Case report: Aberrant fecal microbiota composition of an infant diagnosed with prolonged intestinal botulism.

BACKGROUND: Intestinal botulism is primarily reported in small babies as a condition known as infant botulism. The condition results from the ingestion of environmental or foodborne spores of botulinum neurotoxin (BoNT) producing Clostridia, usually Clostridium botulinum, and subsequent spore germination into active botulinum neurotoxinogenic cultures in the gut. It is generally considered that small babies are susceptible to C. botulinum colonization because of their immature gut microbiota. Yet, it is poorly understood which host factors contribute to the clinical outcome of intestinal botulism. We previously reported a case of infant botulism where the infant recovered clinically in six weeks but continued to secrete C. botulinum cells and/or BoNT in the feces for seven months. CASE PRESENTATION: To further understand the microbial ecology behind this exceptionally long-lasting botulinum neurotoxinogenic colonization, we characterized the infant fecal microbiota using 16S rRNA gene amplicon sequencing over the course of disease and recovery. C. botulinum could be detected in the infant fecal samples at low levels through the acute phase of the disease and three months after recovery. Overall, we observed a temporal delay in the maturation of the infant fecal microbiota associated with a persistently high-level bifidobacterial population and a low level of Lachnospiraceae, Bacteroidaceae and Ruminococcaceae compared to healthy infants over time. CONCLUSION: This study brings novel insights into the infant fecal composition associated with intestinal botulism and provides a basis for a more systematic analysis of the gut microbiota of infants diagnosed with botulism. A better understanding of the gut microbial ecology associated with infant botulism may support the development of prophylactic strategies against this life-threatening disease in small babies.

A Novel Prophage-like Insertion Element within yabG Triggers Early Entry into Sporulation in Clostridium botulinum.

Sporulation is a finely regulated morphogenetic program important in the ecology and epidemiology of Clostridium botulinum. Exogenous elements disrupting sporulation-associated genes contribute to sporulation regulation and introduce diversity in the generally conserved sporulation programs of endospore formers. We identified a novel prophage-like DNA segment, termed the yin element, inserted within yabG, encoding a sporulation-specific cysteine protease, in an environmental isolate of C. botulinum. Bioinformatic analysis revealed that the genetic structure of the yin element resembles previously reported mobile intervening elements associated with sporulation genes. Within a pure C. botulinum culture, we observed two subpopulations of cells with the yin element either integrated into the yabG locus or excised as a circular DNA molecule. The dynamics between the two observed conformations of the yin element was growth-phase dependent and likely mediated by recombination events. The yin element was not required for sporulation by C. botulinum but triggered an earlier entry into sporulation than in a related isolate lacking this element. So far, the yin element has not been found in any other C. botulinum strains or other endospore-forming species. It remains to be demonstrated what kind of competitive edge it provides for C. botulinum survival and persistence.

Cellular and population strategies underpinning neurotoxin production and sporulation in Clostridium botulinum type E cultures.

IMPORTANCE: Toxin production and sporulation are key determinants of pathogenesis in Clostridia. Toxins cause the clinical manifestation of clostridial diseases, including diarrhea and colitis, tissue damage, and systemic effects on the nervous system. Spores ensure long-term survival and persistence in the environment, act as infectious agents, and initiate the host tissue colonization leading to infection. Understanding the interplay between toxin production and sporulation and their coordination in bacterial cells and cultures provides novel intervention points for controlling the public health and food safety risks caused by clostridial diseases. We demonstrate environmentally driven cellular heterogeneity in botulinum neurotoxin and spore production in Clostridium botulinum type E populations and discuss the biological rationale of toxin and spore production in the pathogenicity and ecology of C. botulinum. The results invite to reassess the epidemiology of botulism and may have important implications in the risk assessment and risk management strategies in food processing and human and animal health.

Specific Isolation of Clostridium botulinum Group I Cells by Phage Lysin Cell Wall Binding Domain with the Aid of S-Layer Disruption.

