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Yersinia (Y.) enterocolitica , an etiological agent of yersiniosis, is a bacterium whose pathogenicity is determined, among other things, by its ability to produce toxins. The aim of this article was to present the most important toxins that are produced by biotype 1A strains of Y. enterocolitica , and to discuss their role in the pathogenesis of yersiniosis. Y. enterocolitica biotype 1A strains are able to synthesize variants of thermostable YST enterotoxin and play a key role in the pathogenesis of yersiniosis. Biotype 1A strains of Y. enterocolitica also produce Y. enterocolitica pore-forming toxins, YaxA and YaxB. These toxins form pores in the cell membrane of host target cells and cause osmotic lysis, which is of particular importance in systemic infections. Insecticidal toxin complex genes have been detected in some clinical biotype 1A strains of Y. enterocolitica . However, their role has not yet been fully elucidated. Strains belonging to biotype 1A have long been considered non-pathogenic. This view is beginning to change due to the emerging knowledge about the toxigenic potential of these bacteria and their ability to overcome the defense barriers of the host organism.

Purpose: Infective endocarditis (IE) is a major complication in patients with bacteremia of Staphylococcus ( S. ) aureus infection. Our aim was to determine the association of the major Staphylococcal superantigens (SAgs), including Staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1), among hospitalized patients diagnosed with bacteremia and those with IE., Methods: This study was conducted on 88 patients; of these, 84 (95.5%) had two positive blood cultures. Eighteen out of the 84 patients (21.4%) were diagnosed based on the modified Duke criteria by a cardiologist to have IE. The recovered isolates were screened phenotypically using ELISA followed by molecular analysis of sea , seb , sec , sed , see , and tsst-1 , the major SAg coding genes, and the obtained findings were statistically analyzed., Results: Phenotypic screening for SE production of 26 selected Staphylococci (15 isolated from the IE patients (10 S. aureus and 5 coagulase negative staphylococci (CoNS)) and 11 from bacteremic patients (10 S. aureus and 1 CoNS)) using ELISA revealed that 12/26 (46%) isolates were SE producers. PCR analysis showed that 19 (73%) isolates were PCR positive for SAg genes with the highest prevalence of the sea gene (79%), followed by seb (63%) and tsst-1 (21%). The least frequent gene was sed (5.3%). Statistical correlations between bacteremic and IE isolates with respect to prevalence of SAgs showed no significant difference ( P value = 0.139, effect size = 0.572) indicating no specific association between any of the detected SAgs and IE., Conclusion: There is high prevalence of SEs among clinical isolates of Staphylococci recovered from patients suffering bacteremia and those with IE. No significant difference was found among Staphylococcal isolates recovered from patients with bacteremia or IE regarding both phenotypic and genotypic detection of the tested SAgs., Competing Interests: The authors declare that no competing interests exist., (Copyright © 2020 Heba M. Elsherif et al.)

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens for humans and farm animals such as pigs. Porcine ETEC strains induce diarrhea through the production of heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (pSTa/STb). Although LT secretion levels differ between porcine ETEC strains, and this has been linked to virulence, it is unclear whether ST secretion levels also differ between porcine ETEC strains. In addition, the molecular mechanism underlying different LT secretion levels has not been elucidated. In this work, multiple porcine ETEC strains were assessed for their capacity to produce and secrete the enterotoxins LT, pSTa, and STb. The strains differed greatly in their capacity to secrete LT, pSTa, and STb. Remarkably, in some strains, periplasmic production did not correlate with their ability to secrete LT, resulting in high periplasmic production and low LT secretion levels. Furthermore, the results indicated that the type II secretion system (T2SS) protein YghG plays a regulatory role in controlling LT secretion levels. These findings highlight YghG as an important mediator of the secretion of the heat-labile enterotoxin LT by porcine ETEC strains and provide better insights into ETEC enterotoxin secretion. IMPORTANCE Enterotoxigenic E. coli strains are a major health concern. Enterotoxins secreted by enterotoxigenic E. coli are crucial for diarrhea induction. Enterotoxin secretion levels differ between strains; however, it is currently unclear what drives these differences. The discrepancy in the production and secretion capacities of enterotoxins in ETEC is important to clarify their function involved in diarrhea induction. Our results further deepen our understanding of how type II secretion system (T2SS) components of ETEC control enterotoxin secretion levels and may lay the foundation for a better understanding of ETEC molecular pathogenesis., (Copyright © 2020 American Society for Microbiology.)

