Detection of Yersinia enterocolitica, Shigella spp. and Salmonella spp. in Rattus norvegicus captured from Tehran, Iran.

Background: The present study aims to determine the presence of Yersinia spp., Yersinia pestis, Yersinia enterocolitica pathogen, Listeria monocytogenes, Salmonella spp., Shigella spp., Francisella tularensis and Borrelia spp. in brown rats of Tehran, Iran. Methods: PCR was used to detect various bacteria in 100 brown rats, Also, ELISA was used to detect antibodies against the F. tularensis and Borrelia spp. Results: A total of 16% and 13% of fecal samples were positive for Yersinia spp. and Y. enterocolitica pathogen. ELISA results were negative for F. tularensis and Borrelia. No specific antibodies (IgG) were against these bacteria. Conclusion: According to the results of our analysis, rats are significant transmitters and carriers of a variety of illnesses that can spread to both people and other animals.

Seroepidemiological survey of pathogenic Yersinia in domestic pigs in Chiba Prefecture, Japan.

This study aimed to investigate the prevalence of antibodies against pathogenic Yersinia such as Y. enterocolitica and Y. pseudotuberculosis in domestic pigs. A total of 650 serum samples from pigs in nine regions of the Chiba Prefecture in Japan, were tested using plasmid-encoded Yersinia outer membrane protein (Yops) antigen ELISA. The cutoff value was calculated using 20 pathogenic Yersinia-free pig serum samples. According to the cutoff value, 246 (37.8%) pigs from seven regions were considered seropositive for pathogenic Yersinia during the study period. These results indicate that pathogenic Yersinia is widespread in pigs in Chiba, which may become the source of human yersiniosis in this region.

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system.

In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics, and live-cell microscopy, we determined the contribution of the pilotin to the assembly, function, and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. Accordingly, most export apparatus subcomplexes are mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, while absence of the pilotin all but prevents export of early T3SS substrates, such as the needle subunits, it has little effect on secretion of late T3SS substrates, including the virulence effectors. We found that although pilotins interact with other injectisome components such as the secretin in the outer membrane, they mostly localize in transient mobile clusters in the bacterial membrane. Together, these findings provide a new view on the role of pilotins in the assembly and function of type III secretion injectisomes.

Thyroid abscess associated with thyrotoxicosis caused by Yersinia enterocolitica subsp. palearctica in a patient with follicular adenoma of the thyroid gland.

BACKGROUND: Yersinia enterocolitica is a gram-negative zoonotic bacterial pathogen that is typically transmitted via the fecal-oral route. The most common clinical manifestation of a Y. enterocolitica infection is self-limited gastroenteritis. Although various extraintestinal manifestations of Y. enterocolitica infection have been reported, there are no reports of thyroid abscesses. CASE PRESENTATION: An 89-year-old Japanese man with follicular adenoma of the left thyroid gland was admitted to our hospital with a 2-day history of fever and left neck pain. Laboratory tests revealed low levels of thyroid stimulating hormone and elevated levels of free thyroxine 4. Contrast-enhanced computed tomography showed low-attenuation areas with peripheral enhancement in the left thyroid gland. He was diagnosed with thyroid abscess and thyrotoxicosis, and treatment with intravenous piperacillin-tazobactam was initiated after collecting blood, drainage fluid, and stool samples. The isolated Gram-negative rod bacteria from blood and drainage fluid cultures was confirmed to be Y. enterocolitica. He was diagnosed with thyroid abscess and thyrotoxicosis due to be Y. enterocolitica subsp. palearctica. The piperacillin-tazobactam was replaced with levofloxacin. CONCLUSION: We report a novel case of a thyroid abscess associated with thyrotoxicosis caused by Y. enterocolitica subsp. palearctica in a patient with a follicular thyroid adenoma.

Yersinia enterocolitica biovar 1A: An underappreciated potential pathogen in the food chain.