Clostridium botulinum is a notorious pathogen that raises health and food safety concerns by producing the potent botulinum neurotoxin and causing botulism, a potentially fatal neuroparalytic disease in humans and animals. Efficient methods for the identification and isolation of C. botulinum are warranted for laboratory diagnostics of botulism and for food safety risk assessment. The cell wall binding domains (CBD) of phage lysins are recognized by their high specificity and affinity to distinct types of bacteria, which makes them promising for the development of diagnostic tools. We previously identified CBO1751, which is the first antibotulinal phage lysin showing a lytic activity against C. botulinum Group I. In this work, we assessed the host specificity of the CBD of CBO1751 and tested its feasibility as a probe for the specific isolation of C. botulinum Group I strains. We show that the CBO1751 CBD specifically binds to C. botulinum Group I sensu lato (including C. sporogenes) strains. We also demonstrate that some C. botulinum Group I strains possess an S-layer, the disruption of which by an acid glycine treatment is required for efficient binding of the CBO1751 CBD to the cells of these strains. We further developed CBO1751 CBD-based methods using flow cytometry and magnetic separation to specifically isolate viable cells of C. botulinum Group I. These methods present potential for applications in diagnostics and risk assessment in order to control the botulism hazard.

Genomic and Phenotypic Characterization of Clostridium botulinum Isolates from an Infant Botulism Case Suggests Adaptation Signatures to the Gut.

In early life, the immature human gut microbiota is prone to colonization by pathogens that are usually outcompeted by mature microbiota in the adult gut. Colonization and neurotoxin production by a vegetative Clostridium botulinum culture in the gut of an infant can lead to flaccid paralysis, resulting in a clinical outcome known as infant botulism, a potentially life-threatening condition. Beside host factors, little is known of the ecology, colonization, and adaptation of C. botulinum to the gut environment. In our previous report, an infant with intestinal botulism was shown to be colonized by neurotoxigenic C. botulinum culture for 7 months. In an effort to gain ecological and evolutionary insights into this unusually long gut colonization by C. botulinum, we analyzed and compared the genomes of C. botulinum isolates recovered from the infant feces during the course of intoxication and isolates from the infant household dust. A number of observed mutations and genomic alterations pinpointed at phenotypic traits that may have promoted colonization and adaptation to the gut environment and to the host. These traits include motility, quorum-sensing, sporulation, and carbohydrate metabolism. We provide novel perspectives and suggest a tentative model of the pathogenesis of C. botulinum in infant botulism. IMPORTANCE While the clinical aspects of infant botulism and the mode of action of BoNT have been thoroughly investigated, little is known on the pathogenesis and adaptive mechanisms of C. botulinum in the gut. Here, we provide for the first time a comprehensive view on the genomic dynamics and plasticity of C. botulinum over time in a case of infant botulism. The genomic and phenotypic analysis of C. botulinum isolates collected during the disease course offers an unprecedented view of C. botulinum ecology, evolution, and pathogenesis and may be instrumental in developing novel strategies for prevention and treatment of toxicoinfectious botulism.

A European-wide dataset to uncover adaptive traits of Listeria monocytogenes to diverse ecological niches.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

Prudent Antimicrobial Use Is Essential to Prevent the Emergence of Antimicrobial Resistance in Yersinia enterocolitica 4/O:3 Strains in Pigs.

Yersinia enterocolitica is a psychrotrophic zoonotic foodborne pathogen. Pigs are considered the main reservoir of Y. enterocolitica 4/O:3, which is the most commonly isolated bioserotype in many European countries. Consuming pork contaminated with Y. enterocolitica can be a health threat, and antimicrobial-resistant strains may complicate the treatment of the most severe forms of yersiniosis. We analyzed the antimicrobial resistance of 1,016 pathogenic porcine Y. enterocolitica 4/O:3 strains originating from Belgium, Estonia, Finland, Germany, Italy, Latvia, Russia, Spain, and the United Kingdom. Based on available reports, we also compared antimicrobial sales for food production animals in these countries, excluding Russia. Antimicrobial resistance profiles were determined using a broth microdilution method with VetMIC plates for 13 antimicrobial agents: ampicillin, cefotaxime, ceftiofur (CTF), chloramphenicol (CHL), ciprofloxacin, florfenicol, gentamicin, kanamycin, nalidixic acid (NAL), streptomycin (STR), sulfamethoxazole (SME), tetracycline (TET), and trimethoprim (TMP). The antimicrobial resistance of Y. enterocolitica 4/O:3 strains varied widely between the countries. Strains resistant to antimicrobial agents other than ampicillin were rare in Estonia, Finland, Latvia, and Russia, with prevalence of 0.7, 0.4, 0, and 8.3%, respectively. The highest prevalence of antimicrobial resistance was found in Spanish and Italian strains, with 98 and 61% of the strains being resistant to at least two antimicrobial agents, respectively. Resistance to at least four antimicrobial agents was found in 34% of Spanish, 19% of Italian, and 7.1% of English strains. Antimicrobial resistance was more common in countries where the total sales of antimicrobials for food production animals are high and orally administered medications are common. Our results indicate that antimicrobials should be used responsibly to treat infections, and parenteral medications should be preferred to orally administered mass medications.

Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum.

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

Comparative Genome Analysis and Spore Heat Resistance Assay Reveal a New Component to Population Structure and Genome Epidemiology Within Clostridium perfringens Enterotoxin-Carrying Isolates.

Clostridium perfringens causes a variety of human and animal enteric diseases including food poisoning, antibiotic-associated diarrhea, and necrotic enteritis. Yet, the reservoirs of enteropathogenic enterotoxin-producing strains remain unknown. We conducted a genomic comparison of 290 strains and a heat resistance phenotyping of 30 C. perfringens strains to elucidate the population structure and ecology of this pathogen. C. perfringens genomes shared a conserved genetic backbone with more than half of the genes of an average genome conserved in >95% of strains. The cpe-carrying isolates were found to share genetic context: the cpe-carrying plasmids had different distribution patterns within the genetic lineages and the estimated pan genome of cpe-carrying isolates had a larger core genome and a smaller accessory genome compared to that of 290 strains. We characterize cpe-negative strains related to chromosomal cpe-carrying strains elucidating the origin of these strains and disclose two distinct groups of chromosomal cpe-carrying strains with different virulence characteristics, spore heat resistance properties, and, presumably, ecological niche. Finally, an antibiotic-associated diarrhea isolate carrying two copies of the enterotoxin cpe gene and the associated genetic lineage with the potential for the emergence of similar strains are outlined. With C. perfringens as an example, implications of input genome quality for pan genome analysis are discussed. Our study furthers the understanding of genome epidemiology and population structure of enteropathogenic C. perfringens and brings new insight into this important pathogen and its reservoirs.

Mobile Elements Harboring Heavy Metal and Bacitracin Resistance Genes Are Common among Listeria monocytogenes Strains Persisting on Dairy Farms.

Listeria monocytogenes is a foodborne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes, and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements (MGEs) of Listeria strains inhabiting dairy farms. We performed a single-nucleotide polymorphism (SNP)-based phylogenomic analysis to identify 14 monophyletic clades of L. monocytogenes persistent on the farms for ≥6 months. We found that prophages and other mobile genetic elements were, on average, more numerous among isolates in persistent than nonpersistent clades, and we demonstrated that resistance genes against bacitracin, arsenic, and cadmium were significantly more prevalent among isolates in persistent than nonpersistent clades. We identified a diversity of mobile elements among the 250 farm isolates, including three novel plasmids, three novel transposons, and a novel prophage harboring cadmium resistance genes. Several of the mobile elements we identified in Listeria were identical to the mobile elements of enterococci, which is indicative of recent transfer between these genera. Through a genome-wide association study, we discovered that three putative defense systems against invading prophages and plasmids were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements that may spread to the food industry. IMPORTANCE Animal-derived raw materials are an important source of L. monocytogenes in the food industry. Knowledge of the factors contributing to the pathogen's transmission and persistence on farms is essential for designing effective strategies against the spread of the pathogen from farm to fork. An increasing body of evidence suggests that mobile genetic elements support the adaptation and persistence of L. monocytogenes in the food industry, as these elements contribute to the dissemination of genes encoding favorable phenotypes, such as resilience against biocides. Understanding of the role of farms as a potential reservoir of these elements is needed for managing the transmission of mobile elements across the food chain. Because L. monocytogenes coinhabits the farm ecosystem with a diversity of other bacterial species, it is important to assess the degree to which genetic elements are exchanged between Listeria and other species, as such exchanges may contribute to the rise of novel resistance phenotypes.

Phage lysin that specifically eliminates Clostridium botulinum Group I cells.

Clostridium botulinum poses a serious threat to food safety and public health by producing potent neurotoxin during its vegetative growth and causing life-threatening neuroparalysis, botulism. While high temperature can be utilized to eliminate C. botulinum spores and the neurotoxin, non-thermal elimination of newly germinated C. botulinum cells before onset of toxin production could provide an alternative or additional factor controlling the risk of botulism in some applications. Here we introduce a putative phage lysin that specifically lyses vegetative C. botulinum Group I cells. This lysin, called CBO1751, efficiently kills cells of C. botulinum Group I strains at the concentration of 5 µM, but shows little or no lytic activity against C. botulinum Group II or III or other Firmicutes strains. CBO1751 is active at pH from 6.5 to 10.5. The lytic activity of CBO1751 is tolerant to NaCl (200 mM), but highly susceptible to divalent cations Ca2+ and Mg2+ (50 mM). CBO1751 readily and effectively eliminates C. botulinum during spore germination, an early stage preceding vegetative growth and neurotoxin production. This is the first report of an antimicrobial lysin against C. botulinum, presenting high potential for developing a novel antibotulinal agent for non-thermal applications in food and agricultural industries.