Staphylococcus aureus is the causative agent of staphylococcal food poisoning and is a common contaminant in milk. Despite efforts to control S. aureus, recalls and outbreaks continue to occur, highlighting the need for additional interventions. This study determined the potential for protective cultures (PC) that are commercially available to producers to control S. aureus growth in raw milk and attenuate virulence by impeding staphylococcal enterotoxin (SE) production in raw milk and laboratory medium. Cultures of Hafnia alvei and Lactococcus lactis effectively inhibited S. aureus growth in raw milk to counts ~5 log CFU/mL lower than control when cocultured following a cheesemaking time and temperature profile; two cultures of Lactobacillus plantarum inhibited growth to ~1.5 log CFU/mL less than control. Cocultures of S. aureus with Lc. lactis, H. alvei and Lb. plantarum in raw milk reduced SE levels by 24.9%, 62.4%, and 76%, respectively. Lc. lactis also decreased SE production in raw milk in the absence of PC-mediated growth inhibition. Significant reductions in SE production in the absence of pathogen growth inhibition were also achieved in laboratory medium. Together, these results demonstrate the potential for PCs to inhibit S. aureus growth and impede SE production in the absence of growth inhibition., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Artisan fresh cheese producing farms from six provinces of Cuba were studied to identify the presence of bacterial hazards and the results are presented in this research communication. The bacterial hazards identified in milk and cheese respectively were: Listeria spp. (9.5 and 18.9%), Bacillus cereus (23.2 and 24.2%), Escherichia coli O157 (12.6 and 13.7%), Salmonella spp. (10.5 and 17.9%), and Staphylococcus aureus (29.5 and 51.6%). Listeria monocytogenes was not detected. Nine Salmonella serotypes corresponding to Salmonella enterica subsp. enterica and Salmonella enterica subsp. arizonae were isolated, whereas Salmonella Anatum was present most often. Biofilm formation by the isolated species and enterotoxin production by S. aureus strains demonstrated the pathogenic potential of the identified bacterial hazards. Results proved the presence of bacterial hazards in the raw milk and cheeses analyzed, so that good manufacturing practices must be accomplished throughout the entire production process in order to avoid the occurrence of foodborne diseases in the population.

Background: Superantigens of Staphylococcus aureus namely enterotoxin A, exfoliative toxin A, and Toxic shock syndrome toxin-1 cause detrimental effects on the cells of the immune system., Methods: In this work, the toxins were downloaded from the Protein DataBank database and energies were minimized using KoBaMIN server. Forty flavonoids compounds were identified by pubchem compound database through extensive literature study and their 3D structures were obtained by submitting SMILES to CORINA tool. Based on Lipinski's rule of five, the molecules were filtered that resulted in 27 compounds. Molecular docking was performed for identifying the binding and interaction sites of flavonoids with the toxins using Autodock 4., Results and Conclusion: The docked complexes were then subjected to molecular dynamics simulation using Gromacs. The analysis revealed the stability of the complexes as indicated by three hydrogen bonds formed during the simulation time period of 20 ns., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

This article describes the antimicrobial resistance to date of the most frequently encountered anaerobic bacterial pathogens of animals. The different sections show that antimicrobial resistance can vary depending on the antimicrobial, the anaerobe, and the resistance mechanism. The variability in antimicrobial resistance patterns is also associated with other factors such as geographic region and local antimicrobial usage. On occasion, the same resistance gene was observed in many anaerobes, whereas some were limited to certain anaerobes. This article focuses on antimicrobial resistance data of veterinary origin.

Biocontrol of Staphylococcus aureus by lactic acid bacteria can be considered as a feasible alternative to the use of chemicals in foods, but the mechanisms underlying this antagonistic interaction remains poorly understood. This study aimed to evaluate the impact of a LAB species (Enterococcus faecalis) over the growth, enterotoxin production and gene expression of S. aureus under experimental conditions. E. faecalis 41FL1 and S. aureus ATCC 29213 were inoculated isolated and together in brain heart infusion (BHI) at 30 °C and in a fresh cheese model at 15 °C: microbial populations were monitored by culture plating, production of classical staphylococcal enterotoxins (SEs) was verified by an ELISA assay, expression of S. aureus genes (virulence, transcriptional regulation and central carbon metabolism) was investigated by quantitative real-time PCR, and pH and contents of water-soluble metabolites in both matrices were measured. S. aureus growth was inhibited in co-cultures assays, with a 2.02-log reduction in BHI and a 3.39-log reduction in cheese model compared to respective single cultures. Classical SEs were detected in S. aureus single culture assays (BHI and cheese), in BHI inoculated with both strains after 48 h of incubation, but not detected in co-inoculated cheeses. pH in all matrices containing E. faecalis reached lower values than in matrices containing S. aureus alone, due to lactate production by E. faecalis. Expression of genes coding for transcription regulators (ccpA and rex) and enzymes involved in central carbon metabolism (alsD and citZ) was mostly upregulated in co-inoculated cheeses, whereas expression of several virulence determinants (agrC, hld, hla, entA and spa) was strongly downregulated. This study provides relevant data on the behaviour of S. aureus in the presence of competing microbiota and support the use of controlled population dominance by LAB as an effective biopreservation strategy to ensuring food safety., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Food poisoning caused by Staphylococcus aureus is responsible for staphylococcal enterotoxin (SE) produced in foods. Staphylococcal food poisoning is mostly caused by staphylococcal enterotoxin type A (SEA) among SEs. Growth/no growth for S. aureus under various environmental conditions was well studied with a logistic regression model so far. Recently we successfully described the boundaries of SEA production and growth of S. aureus in broth at various temperatures and salt concentrations with the model. In this study, the effects of lactic acid and salt on SEA production and growth of S. aureus was quantitatively studied. Consequently the boundaries of SEA production and growth of S. aureus cocktail in broth at various combinations of salt concentrations and pH values that were adjusted with lactic acid were successfully described with a logistic regression model. Here the cocktail was incubated in stationary culture at 30 °C and 10 °C. The maximum toxin production and cell growth of the cocktail were observed both at 5% salt in the pH range from 4.5 to 7.0. Also, the characteristics of individual strains of the cocktail in SEA production and growth at 30 °C and 10 °C were found to be specific to the strains. The present study revealed the effect of lactic acid and salt on SEA production and growth of S. aureus as well as the variety of SEA production and growth of S. aureus strains. These results would become useful information in food industry to prevent staphylococcal food poisoning. PRACTICAL APPLICATION: Boundaries of enterotoxin A production/no production and growth/no growth of staphylococcal cocktail at various combinations of pHs adjusted with lactic acid and salt concentrations were well described with a logistic regression model. The maximum toxin production and cell growth were observed both at 5% salt in the pH range from 4.5 to 7.0. A variety of the toxin production and cell growth were observed in terms of pH and salt concentration among individual strains of the cocktail., (© 2019 Institute of Food Technologists®.)

