic activity only at room temperature. Whole-genome sequencing of the phage genomes revealed that they belong to three groups, which, however, are not closely related to known phages. Group 1 is composed of five phages (type phage: vB_YenM_06.16.1) with genome sizes of 43.8 to 44.9 kb, whereas the four group 2 phages (type phage: vB_YenM_06.16.2) possess smaller genomes of 29.5 to 33.2 kb. Group 3 contains only one phage (vB_YenM_42.18) whose genome has a size of 36.5 kb, which is moderately similar to group 2. The host range of the phages differed significantly. While group 1 phages almost exclusively lysed strains of B2/O:5,27, phages of group 2 and 3 were additionally able to lyse B4/O:3, and some of them even B2/O:9 and 1B/O:8 strains.

Grey wolf genomic history reveals a dual ancestry of dogs.

The grey wolf (Canis lupus) was the first species to give rise to a domestic population, and they remained widespread throughout the last Ice Age when many other large mammal species went extinct. Little is known, however, about the history and possible extinction of past wolf populations or when and where the wolf progenitors of the present-day dog lineage (Canis familiaris) lived1-8. Here we analysed 72 ancient wolf genomes spanning the last 100,000 years from Europe, Siberia and North America. We found that wolf populations were highly connected throughout the Late Pleistocene, with levels of differentiation an order of magnitude lower than they are today. This population connectivity allowed us to detect natural selection across the time series, including rapid fixation of mutations in the gene IFT88 40,000-30,000 years ago. We show that dogs are overall more closely related to ancient wolves from eastern Eurasia than to those from western Eurasia, suggesting a domestication process in the east. However, we also found that dogs in the Near East and Africa derive up to half of their ancestry from a distinct population related to modern southwest Eurasian wolves, reflecting either an independent domestication process or admixture from local wolves. None of the analysed ancient wolf genomes is a direct match for either of these dog ancestries, meaning that the exact progenitor populations remain to be located.

Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians.

BACKGROUND: The 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence (p-distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths. RESULTS: Using empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwise p-distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content. CONCLUSIONS: Taken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost.

Properties of Two Broad Host Range Phages of Yersinia enterocolitica Isolated from Wild Animals.

Yersinia (Y.) enterocolitica and Y. pseudotuberculosis are important zoonotic agents which can infect both humans and animals. To combat these pathogens, the application of strictly lytic phages may be a promising tool. Since only few Yersinia phages have been described yet, some of which demonstrated a high specificity for certain serotypes, we isolated two phages from game animals and characterized them in terms of their morphology, host specificity, lytic activity on two bio-/serotypes and genome composition. The T7-related podovirus vB_YenP_Rambo and the myovirus vB_YenM_P281, which is very similar to a previously described phage PY100, showed a broad host range. Together, they lysed all the 62 tested pathogenic Y. enterocolitica strains belonging to the most important bio-/serotypes in Europe. A cocktail containing these two phages strongly reduced cultures of a bio-/serotype B4/O:3 and a B2/O:9 strain, even at very low MOIs (multiplicity of infection) and different temperatures, though, lysis of bio-/serotype B2/O:9 by vB_YenM_P281 and also by the related phage PY100 only occurred at 37 °C. Both phages were additionally able to lyse various Y. pseudotuberculosis strains at 28 °C and 37 °C, but only when the growth medium was supplemented with calcium and magnesium cations.

Unexpectedly high levels of lineage diversity in Sundaland puddle frogs (Dicroglossidae: Occidozyga Kuhl and van Hasselt, 1822).