Yersinia enterocolitica is an underreported cause of foodborne gastroenteritis. Little is known of the diversity of Y. enterocolitica isolated from food and which food commodities contribute to human disease. In this study, Y. enterocolitica was isolated from 37/50 raw chicken, 8/10 pork, 8/10 salmon and 1/10 leafy green samples collected at retail in the UK. Up to 10 presumptive Y. enterocolitica isolates per positive sample underwent whole genome sequencing (WGS) and were compared with publicly available genomes. In total, 207 Y. enterocolitica isolates were analyzed and belonged to 38 sequence types (STs). Up to five STs of Y. enterocolitica were isolated from individual food samples and isolates belonging to the same sample and ST differed by 0-74 single nucleotide polymorphisms (SNPs). Biotype was predicted for 205 (99 %) genomes that all belonged to biotype 1A, previously described as non-pathogenic. However, around half (51 %) of food samples contained isolates belonging to the same ST as previously isolated from UK human cases. The closest human-derived isolates shared between 17 and 7978 single nucleotide polymorphisms (SNPs) with the food isolates. Extensive food surveillance is required to determine what food sources are responsible for Y. enterocolitica infections and to re-examine the role of biotype 1A as a human pathogen.

Cytosolic sorting platform complexes shuttle type III secretion system effectors to the injectisome in Yersinia enterocolitica.

Bacteria use type III secretion injectisomes to inject effector proteins into eukaryotic target cells. Recruitment of effectors to the machinery and the resulting export hierarchy involve the sorting platform. These conserved proteins form pod structures at the cytosolic interface of the injectisome but are also mobile in the cytosol. Photoactivated localization microscopy in Yersinia enterocolitica revealed a direct interaction of the sorting platform proteins SctQ and SctL with effectors in the cytosol of live bacteria. These proteins form larger cytosolic protein complexes involving the ATPase SctN and the membrane connector SctK. The mobility and composition of these mobile pod structures are modulated in the presence of effectors and their chaperones, and upon initiation of secretion, which also increases the number of injectisomes from ~5 to ~18 per bacterium. Our quantitative data support an effector shuttling mechanism, in which sorting platform proteins bind to effectors in the cytosol and deliver the cargo to the export gate at the membrane-bound injectisome.

A poly-proline II helix in YadA from Yersinia enterocolitica serotype O:9 facilitates heparin binding through electrostatic interactions.

Poly-proline II helices are secondary structure motifs frequently found in ligand-binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen-bonded α-helices or ß-strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly-proline II helix interaction motif in the N-terminal region. The motif is involved in the interaction of YadAO:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N-terminal motif of YadA are required for electrostatic interactions with the sulfate groups of heparin. Biophysical methods including CD spectroscopy, solution-state NMR and SAXS all independently support the presence of a poly-proline helix allowing YadAO:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulfation of heparin and heparan sulfate are not targeted by YadAO:9 -mediated adhesion. We speculate that the YadAO:9 -heparin interaction plays an important and highly strain-specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.

Molecular characterization of Yersinia enterocolitica strains to evaluate virulence associated genes.

INTRODUCTION: Yersinia enterocolitica (Ye) species is divided into 6 biotypes (BT), 1A, 1B, 2, 3, 4, 5 classified based on biochemical reactions and about 70 serotypes, classified based on the structure of the lipopolysaccharide O-antigen. The BT1A is considered non-pathogenic, while the BT 1B-5 are considered pathogenic. METHODS: Evaluate the distribution of eleven chromosomal and plasmid virulence genes, ail, ystA, ystB, myfA, hreP, fes, fepD, ymoA, sat, virF and yadA, in 87 Ye strains isolated from food, animals and humans, using two SYBR Green real-time PCR platforms. RESULTS: The main results showed the presence of the ail and ystA genes in all the pathogenic bioserotypes analyzed. The ystB, on the other hand, was identified in all non-pathogenic strains biotype 1A. The target fes, fepD, sat and hreP were found in both pathogenic biotypes and in BT1A strains. The myfA gene was found in all pathogenic biotype and in some Ye BT1A strains. The virF and yadA plasmid genes were mainly detected in bioserotype 4/O:3 and 2/O:9, while ymoA was identified in all strains. CONCLUSIONS: The two molecular platforms could be used to better define some specific molecular targets for the characterization and rapid detection of Ye in different sources which important implications for food safety and animal and human health.

One-step preparation of a self-assembled bioconjugate nanovaccine against Brucella.