Development of long-term memory is crucial for vaccine-induced adaptive immunity against infectious diseases such as Staphylococcus aureus infection. Toxic shock syndrome toxin 1 (TSST-1), one of the superantigens produced by S. aureus , is a possible vaccine candidate against infectious diseases caused by this pathogen. We previously reported that vaccination with less toxic mutant TSST-1 (mTSST-1) induced T helper 17 (Th17) cells and elicited interleukin-17A (IL-17A)-mediated protection against S. aureus infection 1 week after vaccination. In the present study, we investigated the host immune response induced by mTSST-1 vaccination in the memory phase, 12 weeks after the final vaccination. The protective effect and IL-17A production after vaccination with mTSST-1 were eliminated because of IL-10 production. In the presence of IL-10-neutralizing monoclonal antibody (mAb), IL-17A production was restored in culture supernatants of CD4 + T cells and macrophages sorted from the spleens of vaccinated mice. Vaccinated mice treated with anti-IL-10 mAb were protected against systemic S. aureus infection in the memory phase. From these results, it was suggested that IL-10 produced in the memory phase suppresses the IL-17A-dependent vaccine effect through downregulation of IL-17A production., (Copyright © 2019 American Society for Microbiology.)

Aims: This investigation was undertaken to study the prevalence, enterotoxin gene profile and molecular epidemiology of Aeromonads from various sources of water (182) and fish (173)., Methods and Results: A total of 116 Aeromonas sp. were isolated, of which 48 (26·37%) were from water and 68 (34·62%) were from fish samples collected from retail markets and fish farms. The Aeromonads were recovered from all types of water sources viz. drinking water (13%), surface waters (26%) and fish ponds (69%). The most prevalent species recovered from drinking water was A. hydrophila, from fish ponds it was A. caviae, from surface water sources A. hydrophila and A. caviae were recovered more frequently, and A. hydrophila and A. veronii bv. sobria were isolated predominantly from gills of fish samples. On multiplex PCR analysis for the detection of enterotoxin genes (act, alt, ast), the above mentioned Aeromonas species frequently contained enterotoxin genes, irrespective of their sources. From isolates across all the sources, act (63%) and alt (57%) genes were encountered more frequently than ast (6%). The enterobacterial repetitive intergenic consensus sequences polymerase chain reaction was used for typing of isolates and most of the isolates from water and fish were related, owing to similar ecosystem., Conclusion: A wide distribution of enterotoxin genes in Aeromonads from water and fish is a potential public health threat and molecular genotyping can be helpful to study epidemiology of the pathogen., Significance and Impact of the Study: A high proportion of isolates recovered from diverse water sources, particularly potable drinking water and fish samples carried one or more enterotoxin genes thereby indicating a potential pathogenic nature of isolates from these sources. The genetic relatedness was detected amongst many isolates recovered from water sources and fish samples indicating circulation of familiar virulent clones in the aquatic environments., (© 2019 The Society for Applied Microbiology.)