Transcriptomic and Phenotypic Analyses of the Sigma B-Dependent Characteristics and the Synergism between Sigma B and Sigma L in Listeria monocytogenes EGD-e.

Numerous gene expression and stress adaptation responses in L. monocytogenes are regulated through alternative sigma factors σB and σL. Stress response phenotypes and transcriptomes were compared between L. monocytogenes EGD-e and its ΔsigB and ΔsigBL mutants. Targeted growth phenotypic analysis revealed that the ΔsigB and ΔsigBL mutants are impaired during growth under cold and organic-acid stress conditions. Phenotypic microarrays revealed increased sensitivity in both mutants to various antimicrobial compounds. Genes de-regulated in these two mutants were identified by genome-wide transcriptome analysis during exponential growth in BHI. The ΔsigB and ΔsigBL strains repressed 198 and 254 genes, respectively, compared to the parent EGD-e strain at 3 °C, whereas 86 and 139 genes, respectively, were repressed in these mutants during growth at 37 °C. Genes repressed in these mutants are involved in various cellular functions including transcription regulation, energy metabolism and nutrient transport functions, and viral-associated processes. Exposure to cold stress induced a significant increase in σB and σL co-dependent genes of L. monocytogenes EGD-e since most (62%) of the down-regulated genes uncovered at 3 °C were detected in the ΔsigBL double-deletion mutant but not in ΔsigB or ΔsigL single-deletion mutants. Overall, the current study provides an expanded insight into σB and σL phenotypic roles and functional interactions in L. monocytogenes. Besides previously known σB- and σL-dependent genes, the transcriptomes defined in ΔsigB and ΔsigBL mutants reveal several new genes that are positively regulated by σB alone, as well as those co-regulated through σB- and σL-dependent mechanisms during L. monocytogenes growth under optimal and cold-stress temperature conditions.

High prevalence of Clostridium botulinum in vegetarian sausages.

Clostridium botulinum is a significant food safety concern due to its ability to produce highly potent neurotoxin and resistant endospores. Vegetarian sausages have become a popular source of plant protein and alternative for meat products. While vegetarian sausages have not been linked to botulism, numerous outbreaks due to preserved vegetables suggest a frequent occurrence of C. botulinum spores in the raw material. The product formulation of vegetarian sausages involves limited NaCl and preservatives, and shelf-lives may be several months. The safety of vegetarian sausages thus relies mainly on heat treatment and chilled storage. The main food safety concern is C. botulinum Group II that can grow and produce toxin at refrigeration temperatures. Here we show a high overall prevalence (32%) of C. botulinum in 74 samples of vegetarian sausages from seven producers. Both Groups I and II strains and genes for neurotoxin types A, B, E and F were detected in the products. The highest cell counts (1200 spores/kg) were observed for C. botulinum Group II in products with remaining shelf-lives of 6 months at the time of purchase. We conclude that vacuum-packaged vegetarian sausage products frequently contain C. botulinum spores and may possess a high risk of C. botulinum growth and toxin production. Chilled storage below 3°C and thorough reheating before consumption are warranted.

Strain Variability of Listeria monocytogenes under NaCl Stress Elucidated by a High-Throughput Microbial Growth Data Assembly and Analysis Protocol.