Brucellosis, caused by Brucella, is a severe zoonosis, and the current Brucella live attenuated vaccine cannot be used in humans due to major safety risks. Although polysaccharide antigens can be used to prepare the Brucella vaccine, their lower immunogenicity limits them from producing efficient and broad protection. In this study, we produced a high-performance bioconjugate nanovaccine against different species of Brucella by introducing a self-assembly nanoparticle platform and an O-linked glycosylation system into Yersinia enterocolitica serotype O:9, which has an O-polysaccharide composed of the same unit as Brucella. After successfully preparing the vaccine and confirming its stability, we subsequently demonstrated the safety of the vaccine in mice by high-dose immunization. Then, by a series of mouse experiments, we found that the nanovaccine greatly promoted antibody responses. In particular, the increase of IgG2a was more obvious than that of IgG1. Most importantly, this nanovaccine could provide cross-protection against B. abortus, B. melitensis, and B. suis strains by lethal dose challenged models, and could improve the clearance of B. melitensis, the most common pathogenic species in human brucellosis, by non-lethal dose infection. Overall, for the first time, we biocoupled polysaccharide antigens with nano carriers to prepare a Brucella vaccine, which showed pronounced and extensive protective effects in mice. Thus, we provided a potential candidate vaccine and a new direction for Brucella vaccine design.

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.

Isolation, characterization and antimicrobial resistance of Yersinia enterocolitica from Polish cattle and their carcasses.

BACKGROUND: Yersinia enterocolitica is a heterogeneous bacterial species that has been divided into six biotypes and more than 70 serotypes. Each year, the European Food Safety Authority classifies yersiniosis caused by Y. enterocolitica as one of the most important zoonotic diseases. The prevalence of Y. enterocolitica in cattle has not been thoroughly analyzed in Poland, and beef and bovine carcasses contaminated with antimicrobial resistant Y. enterocolitica pose a health risk for both, farm workers and consumers. Therefore, the aim of this study was to evaluate the prevalence of Y. enterocolitica in cattle and to determine the antimicrobial susceptibility of the isolated strains. RESULTS: A total of 1020 samples were analyzed, including 660 rectal swabs collected from live cattle and 360 swabs from cold-stored beef carcasses. The results of this study indicate that Y. enterocolitica was isolated from three of the 15 examined cattle herds and the prevalence within these herds ranged from 0% to nearly 32%. Y. enterocolitica was isolated from 14.7% of the examined heifers, 7.4% of calves and 5.5% of adult cows. More than 65% of the strains were isolated from cold enrichment. The strains isolated from live cattle tested positive for the ystB gene, while ail and ystA genes were not found. Most of the isolated strains belonged to bioserotype 1A/NT. The majority of the isolated strains were resistant to ampicillin, cefalexin and amoxicillin with clavulanic acid, however these are expected phenotypes for Y. enterocolitica. CONCLUSIONS: The results of this study indicate that Y. enterocolitica is present in cattle herds in Poland. The strains isolated from live cattle were ystB-positive, most of them belonged to bioserotype 1A/NT. The prevalence of Y. enterocolitica strains was generally low in cold-stored beef carcasses.

The implication of viability and pathogenicity by truncated lipopolysaccharide in Yersinia enterocolitica.

The fast envelope stress responses play a key role in the transmission and pathogenesis of Yersinia enterocolitica, one of the most common foodborne pathogens. Our previous study showed that deletion of the waaF gene, essential for the biosynthesis of lipopolysaccharide (LPS) core polysaccharides, led to the formation of a truncated LPS structure and induced cell envelope stress. This envelope stress may disturb the intracellular signal transduction, thereby affecting the physiological functions of Y. enterocolitica. In this study, truncated LPS caused by waaF deletion was used as a model of envelope stress in Y. enterocolitica. We investigated the mechanisms of envelope stress responses and the cellular functions affected by truncated LPS. Transcriptome analysis and phenotypic validation showed that LPS truncation reduced flagellar assembly, bacterial chemotaxis, and inositol phosphate metabolism, presenting lower pathogenicity and viability both in vivo and in vitro environments. Further 4D label-free phosphorylation analysis confirmed that truncated LPS perturbed multiple intracellular signal transduction pathways. Specifically, a comprehensive discussion was conducted on the mechanisms by which chemotactic signal transduction and Rcs system contribute to the inhibition of chemotaxis. Finally, the pathogenicity of Y. enterocolitica with truncated LPS was evaluated in vitro using IPEC-J2 cells as models, and it was found that truncated LPS exhibited reduced adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. Our research provides an understanding of LPS in the regulation of Y. enterocolitica viability and pathogenicity and, thus, opening new avenues to develop novel food safety strategies or drugs to prevent and control Y. enterocolitica infections. KEY POINTS: • Truncated LPS reduces flagellar assembly, chemotaxis, and inositol phosphate metabolism in Y. enterocolitica. • Truncated LPS reduces adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. • Truncated LPS regulates intracellular signal transduction of Y. enterocolitica.