One of the most urgent contemporary tasks for taxonomists and evolutionary biologists is to estimate the number of species on earth. Recording alpha diversity is crucial for protecting biodiversity, especially in areas of elevated species richness, which coincide geographically with increased anthropogenic environmental pressures - the world's so-called biodiversity hotspots. Although the distribution of Puddle frogs of the genus Occidozyga in South and Southeast Asia includes five biodiversity hotspots, the available data on phylogeny, species diversity, and biogeography are surprisingly patchy. Samples analyzed in this study were collected throughout Southeast Asia, with a primary focus on Sundaland and the Philippines. A mitochondrial gene region comprising ~ 2000 bp of 12S and 16S rRNA with intervening tRNA Valine and three nuclear loci (BDNF, NTF3, POMC) were analyzed to obtain a robust, time-calibrated phylogenetic hypothesis. We found a surprisingly high level of genetic diversity within Occidozyga, based on uncorrected p-distance values corroborated by species delimitation analyses. This extensive genetic diversity revealed 29 evolutionary lineages, defined by the > 5% uncorrected p-distance criterion for the 16S rRNA gene, suggesting that species diversity in this clade of phenotypically homogeneous forms probably has been underestimated. The comparison with results of other anuran groups leads to the assumption that anuran species diversity could still be substantially underestimated in Southeast Asia in general. Many genetically divergent lineages of frogs are phenotypically similar, indicating a tendency towards extensive morphological conservatism. We present a biogeographic reconstruction of the colonization of Sundaland and nearby islands which, together with our temporal framework, suggests that lineage diversification centered on the landmasses of the northern Sunda Shelf. This remarkably genetically structured group of amphibians could represent an exceptional case for future studies of geographical structure and diversification in a widespread anuran clade spanning some of the most pronounced geographical barriers on the planet (e.g., Wallace's Line). Studies considering gene flow, morphology, ecological and bioacoustic data are needed to answer these questions and to test whether observed diversity of Puddle frog lineages warrants taxonomic recognition.

Anatomical features of the phytotelma dwelling, egg-eating, fanged tadpoles of Rhacophorus vampyrus (Anura: Rhacophoridae).

Tadpoles of the Vampire tree frog Rhacophorus vampyrus differ substantially from other rhacophorid tadpoles, by having profound modifications in external morphology. The morphological peculiarities of this species likely correlate with their arboreal microhabitat and strict oophagous diet. In this work, we examine buccal and musculoskeletal anatomy and compare them to other rhacophorid and egg-eating larvae. The shape and arrangement of cartilages of the lower jaw are unique among tadpoles, and the lack of a palatoquadrate suspensorium is only known in the distantly related macrophagous tadpoles of the dicroglossid Occidozyga baluensis. The cranial musculature is massive, and the morphology of several mandibular, hyoid, and abdominal muscles could be related to the ingestion and transit of large eggs. In the buccal cavity, conspicuous aspects are the absence of ridges and papillae, and the development of a unique glandular zone in the buccal floor. Finally, observations of the skeletal support of keratinized mouthparts allow us to present a topography-based hypothesis of homology of the conspicuous fangs of these tadpoles.

Birds Kept in the German Zoo "Tierpark Berlin" Are a Common Source for Polyvalent Yersinia pseudotuberculosis Phages.

Yersinia pseudotuberculosis is an important animal pathogen, particularly for birds, rodents, and monkeys, which is also able to infect humans. Indeed, an increasing number of reports have been published on zoo animals that were killed by this species. One option to treat diseased animals is the application of strictly lytic (virulent) phages. However, thus far relatively few phages infecting Y. pseudotuberculosis have been isolated and characterized. To determine the prevalence of Y. pseudotuberculosis phages in zoo animals, fecal samples of birds and some primates, maras, and peccaries kept in the Tierpark Berlin were analyzed. Seventeen out of 74 samples taken in 2013 and 2017 contained virulent phages. The isolated phages were analyzed in detail and could be allocated to three groups. The first group is composed of 10 T4-like phages (PYps2T taxon group: Myoviridae; Tevenvirinae; Tequatrovirus), the second group (PYps23T taxon group: Chaseviridae; Carltongylesvirus; Escherichia virus ST32) consists of five phages encoding a podovirus-like RNA polymerase that is related to an uncommon genus of myoviruses (e.g., Escherichia coli phage phiEcoM-GJ1), while the third group is comprised of two podoviruses (PYps50T taxon group: Autographiviridae; Studiervirinae; Berlinvirus) which are closely related to T7. The host range of the isolated phages differed significantly. Between 5.5 and 86.7% of 128 Y. pseudotuberculosis strains belonging to 20 serotypes were lysed by each phage. All phages were additionally able to lyse Y. enterocolitica B4/O:3 strains, when incubated at 37°C. Some phages also infected Y. pestis strains and even strains belonging to other genera of Enterobacteriaceae. A cocktail containing two of these phages would be able to lyse almost 93% of the tested Y. pseudotuberculosis strains. The study indicates that Y. pseudotuberculosis phages exhibiting a broad-host range can be isolated quite easily from zoo animals, particularly birds.