Listeria monocytogenes causes the severe foodborne illness listeriosis and survives in food-associated environments due to its high stress tolerance. A data assembly and analysis protocol for microbial growth experiments was compiled to elucidate the strain variability of L. monocytogenes stress tolerance. The protocol includes measurement of growth ability under stress (step 1), selection of a suitable method for growth parameter calculation (step 2), comparison of growth patterns between strains (step 3), and biological interpretation of the discovered differences (step 4). In step 1, L. monocytogenes strains (n = 388) of various serovars and origins grown on media with 9.0% NaCl were measured using a Bioscreen C microbiology reader. Technical variability of the growth measurements was assessed and eliminated. In step 2, the growth parameters determined by Gompertz, modified-Gompertz, logistic, and Richards models and model-free splines were compared, illustrating differences in the suitability of these methods to describe the experimental data. In step 3, hierarchical clustering was used to describe the NaCl tolerance of L. monocytogenes measured by strain-specific variation in growth ability; tolerant strains had higher growth rates and maximum optical densities and shorter lag phases than susceptible strains. The spline parameter area under the curve best classified "poor," "average," and "good" growers. In step 4, the tested L. monocytogenes lineage I strains (serovars 4b and 1/2b) proved to be significantly more tolerant toward 9.0% NaCl than lineage II strains (serovars 1/2a, 1/2c, and 3a). Our protocol provides systematic tools to gain comparable data for investigating strain-specific variation of bacterial growth under stress.IMPORTANCE The pathogen Listeria monocytogenes causes the foodborne disease listeriosis, which can be fatal in immunocompromised individuals. L. monocytogenes tolerates several environmental stressors and can persist in food-processing environments and grow in foodstuffs despite traditional control measures such as high salt content. Nonetheless, L. monocytogenes strains differ in their ability to withstand stressors. Elucidating the intraspecies strain variability of L. monocytogenes stress tolerance is crucial for the identification of particularly tolerant strains. To enhance reliable identification of variability in bacterial stress tolerance phenotypes, we compiled a large-scale protocol for the entire data assembly and analysis of microbial growth experiments, providing a systematic approach and checklist for experiments on strain-specific growth ability. Our study illustrated the diversity and strain-specific variation of L. monocytogenes stress tolerance with an unprecedented scope and discovered biologically relevant serovar- and lineage-dependent phenotypes of NaCl tolerance.

Shopping Detail Information and Home Freezer Sampling Confirmed the Role of Commercial, Modified-Atmosphere Packaged Meatballs as a Vehicle for Listeriosis in Finland.

In November 2016, an elderly patient was diagnosed with Listeria monocytogenes bacteremia in Finland. Grocery store loyalty card records and microbiological investigation of foods found in the home fridge and freezer of the patient revealed commercial, modified-atmosphere packaged meatballs as the source of the infection. Investigation of the meatball production plant revealed that the floor drain samples were contaminated with the same L. monocytogenes strain as those isolated from the patient and meatballs. Ready-to-eat meatballs were likely contaminated after heat treatment from the production environment before packaging. Long-term cold storage, modified-atmosphere conditions, and the absence of competing bacteria presumably enhanced the growth of L. monocytogenes. We recommend that collection of shopping details and home fridge and freezer sampling should be part of surveillance of all cases of L. monocytogenes infections to complement information obtained from in-depth interviews.

Insights into the Phylogeny and Evolution of Cold Shock Proteins: From Enteropathogenic Yersinia and Escherichia coli to Eubacteria.

Psychrotrophic foodborne pathogens, such as enteropathogenic Yersinia, which are able to survive and multiply at low temperatures, require cold shock proteins (Csps). The Csp superfamily consists of a diverse group of homologous proteins, which have been found throughout the eubacteria. They are related to cold shock tolerance and other cellular processes. Csps are mainly named following the convention of those in Escherichia coli. However, the nomenclature of certain Csps reflects neither their sequences nor functions, which can be confusing. Here, we performed phylogenetic analyses on Csp sequences in psychrotrophic enteropathogenic Yersinia and E. coli. We found that representative Csps in enteropathogenic Yersinia and E. coli can be clustered into six phylogenetic groups. When we extended the analysis to cover Enterobacteriales, the same major groups formed. Moreover, we investigated the evolutionary and structural relationships and the origin time of Csp superfamily members in eubacteria using nucleotide-level comparisons. Csps in eubacteria were classified into five clades and 12 subclades. The most recent common ancestor of Csp genes was estimated to have existed 3585 million years ago, indicating that Csps have been important since the beginning of evolution and have enabled bacterial growth in unfavorable conditions.

Prevalence and Dynamics of Pathogenic Yersinia enterocolitica 4/O:3 Among Finnish Piglets, Fattening Pigs, and Sows.