Stringent Response Factor DksA Contributes to Fatty Acid Degradation Function to Influence Cell Membrane Stability and Polymyxin B Resistance of Yersinia enterocolitica.

DksA is a proteobacterial regulator that binds directly to the secondary channel of RNA polymerase with (p)ppGpp and is responsible for various bacterial physiological activities. While (p)ppGpp is known to be involved in the regulation and response of fatty acid metabolism pathways in many foodborne pathogens, the role of DksA in this process has yet to be clarified. This study aimed to characterize the function of DksA on fatty acid metabolism and cell membrane structure in Yersinia enterocolitica. Therefore, comparison analysis of gene expression, growth conditions, and membrane permeabilization among the wide-type (WT), DksA-deficient mutant (YEND), and the complemented strain was carried out. It confirmed that deletion of DksA led to a more than four-fold decrease in the expression of fatty acid degradation genes, including fadADEIJ. Additionally, YEND exhibited a smaller growth gap compared to the WT strain at low temperatures, indicating that DksA is not required for the growth of Y. enterocolitica in cold environments. Given that polymyxin B is a cationic antimicrobial peptide that targets the cell membrane, the roles of DksA under polymyxin B exposure were also characterized. It was found that DksA positively regulates the integrity of the inner and outer membranes of Y. enterocolitica under polymyxin B, preventing the leakage of intracellular nucleic acids and proteins and ultimately reducing the sensitivity of Y. enterocolitica to polymyxin B. Taken together, this study provides insights into the functions of DksA and paves the way for novel fungicide development.

Interplay between the RNA Chaperone Hfq, Small RNAs and Transcriptional Regulator OmpR Modulates Iron Homeostasis in the Enteropathogen Yersinia enterocolitica.

Iron is both essential for and potentially toxic to bacteria, so the precise maintenance of iron homeostasis is necessary for their survival. Our previous study indicated that in the human enteropathogen Yersinia enterocolitica, the regulator OmpR directly controls the transcription of the fur, fecA and fepA genes, encoding the ferric uptake repressor and two transporters of ferric siderophores, respectively. This study was undertaken to determine the significance of the RNA chaperone Hfq and the small RNAs OmrA and RyhB1 in the post-transcriptional control of the expression of these OmpR targets. We show that Hfq silences fur, fecA and fepA expression post-transcriptionally and negatively affects the production of FLAG-tagged Fur, FecA and FepA proteins. In addition, we found that the fur gene is under the negative control of the sRNA RyhB1, while fecA and fepA are negatively regulated by the sRNA OmrA. Finally, our data revealed that the role of OmrA results from a complex interplay of transcriptional and post-transcriptional effects in the feedback circuit between the regulator OmpR and the sRNA OmrA. Thus, the expression of fur, fecA and fepA is subject to complex transcriptional and post-transcriptional regulation in order to maintain iron homeostasis in Y. enterocolitica.

Development of detection methods by multiplex real-time PCR for highly pathogenic Yersinia enterocolitica, low pathogenic Yersinia enterocolitica, and Yersinia pseudotuberculosis based on SYBR Green and TaqMan probes.

This study aimed to develop multiplex real-time PCR methods using SYBR Green and TaqMan probes for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis. Specific primer and probe combinations for differentiating three pathogenic Yersinia groups were designed from three chromosomally encoded genes (ail, fyuA, and inv). Twenty-six stains of pathogenic Yersinia species including 6 strains of low pathogenic Y. enterocolitica serotypes, 7 strains of highly pathogenic Y. enterocolitica serotypes, and 13 strains of pathogenic Y. pseudotuberculosis were used for specificity testing. Specific patterns of real-time amplification signals distinguished three pathogenic Yersinia groups. A detection limit of approximately 101 colony forming units (CFU) /reaction of genomic DNA was determined based on plate counts. Furthermore, the multiplex real-time PCR methods also detected Y. enterocolitica O:8 from the DNA extracted from spiked rabbit blood samples and potentially infected wild rodent fecal samples. These results demonstrated that the multiplex real-time PCR methods developed in this study are useful for rapid detection and differentiation of three pathogenic Yersinia groups. Therefore, these methods provide a new monitoring and detection capability to understand the epidemiology of pathogenic Yersinia and to diagnose three pathogenic Yersinia groups.

Characterization of phenyl propiolic acid from Proteus mirabilis DMTMMR-11 and Evaluation of its mode of action against Yersinia enterocolitica (MTCC-840) an in-Vitro and in-Vivo based approach.