Bifidobacterium breve (YH68) is widely used in the fields of food fermentation and biomedicine. In this study, we explored the antibacterial activity of the cell free culture supernatant (CFCS) of YH68 against Clostridioides difficile ATCC 9689 (CD) by measuring multiple indexes, including the growth, spores production, toxin A/B production, and the expression levels of the tcdA and tcdB genes of CD. In addition, we examined the changes in major cellular functional groups, structures, permeability, integrity, and the proton motive force (PMF) of the cytoplasmic membrane. The results showed that double-dilution ratio of YH68-CFCS (3 × 10 9 CFU/mL) was the MIC value. The cell density, spores production, and the toxin production of CD treated with YH68-CFCS were lower than that of the control ( p < 0.05). In addition, the gene expression levels of tcdA and tcdB in CD treated with YH68-CFCS were significant downregulated ( p < 0.05). Marked differences were observed in the cell membrane and cell wall by a FT-IR spectroscopy and SEM. Analysis of the cell membrane permeability and integrity of the CD cells revealed that YH68-CFCS induced the leakage of a large amount of intracellular K + , inorganic phosphate, ATP, nucleic acids and proteinaceous substances. Furthermore, PMF analysis indicated that there was a significant change in Δψ and ΔpH. These findings demonstrated that the antibacterial activity of YH68-CFCS against CD involved the inhibition of growth, spore production, toxin production, and virulence genes expression; a consumption of PMF in the cytoplasmic membrane, the formation of pore in the cell membrane, together with the enhanced cell membrane permeability; and, eventually, cell completely disintegration.

We report a case of community-acquired fulminant colitis caused by Clostridium difficile in Japan. A 46-year-old woman was diagnosed with severe infectious enterocolitis and was admitted at another hospital. The stool culture was positive for toxigenic C. difficile. Since the patient presented with fulminant C. difficile infection (CDI) with toxic megacolon, respiratory insufficiency, and circulatory failure, she was transferred to Kyorin University Hospital for intensive care. Intubation and antibiotic therapy were performed. The general condition improved with conservative treatment, and she was discharged without sequelae. While the recovered isolate was toxin A and B-positive and binary toxin-positive, it was identified as polymerase chain reaction (PCR) ribotype ts0592 and slpA sequence type ts0592. The isolate was different from PCR ribotype 027 epidemic in Europe and North America. In Japan, binary toxin-producing strains are rare and have not caused an epidemic to date. Furthermore, there are few data on community-acquired CDI in Japan. In this case, a non-elderly woman with no major risk factors such as antibiotic use, administration of proton pump inhibitor and history of gastrointestinal surgery developed community-acquired fulminant CDI caused by the binary toxin-positive strain, and ICU treatment was required. Further studies focusing on the role of binary toxin-positive C. difficile in the severity of community-acquired CDI are necessary.

Clostridioides difficile is a spore-forming, Gram-positive, anaerobic pathogen that caused gastrointestinal illness. During dysbiosis, overgrowth of C. difficile resulting in higher levels of toxin production. Since Lactobacillus has been commonly used to alleviate gastrointestinal discomfort, this study aimed to investigate the effects of Lactobacillus isolated from kimchi on the quorum-sensing and virulence factors of C. difficile 027. Among the isolated Lactobacillus strains, the acid and bile tolerant L. fermentum Lim2 was only able to reduce C. difficile 027 growth by one log 10 CFU per ml. In keeping with this finding, C. difficile 027 growth was unaffected by either untreated or heat-inactivated cell extracts from L. fermentum Lim2. Both untreated and heat-inactivated cell extracts did, however, significantly reduce the autoinducer-2 (AI-2) activity of C. difficile 027, with the most prominent suppression effect (654-fold) being found from 100 mg ml -1 of heat-inactivated cell extract. A gene expression analysis indicated that in the presence of 100 mg ml -1 heat-inactivated cell extract, the quorum-sensing (luxS) and the virulence factors (tcdA, tcdB and tcdE) were significantly suppressed, whereas the negative regulator gene (tcdC) was significantly up-regulated. Taken together, the significant anti-pathogenic effect from L. fermentum Lim2 could potentially be used to treat C. difficile-infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridioides difficile is a Gram-positive pathogenic bacteria that caused gastrointestinal illness via toxic production. The emergence of highly virulence and foodborne C. difficile strains has further increased the incident and severity of C. difficile-infections (CDIs). Numerous studies have reported the immunomodulatory activity of Lactobacillus, a member of healthy gut microbiota, to maintain gastrointestinal health. Here, we successfully isolated L. fermentum Lim2 from kimchi, and identified a promising anti-pathogenic effect against C. difficile 027, from the heat-inactivated L. fermentum cell extract via suppression on the C. difficile 027 quorum-sensing system and toxin production, which could potentially be used to treat and prevent CDIs., (© 2019 The Society for Applied Microbiology.)