The Burkholderia thailandensis Phages ΦE058 and ΦE067 Represent Distinct Prototypes of a New Subgroup of Temperate Burkholderia Myoviruses.

Burkholderia mallei and B. pseudomallei are highly pathogenic species which are closely related, but diverse regarding their prophage content. While temperate phages have not yet been isolated from B. mallei, several phages of B. pseudomallei, and its non-pathogenic relative B. thailandensis have been described. In this study we isolated two phages from B. pseudomallei and three phages from B. thailandensis and determined their morphology, host range, and relationship. All five phages belong to the family Myoviridae, but some of them revealed different host specificities. DNA-DNA hybridization experiments indicated that the phages belong to two groups. One group, composed of ΦE058 (44,121 bp) and ΦE067 (43,649 bp), represents a new subgroup of Burkholderia myoviruses that is not related to known phages. The genomes of ΦE058 and ΦE067 are similar but also show some striking differences. Repressor proteins differ clearly allowing the phages to form plaques on hosts containing the respective other phage. The tail fiber proteins exhibited some minor deviations in the C-terminal region, which may account for the ability of ΦE058, but not ΦE067, to lyse B. mallei, B. pseudomallei, and B. thailandensis. In addition, the integrases and attachment sites of the phages are not related. While ΦE058 integrates into the Burkholderia chromosome within an intergenic region, the ΦE067 prophage resides in the selC tRNA gene for selenocysteine. Experiments on the structure of phage DNA isolated from particles suggest that the ΦE058 and ΦE067 genomes have a circular conformation.

Campylobacter Phage Isolation and Characterization: What We Have Learned So Far.

Lytic Campylobacter phages, which can be used to combat this pathogen in animals and on food products, have been studied for more than 30 years. Though, due to some peculiarities of the phages, which hampered their isolation and particularly their molecular analysis for a long time, progress in this research field was rather slow. Meanwhile, the situation has changed and much more is known about the biology and genetics of those phages. In this article, we address specific issues that should be considered when Campylobacter phages are studied, starting with the isolation and propagation of the phages and ending with a thorough characterization including whole-genome sequencing. The basis for advice and recommendations given here is a careful review of the scientific literature and experiences that we have had ourselves with Campylobacter phages.

Validation of EN ISO method 10273 - Detection of pathogenic Yersinia enterocolitica in foods.