Pigs are considered the main reservoir of Yersinia enterocolitica, and hence, understanding the ecology of this foodborne pathogen at the farm level is crucial. We calculated Bayesian estimates for the ability of a commercial enzyme-linked immunosorbent assay (ELISA) diagnostic test kit to detect antibodies against pathogenic Yersinia in pigs. The sensitivity and specificity of the test were 75.4% and 98.1%, respectively. We also studied the dynamics of Y. enterocolitica infection in 3 farrow-to-finish pig farms by following the same 30 pens of pigs through their lifetime from farrowing unit to slaughterhouse. Each farm was sampled 4 times, and 864 fecal and 730 serum samples were collected altogether. Pathogenic Y. enterocolitica 4/O:3 was isolated from 31.6% of the fecal samples by culturing, and Yersinia antibodies were detected in 38.2% of the serum samples with the commercial ELISA test. The pathogen was not isolated from farrowing units or all-in/all-out weaning units. However, in the weaning and fattening units using continuous management systems, the pathogen was isolated from every pen at some point of the study. After the pigs were transported into slaughterhouse, 150 tonsils were collected and 96.7% were positive by culturing. Among the strains isolated from feces and tonsils, 56 different genotypes of pathogenic Y. enterocolitica 4/O:3 were found by multilocus variable-number tandem-repeat analysis (MLVA). Finally, we collected tonsils of 266 sows from 115 farrowing farms, and Y. enterocolitica 4/O:3 was detected in 6.0% of the samples by the culture method, whereas 77.1% of the tonsils were serologically positive; the estimate for true seroprevalence was 95.8%. In conclusion, sows may not be the main source of Y. enterocolitica for piglets, although sows may still play a role in maintaining Y. enterocolitica in pig farms. Instead, pigs appear to get this foodborne pathogen mainly during the fattening period, especially if continuous management is applied.

Role of DEAD-box RNA helicase genes in the growth of Yersinia pseudotuberculosis IP32953 under cold, pH, osmotic, ethanol and oxidative stresses.

Yersinia pseudotuberculosis is an important foodborne pathogen threatening modern food safety due to its ability to survive and grow at low temperatures. DEAD-box RNA helicase CsdA has been shown to play an important role in the low-temperature growth of psychrotrophic Y. pseudotuberculosis. A total of five DEAD-box RNA helicase genes (rhlB, csdA, rhlE, dbpA, srmB) have been identified in Y. pseudotuberculosis IP32953. However, their role in various stress conditions used in food production is unclear. We studied the involvement of the DEAD-box RNA helicase-encoding genes in the cold tolerance of Y. pseudotuberculosis IP32953 using quantitative real-time reverse transcription (RT-qPCR) and mutational analysis. Quantitative RT-PCR revealed that mRNA transcriptional levels of csdA, rhlE, dbpA and srmB were significantly higher after cold shock at 3°C compared to non-shocked culture at 28°C, suggesting the involvement of these four genes in cold shock response at the transcriptional level. The deletion of csdA ceased growth, while the deletion of dbpA or srmB significantly impaired growth at 3°C, suggesting the requirement of these three genes in Y. pseudotuberculosis at low temperatures. Growth of each DEAD-box RNA helicase mutant was also investigated under pH, osmotic, ethanol and oxidative stress conditions. The five helicase-encoding genes did not play major roles in the growth of Y. pseudotuberculosis IP32953 under pH, osmotic, ethanol or oxidative stress.

Genomic Epidemiology and Phenotyping Reveal on-Farm Persistence and Cold Adaptation of Raw Milk Outbreak-Associated Yersinia pseudotuberculosis.

Packaged raw milk contaminated with Yersinia pseudotuberculosis mediated a large yersiniosis outbreak in southern Finland in 2014. The outbreak was traced back to a single dairy farm in southern Finland. Here we explore risk factors leading to the outbreak through epidemiologic investigation of the outbreak farm and through genomic and phenotypic characterization of the farm's outbreak and non-outbreak associated Y. pseudotuberculosis strains. We show that the outbreak strain persisted on the farm throughout the 7-month study, whereas the non-outbreak strains occurred sporadically. Phylogenomic analysis illustrated that the outbreak strain was related to previously published genomes of wild animal isolates from Finland, implying that wild animals were a potential source of the outbreak strain to the farm. We observed allelic differences between the farm's outbreak and non-outbreak strains in several genes associated with virulence, stress response and biofilm formation, and found that the outbreak strain formed biofilm in vitro and maintained better growth fitness during cold stress than the non-outbreak strains. Finally, we demonstrate the rapid growth of the outbreak strain in packaged raw milk during refrigerated storage. This study provides insight of the risk factors leading to the Y. pseudotuberculosis outbreak, highlights the importance of pest control to avoid the spread of pathogens from wild to domestic animals, and demonstrates that the cold chain is insufficient as the sole risk management strategy to control Y. pseudotuberculosis risk associated with raw drinking milk.