The aim of the present study was to evaluate the safety of the probiotic strain Bacillus toyonensis BCT-7112T (active ingredient of Toyocerin) in relation to the enterotoxins haemolysin BL (Hbl) and the non-haemolytic enterotoxin (Nhe) by performing a quantitative reverse transcription (RT) real-time polymerase chain reaction (PCR) and a Western blot assay. The expression levels of the enterotoxin genes hblA, hblD, nheA, nheB and nheC, determined by means of RT real-time PCR in B. toyonensis, were lower than those in B. cereus reference strains. No expression of hblC was detected. The Western blot assays of native and 25-fold concentrated supernatants from B. toyonensis, using monoclonal antibodies directed against the Hbl component L1 and the Nhe component NheB, showed weak bands. The NheC component was not detected in the native supernatant, but weakly in the 25-fold concentrated supernatant. According to the results of the present study, the enterotoxin expression and protein levels of B. toyonensis BCT-7112T were absent or clearly lower compared to the B. cereus reference strains. Thus, their ability to form functional enterotoxins can also be considered to be lower or unlikely compared to the B. cereus reference strains. This experimental approach can be implemented when studying the health and safety as well as harmlessness of probiotic microorganisms., Competing Interests: I have read the journal´s policy. G. Jimenez is an employee of Rubinum, S. A., that has a commercial interest in B. toyonensis BCT-7112T. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The aim of the study was to determine whether the presence of the Yersinia virulence plasmid could affect the production of enterotoxin YstA by Y. enterocolitica strains isolated from pigs which are the main source of infection for humans. The phenotypic features characteristic for the Yersinia virulence plasmid were detected on CRMOX agar in 8 out of 12 strains producing enterotoxin YstA, in 5 out of 12 doubtful strains, and in 11 out of 12 strains not producing YstA. Autoagglutination ability was detected in all 12 Y. enterocolitica strains that were positive in the suckling mice bioassay, in 11 doubtful strains and 10 negative strains. CRMOX+ colonies were generally ystA, myfA, virF and yadA positive, while CRMOX- colonies were only ystA and myfA positive. The amplicons of yadA were not detected in 2 (8.3%) out of 24 CRMOX+ and virF positive strains. The results of this study indicate that the presence of pYV does not affect the enterotoxin-producing ability of Y. enterocolitica strains., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Clostridioides difficile infection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two major C. difficile toxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified a C. difficile regulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, and rstA transcription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds the rstA promoter via the predicted DNA-binding domain. Through mutational analysis of the rstA promoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genes tcdA and tcdB , as well as the promoters for the sigD and tcdR genes, which encode regulators of toxin gene expression. Complementation analyses with the Clostridium perfringens RstA ortholog and a multispecies chimeric RstA protein revealed that the C. difficile C-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production. IMPORTANCE Clostridioides difficile is an anaerobic, gastrointestinal pathogen of humans and other mammals. C. difficile produces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link between C. difficile sporulation and toxin production. Further, our data suggest that C. difficile toxin production is regulated through a direct, species-specific sensing mechanism., (Copyright © 2019 Edwards et al.)

The present study was to investigate the utilization of prebiotics by Lactobacillus rhamnosus NCDC 298 and its synergistic adversary effect on both population and production of heat-labile (LT) toxin in enterotoxigenic Escherichia coli (ETEC). To select suitable prebiotic in order to enhance functionality, its utilization and the prebiotic activity score was examined. Antivirulence effect on ETEC was inspected by its inactivation rate and heat-labile toxin production in presence of different synbiotic combination. L. rhamnosus NCDC 298 strain grown well on media supplemented with fructooligosaccharides (FOS) and galactooligosaccharides (GOS), whereas significant inactivation of ETEC was observed when FOS was added to the co-culture medium. Significant decrease in LT enterotoxin was seen through GM1 ganglioside enzyme linked immunoassay (GM1 ELISA), when ETEC has grown with L. rhamnosus NCDC 298 and FOS. Short-chain FOS proved to be the most effective substrate, improving antagonistic activity for L. rhamnosus NCDC 298. Both L. rhamnosus NCDC 298 with FOS can be used as an effective synbiotic combination for secretory antidiarrheal fermented dairy formulations.

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells-a hallmark feature of AAHC-by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The aim of this study was to characterize enterotoxigenic Staphylococcus aureus recovered from raw cow milk from two geographical regions of Poland using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Among 610 samples tested, 229 (37.5%) were positive for S. aureus and 30 (13.1%) of them possessed at least one gene encoding enterotoxins. The sec marker was the most commonly identified (12; 40.0% isolates), followed by the sed (9; 30.0%), sea (6; 20.0%), and seb (1; 3.3%) genes. Some S. aureus possessed a combination of the sea and sec or sea and seb toxin markers. Only two (6.7%) of the enterotoxin gene-positive isolates were not able to produce enterotoxins in vitro. Genotypic analysis with the PFGE method of a total of 50 toxigenic S. aureus isolates from the present and previous studies identified 16 clonal groups. Furthermore, MLST revealed the presence of 15 sequence types with the most common being ST45 and ST1. The results of this study indicate that raw cow milk may be a source of S. aureus with classical enterotoxin genes, which may pose a potential threat for the consumers' safety.