Foodborne illnesses are pervasive in raising public health concerns in both developed and developing nations. Yersinia enterocolitica a zoonotic bacterial species that causes food-transmitted infections, and gastroenteritis, is its most prevalent clinical manifestation. This study aims to investigate the differences, dependencies, and inhibitory mechanisms between the host and the microbiome. Proteus mirabilis DMTMMR-11, the bacterium found in the human gastrointestinal tract was used for the extraction of intracellular metabolite, because of its beneficial effects on the normal flora of the human gut. Phenyl propiolic acid was identified as the dominant compound in the metabolite after characterization using FT-IR, NMR, and LC-MS-MS. To assess its inhibitory mechanism against Yersinia enterocolitica, the pathogen was subjected to biological characterization by MBC and MIC, resulting in the rate of inhibition at 50 µg/ml. Anti-bacterial curve supports the inhibited growth of Y. enterocolitica. Mechanism of inhibition at its cellular level was indicated by the increase in alkaline phosphate content, which drastically reduced the cell membrane and cell wall potential expanding its permeability by intruding the membrane proteins, which was observed in SEM Imaging. Phenyl propiolic acid efficiently disrupts the biofilm formation by reducing the adherence and increasing the eradication property of the pathogen by exhibiting 65% of inhibition at the minimal duration of 12h. In-vivo study was carried out through host-pathogen interaction in C. elegans, an efficient model organism assessed for its life-span, physiological, and behavioral assays.

An ultrasensitive aptamer-based fluorescent on/off system for trace amount evaluation of Yersinia enterocolitica in food samples.

An innovative aptamer labeled with 5-FAM has been developed with a high affinity for Yersinia enterocolitica (Y. enterocolitica) using graphene oxide (GO) as a quenching platform. The selectivity of the prepared system was evaluated in the presence of common coexisted bacteria like Yersinia pseudotuberculosis, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella typhimurium. Some experimental factors like pH and stability were investigated. The results showed that in the absence of Y. enterocolitica, aptamer labeled with 5-FAM was bonded with GO, causing fluorescence to be relatively weak. After the addition of Y. enterocolitica, the aptamer is released from the GO surface and binds to the target bacteria, and significantly increases the fluorescence intensity with an excitation wavelength of 410 nm and an emission wavelength of 530 nm. After optimizing all conditions, the system exhibited a wide linear response for Y. enterocolitica in the concentration range 10 to 1.0 × 109 CFU•mL-1 and the limit of detection (LOD) was 3 CFU•mL-1. This system demonstrated that GO-designed aptamers can be successful in detecting Y. enterocolitica in whole-cell forms, making them potentially useful for screening and rapid detection.

The biochemical characteristics of viable but nonculturable state Yersinia enterocolitica induced by lactic acid stress and its presence in food systems.

The viable but nonculturable (VBNC) state is adopted by many foodborne pathogenic bacteria to survive in adverse conditions. This study found that lactic acid, a widely used food preservative, can induce Yersinia enterocolitica to enter a VBNC state. Y. enterocolitica treated with 2 mg/mL lactic acid completely lost culturability within 20 min, and 10.137 ± 1.693 % of the cells entered a VBNC state. VBNC state cells could be recovered (resuscitated) in tryptic soy broth (TSB), 5 % (v/v) Tween80-TSB, and 2 mg/mL sodium pyruvate-TSB. In the VBNC state of Y. enterocolitica induced by lactic acid, the intracellular adenosine triphosphate (ATP) concentration and various enzyme activities were decreased, and the reactive oxygen species (ROS) level was elevated, compared with uninduced cells. The VBNC state cells were significantly more resistant to heat and simulated gastric fluid than uninduced cells, but their ability to survive in a high-osmotic-pressure environment was significantly less than that of uninduced cells. The VBNC state cells induced by lactic acid changed from long rod-like to short rod-like, with small vacuoles at the cell edges; the genetic material was loosened and the density of cytoplasm was increased. The VBNC state cells had decreased ability to adhere to and invade Caco-2 (human colorectal adenocarcinoma) cells. The transcription levels of genes related to adhesion, invasion, motility, and resistance to adverse environmental stress were downregulated in VBNC state cells relative to uninduced cells. In meat-based broth, all nine tested strains of Y. enterocolitica entered the VBNC state after lactic acid treatment; among these strains, only VBNC state cells of Y. enterocolitica CMCC 52207 and Isolate 36 could not be recovered. Therefore, this study is a wake-up call for food safety problems caused by VBNC state pathogens induced by lactic acid.