EN ISO 10273 method for the detection of pathogenic Yersinia enterocolitica in foods was validated in the project Mandate M/381 funded by European Commission. A total of 14 laboratories from five European countries participated in the interlaboratory study (ILS) organized during 2013 and 2014. Before the ILS, the method was revised by an international group of experts and the performance of the revised method was assessed in an ILS study. The results are published as a part of the standard EN ISO 10273 revision. The study included three rounds with different sample types; raw milk, iceberg lettuce and minced meat, inoculated with a low and high level of pathogenic Y. enterocolitica strains representing major pathogenic bioserotypes 4/O:3 and 2/O:9. The homogeneity and stability of the samples were verified before dispatching them to the laboratories. The results demonstrated the method sensitivity of 96% in raw milk, 97% in minced meat, and 98% in lettuce at high inoculation level of pathogenic Y. enterocolitica. The specificity was 100% in raw milk, 96% in minced meat, and 98% in lettuce. The level of detection, LOD50, varied between study rounds, being 9.4 CFU/25 ml in raw milk, 9.9 CFU/25 g in minced meat and 63 CFU/25 g in lettuce samples. During the study, confirmation by using real-time PCR method ISO/TS 18867 together with pyrazinamidase testing was also validated, as alternative to conventional biochemical confirmation. When comparing different isolation steps used in the revised method during the study rounds, PSB enrichment and plating on CIN after alkaline (KOH) treatment showed the highest sensitivity (52-92%) in raw milk and minced meat samples. In lettuce samples, however, ITC with KOH treatment before plating on CIN showed higher sensitivity (64% at low level; 82% at high level) than plating on CIN from PSB with KOH treatment (44% at low level; 74% at high level). Statistical analysis of different isolation steps supported the use of two enrichment media, PSB and ITC, in the revised method. Recovery of pathogenic Y. enterocolitica on CIN was most efficient after KOH treatment and, based on the analysis, plating on CIN agar without KOH treatment could be left as optional procedure in the method.

Yersinia pseudotuberculosis Prevalence and Diversity in Wild Boars in Northeast Germany.

In this study, the prevalence of Yersinia pseudotuberculosis in wild boars in northeast Germany was determined. For that purpose, the tonsils of 503 wild boars were sampled. The presence of Y. pseudotuberculosis was studied by diagnostic PCR. Positive samples were analyzed by cultural detection using a modified cold enrichment protocol. Ten Y. pseudotuberculosis isolates were obtained, which were characterized by biotyping, molecular serotyping, and multilocus sequence typing (MLST). In addition, whole-genome sequences and the antimicrobial susceptibility of the isolates were analyzed. Yersinia pseudotuberculosis was isolated from male and female animals, most of which were younger than 1 year. A prevalence of 2% (10/503) was determined by cultural detection, while 6.4% (32/503) of the animals were positive by PCR. The isolates belonged to the biotypes 1 and 2 and serotypes O:1a (n = 7), O:1b (n = 2), and O:4a (n = 1). MLST analysis revealed three sequence types, ST9, ST23, and ST42. Except one isolate, all isolates revealed a strong resistance to colistin. The relationship of the isolates was studied by whole-genome sequencing demonstrating that they belonged to four clades, exhibiting five different pulsed-field gel electrophoresis (PFGE) restriction patterns and a diverse composition of virulence genes. Six isolates harbored the virulence plasmid pYV. Besides two isolates, all isolates contained ail and inv genes and a complete or incomplete high-pathogenicity island (HPI). None of them possessed a gene for the superantigen YPM. The study shows that various Y. pseudotuberculosis strains exist in wild boars in northeast Germany, which may pose a risk to humans.IMPORTANCEYersinia pseudotuberculosis is a foodborne pathogen whose occurrence is poorly understood. One reason for this situation is the difficulty in isolating the species. The methods developed for the isolation of Yersinia enterocolitica are not well suited for Y. pseudotuberculosis We therefore designed a protocol which enabled the isolation of Y. pseudotuberculosis from a relatively high proportion of PCR-positive wild boar tonsils. The study indicates that wild boars in northeast Germany may carry a variety of Y. pseudotuberculosis strains, which differ in terms of their pathogenic potential and other properties. Since wild boars are widely distributed in German forests and even populate cities such as Berlin, they may transmit yersiniae to other animals and crop plants and may thus cause human infections through the consumption of contaminated food. Therefore, the prevalence of Y. pseudotuberculosis should be determined also in other animals and regions to learn more about the natural reservoir of this species.

Complete Genome Sequences of 10 Yersinia pseudotuberculosis Isolates Recovered from Wild Boars in Germany.