Purpose: Potent extracellular toxins including alpha-haemolysin, Panton-Valentine leukocidin (PVL) and toxic-shock syndrome toxin 1 (TSST-1) significantly contribute to Staphylococcus aureus pathogenesis, thus, toxin suppression is a primary focus in treatment of staphylococcal disease. S. aureus maintains complex strategies to regulate toxin expression and previous data have demonstrated that subinhibitory concentrations of beta-lactam antibiotics can adversely increase S. aureus exotoxin production. The current study evaluates the effects of subinhibitory concentrations of tedizolid, a second-generation oxazolidinone derivative, on expression of staphylococcal exotoxins in both methicillin-resistant and methicillin-sensitive S. aureus., Methodology: S. aureus exotoxin expression levels were compared at 12 and 24 h following treatment with tedizolid, linezolid, nafcillin or vehicle control., Results: Our findings show that the level of antibiotic required to alter toxin production was strain-dependent and corresponds with the quantity of toxin produced, but both tedizolid and linezolid could effectively reduce expression of alpha-haemolysin, PVL and TSST-1 toxin at subinhibitory concentrations. In contrast, nafcillin showed less attenuation and, in some S. aureus strains, led to an increase in toxin expression. Tedizolid consistently inhibited toxin production at a lower overall drug concentration than comparator agents., Conclusion: Together, our data support that tedizolid has the potential to improve outcomes of infection due to its superior ability to inhibit S. aureus growth and attenuate exotoxin production.

Staphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. In an attempt to determine whether staphylococci causing bovine mastitis could also cause food poisoning, 60 isolates of presumed S. aureus were isolated in the period between March and August 2017 from 3,384 routine, composite, quarter milk samples of individual cows raised on 12 dairy farms in central Italy. Seventeen out of 60 isolates were confirmed as S. aureus after coagulase, thermonuclease, and biochemical tests. These isolates were analyzed by PCR for the presence of the nuc, sea, seb, sec, sed, and see genes. The positive isolates were nuc, 100% (17); sea, 35.29% (6); seb, 5.88% (1); sec, 5.88% (1); sed, 29.41% (5); and see, 47.06% (8). The isolates were also tested with 2 enzyme immunoassay diagnostic kits, one for the screening detection of the production of staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE) and one for the detection of specific enterotoxin produced by each isolate. Seven out of 17 (41.18%) were enterotoxin producers: 7 produced SEA (41.18%), 1 SEB (5.88%), 1 SEC (5.88%), 5 SED (29.41%), and 6 SEE (35.29%). To further characterize the isolates, they were analyzed by the Kirby Bauer test for susceptibility to 13 antimicrobials (ampicillin, ciprofloxacin, kanamycin, tetracycline, gentamicin, methicillin, nalidixic acid, erythromycin, amoxicillin/clavulanic acid, streptomycin, vancomycin, neomycin, and enrofloxacin), and we detected resistance to ampicillin (52.94%), nalidixic acid (70.59%), erythromycin (5.88%), and amoxicillin/clavulanic acid (17.65%). The isolates were sensitive to the main classes of antimicrobials used for the treatment of bovine subclinical mastitis. The presence of enterotoxin-producing isolates of S. aureus in bovine milk means that a temperature abuse or a breakdown in the thermal treatment of the milk could present a food safety risk, particularly if all enterotoxigenic isolates could potentially produce SEA in milk., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Clostridium perfringens type F strains cause a common human foodborne illness and many cases of nonfoodborne human gastrointestinal diseases. Sporulation plays two critical roles during type F enteric disease. First, it produces broadly resistant spores that facilitate type F strain survival in the food and nosocomial environments. Second, production of C. perfringens enterotoxin (CPE), the toxin responsible for causing the enteric symptoms of type F diseases, is restricted to cells in the process of sporulation. While later steps in the regulation of C. perfringens sporulation have been discerned, the process leading to phosphorylation of Spo0A, the master early regulator of sporulation and consequent CPE production, has remained unknown. Using an insertional mutagenesis approach, the current study identified the orphan histidine kinase CPR0195 as an important factor regulating C. perfringens sporulation and CPE production. Specifically, a CPR0195 null mutant of type F strain SM101 made 10 3 -fold fewer spores than its wild-type parent and produced no detectable CPE. In contrast, a null mutant of another putative C. perfringens orphan histidine kinase (CPR1055) did not significantly affect sporulation or CPE production. Studies using a spoIIA operon promoter-driven reporter plasmid indicated that CPR0195 functions early during sporulation, i.e., prior to production of sporulation-associated sigma factors. Furthermore, in vitro studies showed that the CPR0195 kinase domain can autophosphorylate and phosphorylate Spo0A. These results support the idea of CPR0195 as an important kinase that initiates C. perfringens sporulation by directly phosphorylating Spo0A. This kinase could represent a novel therapeutic target to block C. perfringens sporulation and CPE production during type F disease. IMPORTANCE Clostridium perfringens type F enteric diseases, which include a very common form of food poisoning and many cases of antibiotic-associated diarrhea, develop when type F strains sporulate and produce C. perfringens enterotoxin (CPE) in the intestines. Spores are also important for transmission of type F disease. Despite the importance of sporulation for type F disease and the evidence that C. perfringens sporulation begins with phosphorylation of the Spo0A transcriptional regulator, the kinase phosphorylating Spo0A to initiate sporulation and CPE production had not been ascertained. In response, the current report now provides identification of an orphan histidine kinase named CPR0195 that can directly phosphorylate Spo0A. Results using a CPR0195 null mutant indicate that this kinase is very important for initiating C. perfringens sporulation and CPE production. Therefore, the CPR0195 kinase represents a potential target to block type F disease by interfering with intestinal C. perfringens sporulation and CPE production., (Copyright © 2019 Freedman et al.)