Traceability, virulence and antimicrobial resistance of Yersinia enterocolitica in two industrial cheese-making plants.

Between 2018 and 2019, 309 environmental and food samples were collected from two industrial cheese-making plants located in Sardinia, in order to investigate Y. enterocolitica presence and to characterize the isolates. Y. enterocolitica isolates were further compared with isolates detected during a previous investigation from sheep and goat raw milk samples. Y. enterocolitica was detected in 7.4 % of the samples and the prevalence was higher, even if not significantly (P > 0.05) higher in non-food contact surface samples (10.2 %) than in food contact surface samples (3.8 %). The highest prevalence was detected in floor samples (13.5 %), followed by drain samples (7.2 %), which might serve as main harborage sites for further contamination. Y. enterocolitica was also detected in food contact surfaces, namely shelves of the Ricotta cooling room and packaging room, one cheese cutting machine surface and one raw milk filter sample. The biotype 1A isolates identified in this study were classified into six different serotypes. Additionally, a bioserotype 2/O:5,27 isolate was identified in one goat milk sample. All 1A isolates possessed the virulence genes invA and ystB while the 2/O:5,27 isolate showed the presence of ail, ystA, invA and yadA genes, thus confirming a pathogenic potential. The isolates showed intrinsic resistance to amoxicillin-clavulanic acid, ticarcillin and cefoxitin due to the presence of the blaA gene. Whole genome sequencing allowed to identify seven different sequence types among the 1A isolates, thus showing a high genetic diversity. The same Y. enterocolitica sequence type (ST3) was detected from three different areas of the same cheese-making plant, indicating a possible transfer of the microorganism along the processing lines. Y. enterocolitica contamination in cheese-making plants can pose a risk to human health. Preventive measures include the hygienic design of the plant layout and equipment, in association with proper cleaning and disinfection programmes.

Regulation and Functionality of a Holin/Endolysin Pair Involved in Killing of Galleria mellonella and Caenorhabditis elegans by Yersinia enterocolitica.

The insecticidal toxin complex (Tc) proteins are produced by several insect-associated bacteria, including Yersinia enterocolitica strain W22703, which oscillates between two distinct pathogenicity phases in invertebrates and humans. The mechanism by which this high-molecular-weight toxin is released into the extracellular surrounding, however, has not been deciphered. In this study, we investigated the regulation and functionality of a phage-related holin/endolysin (HE) cassette located within the insecticidal pathogenicity island Tc-PAIYe of W22703. Using the Galleria mellonella infection model and luciferase reporter fusions, we revealed that quorum sensing contributes to the insecticidal activity of W22703 upon influencing the transcription of tcaR2, which encodes an activator of the tc and HE genes. In contrast, a lack of the Yersinia modulator, YmoA, stimulated HE gene transcription, and mutant W22703 ΔymoA exhibited a stronger toxicity toward insect larvae than did W22703. A luciferase reporter fusion demonstrated transcriptional activation of the HE cassette in vivo, and a significantly larger extracellular amount of subunit TcaA was found in W22703 ΔymoA relative to its ΔHE mutant. Using competitive growth assays, we demonstrated that at least in vitro, the TcaA release upon HE activity is not mediated by cell lysis of a significant part of the population. Oral infection of Caenorhabditis elegans with a HE deletion mutant attenuated the nematocidal activity of the wild type, similar to the case with a mutant lacking a Tc subunit. We conclude that the dual holin/endolysin cassette of yersiniae is a novel example of a phage-related function adapted for the release of a bacterial toxin. IMPORTANCE Members of the genus Yersinia cause gastroenteritis in humans but also exhibit toxicity toward invertebrates. A virulence factor required for this environmental life cycle stage is the multisubunit toxin complex (Tc), which is distinct from the insecticidal toxin of Bacillus thuringiensis and has the potential to be used in pest control. The mechanism by which this high-molecular-weight Tc is secreted from bacterial cells has not been uncovered. Here, we show that a highly conserved phage-related holin/endolysin pair, which is encoded by the genes holY and elyY located between the Tc subunit genes, is essential for the insecticidal activity of Y. enterocolitica and that its activation increases the amount of Tc subunits in the supernatant. Thus, the dual holY-elyY cassette of Y. enterocolitica constitutes a new example for a type 10 secretion system to release bacterial toxins.