We report here the draft genome sequences of 10 Yersinia pseudotuberculosis isolates recovered from tonsils of wild boars hunted between 2015 and 2016 in Germany. Whole-genome sequencing and bioinformatic analyses were performed to assess the diversity of Y. pseudotuberculosis, which may result in human infections caused by the consumption of game meat.

Prevalence and Antimicrobial Resistance of Yersinia enterocolitica in Retail Seafood.

Yersinia enterocolitica is a zoonotic enteropathogenic bacterium that can cause acute gastroenteritis and mesenteric lymphadenitis. Although Y. enterocolitica is common in animals, food, and the environment, the reservoirs and transmission routes of this pathogen are still not fully understood. The aim of our study was to determine the prevalence of Y. enterocolitica in seafood in Germany, because only limited data are available on that topic. Seafood samples were purchased from retail shops in Berlin, Germany and examined for the presence of Y. enterocolitica by cold enrichment followed by cultivation on selective agar. Presumptive Y. enterocolitica isolates were analyzed by biotyping, serotyping, and antimicrobial susceptibility testing. The total prevalence of Y. enterocolitica in seafood samples was 2.7% (6 of 220 samples). Mussel (2 of 90), shrimp (1 of 89), and cephalopod (3 of 41) samples were positive for Y. enterocolitica. Three isolates were identified as serotype O:8, one was identified as serotype O:5,27, and two samples did not belong to any investigated serotypes. The presence of the virulence-associated genes ail, inv, and ystB was studied by multiplex PCR. Four of the six isolates contained inv and ystB, one produced no positive results for the analyzed genes, and one contained only ystB. All Y. enterocolitica isolates were susceptible to cefotaxime, cefuroxime, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, tetracycline, and trimethoprim. Resistance was observed to cephalothin (83.3% of isolates), amoxicillin (83.3%), and ampicillin (50.0%). This study provides the first comprehensive analysis of Y. enterocolitica in retail seafood in Germany. The prevalence found in these seafood samples was comparatively low, and all isolates belonged to biotype 1A. However, seafood contaminated with Y. enterocolitica may pose a risk to consumer health because the pathogenic potential of biotype 1A strains is still being debated.

A multiplex real-time PCR for the detection and differentiation of Campylobacter phages.

Campylobacter jejuni and C. coli are important food-borne pathogens that are widespread in animal husbandry. To combat Campylobacter along the food chain, the application of lytic phages has been shown to be a promising tool. Campylobacter phages are currently classified into three groups, of which group II and group III phages are the most common. Members of each group are closely related, whereas the two groups share only little DNA similarity. Moreover, while group III phages are specific for C. jejuni, group II phages additionally infect C. coli. Phage cocktails intended to be used for applications should be composed of various phages that differ in their host range and growth kinetics. The isolation of phages is generally performed by plaque assays. This approach has the limitation that phages are merely identified by their lytic activity on certain indicator strains and that relatively high numbers of phages must be present in a tested sample. Therefore, a more sensitive molecular detection system would be beneficial, which allows a pre-screening of samples and the quick detection and discrimination of group II and group III phages. New phages can then be isolated by use of indicator strains that may be different from those typically applied. On the basis of available Campylobacter phage genome sequences, we developed a multiplex PCR system for group II and group III phages selecting the tail tube gene and the gene for the base plate wedge, respectively, as target. Phages of both groups could be identified with primers deduced from the putative tail fiber gene. Efficient release of phage DNA from capsids was achieved by an extended heat treatment or digestion of phage particles with proteinase K/SDS yielding a detection limit of 1 pfu/ml. Individual detection of group II phages, group III phages and of both groups was studied with artificially contaminated chicken skin. To recover phages that had strongly adhered to the skin, stomaching was the most efficient technique. The developed PCR protocol was employed to detect Campylobacter phages in food and environmental samples. In 50 out of 110 samples group II and/or group III phages were identified.