Staphylococcus aureus food poisoning, which is still a serious health problem worldwide, is caused by the intoxication of staphylococcal enterotoxin (SE). Among many types of SE, staphylococcal enterotoxin type A (SEA) is known to be the most responsible for S. aureus food poisoning worldwide. Several researchers have reported the modeling of growth/no growth boundaries for S. aureus with a logistic regression model. In this study, the boundaries of SEA production and S. aureus growth in broth were first performed with the logistic regression model, to evaluate the effects of environmental factors of temperature and salt. Consequently, the boundaries of SEA production and growth of S. aureus in temperature and salt concentration could be produced with the model in shaking and stationary cultures. The area where S. aureus cells would grow, but not produce SEA could be shown between the boundaries of SEA production and growth. Internal and external validations showed that the model could well describe and predict experimental results. Further, the maximum concentrations of SEA and cell population under various conditions of temperature and salt concentration were also compared between the shaking and stationary cultures. These results obtained in this study would become useful information in food industry to prevent S. aureus food poisoning outbreaks. PRACTICAL APPLICATION: Production/no production of staphylococcal enterotoxin A at various temperatures and salt concentrations in shaking and stationary cultures could be estimated well with a logistic regression model. Growth/no growth of staphylococcal cells under the same conditions was also well estimated with the model. The area where S. aureus cells would grow, but not produce SEA could be shown with the model. This present analysis would provide useful information in food industry to prevent staphylococcal food poisoning outbreaks., (© 2018 Institute of Food Technologists®.)

Bacillus cereus strains were isolated from dried foods, which included international brands of spices from South East Asia, Mexico and India purchased from several retail stores, samples of powdered infant formula (PIF), medicated fish feed and dietary supplements. The genetic diversity of 64 strains from spices and PIF was determined using a multiplex endpoint PCR assay designed to identify hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Thirteen different B. cereus toxigenic gene patterns or profiles were identified among the strains. Randomly selected B. cereus strains were sequenced and compared with reference Genomic Groups from National Center Biotechnology Information using bioinformatics tools. A comprehensive multi-loci sequence analysis (MLSA) was designed using alleles from 25 known MLST genes specifically tailored for use with whole genome assemblies. A cohort of representative genomes of strains from a few FDA regulated commodities like dry foods and medicated fish feed was used to demonstrate the utility of the 25-MLSA approach for rapid clustering and identification of Genome Groups. The analysis clustered the strains from medicated fish feed, dry foods, and dietary supplements into phylogenetically-related groups. 25-MLSA also pointed to a greater diversity of B. cereus strains from foods and feed than previously recognized. Our integrated approach of toxin gene PCR, and to our knowledge, whole genome sequencing (WGS) based sequence analysis, may be the first of its kind that demonstrates enterotoxigenic potential and genomic diversity in parallel., (Published by Elsevier B.V.)

Clostridioides (formerly Clostridium) difficile produces two major toxins, TcdA and TcdB, upon entry into stationary phase. Transcription of tcdA and tcdB requires the specialized sigma factor, σ TcdR , which also directs RNA Polymerase to transcribe tcdR itself. We fused a gene for a red fluorescent protein to the tcdA promoter to study toxin gene expression at the level of individual C. difficile cells. Surprisingly, only a subset of cells became red fluorescent upon entry into stationary phase. Breaking the positive feedback loop that controls σ TcdR production by engineering cells to express tcdR from a tetracycline-inducible promoter resulted in uniform fluorescence across the population. Experiments with two regulators of tcdR expression, σ D and CodY, revealed neither is required for bimodal toxin gene expression. However, σ D biased cells toward the Toxin-ON state, while CodY biased cells toward the Toxin-OFF state. Finally, toxin gene expression was observed in sporulating cells. We conclude that (i) toxin production is regulated by a bistable switch governed by σ TcdR , which only accumulates to high enough levels to trigger toxin gene expression in a subset of cells, and (ii) toxin production and sporulation are not mutually exclusive developmental programs., (© 2018 John Wiley & Sons Ltd.)

Clostridium perfringens type F strains, which produce C. perfringens enterotoxin (CPE), are a major cause of gastrointestinal infections, including the second most prevalent bacterial foodborne illness and 5 to 10% cases of antibiotic-associated diarrhea. Virulence of type F strains is primarily ascribable to CPE, which is synthesized only during sporulation. Many type F strains also produce NanI sialidase and carry a nan operon that likely facilitates uptake and metabolism of sialic acid liberated from glycoconjugates by NanI. During vegetative growth of type F strain F4969, NanR can regulate expression of nanI Given their importance for type F disease, the current study investigated whether NanR can also influence sporulation and CPE production when F4969 or isogenic derivatives are cultured in modified Duncan-Strong sporulation (MDS) medium. An isogenic F4969 nanR null mutant displayed much less sporulation and CPE production but more NanI production than wild-type F4969, indicating that NanR positively regulates sporulation and CPE production but represses NanI production in MDS. Results for the nanR mutant also demonstrated that NanR regulates expression of the nan operon. A nanI nanR double null mutant mirrored the outcome of the nanR null mutant strain but with a stronger inhibition of sporulation and CPE production, even after overnight incubation. Coupled with results using a nanI null mutant, which had no impairment of sporulation or CPE production, NanR appears to carefully modulate the availability of NanI, nan operon-encoded proteins and sialic acid to provide sufficient nutrients to sustain sporulation and CPE production when F4969 is cultured in MDS medium., (Copyright © 2018 American Society for Microbiology.)

Background: S. aureus strains, with the capability of producing toxic shock syndrome toxin-1 (TSST-1), are more likely to cause complicated infections. However, due to lack of comprehensive local data on the prevalence of TSST-1, we aimed to determine the prevalence of TSST-1 harboring S. aureus isolates in Iran., Methods: A systematic search was performed by using PubMed and Scopus databases from papers published by Iranian authors from January 2000 to the end of March 2017. Then, 10 publications which were matched with inclusion criteria were selected for data extraction and analysis by Comprehensive Meta-Analysis Software., Results: The overall prevalence of TSST-1 carrying S. aureus in Iran was 21.3% (95% CI: 7.9%-46.1%), ranging from 0% to 68%. Moreover, from the included studies, the pooled prevalence of TSST-1 producing MRSA isolates was estimated to be 25.2% (95% CI: 13.3%-42.5%), ranging from 0% to 69.8%. From those studies which showed the distribution of toxin-harboring S. aureus it was found that the skin and soft tissue, respiratory and bloodstream infections were the common sites of TSST-1 harboring S. aureus., Conclusions: In summary, it seems that emergence of MRSA strains leads to higher prevalence of TSST-1 carrying strains in the north of Iran. However, further research is required to elucidate the interplay between the outcome of diseases and TSST-1 producing strains, especially in our country.

Menstrual toxic shock syndrome is associated with vaginal colonization by Staphylococcus aureus strains that encode toxic shock syndrome toxin 1 (tst+). Interestingly, a small proportion of women are colonized by S. aureus tst+ but do not have symptoms of toxic shock syndrome. Here we sought to determine if differences in the species composition of vaginal bacterial communities reflect a differential risk of colonization by S. aureus capable of producing toxic shock syndrome toxin 1 (TSST-1). The composition of vaginal communities of women that were or were not colonized with S. aureus tst+ were compared based on terminal restriction fragment length polymorphism (T-RFLP) profiles and sequences of cloned 16S rRNA genes. There were no detectable differences in community composition or species rank abundance between communities of women vaginally colonized with S. aureus tst+ as compared to those that were not. Phylogenetic analysis of cloned 16S rRNA gene sequences showed that the predominant members of communities of women colonized with S. aureus tst+ were indistinguishable from those of other healthy women. The data suggest that the numerically dominant members of vaginal communities do not preclude colonization and proliferation of S. aureus tst+ within indigenous microbial communities of the vagina.

Fifteen currently marketed intravaginal protection products (11 types of tampon and 4 types of menstrual cup) were tested by the modified tampon sac method to determine their effect on Staphylococcus aureus growth and toxic shock syndrome toxin 1 (TSST-1) production. Most tampons reduced S. aureus growth and TSST-1 production, with differences based on brand and composition, and the level of S. aureus growth was higher in destructured than in unaltered tampons. We observed higher levels of S. aureus growth and toxin production in menstrual cups than in tampons, potentially due to the additional air introduced into the bag by cups, with differences based on cup composition and size. IMPORTANCE Menstrual toxic shock syndrome is a rare but severe disease. It occurs in healthy women vaginally colonized by Staphylococcus aureus producing toxic shock syndrome toxin 1 using intravaginal protection, such as tampons or menstrual cups. Intravaginal protection induces TSS by the collection of catamenial products, which act as a growth medium for S. aureus Previous studies evaluated the impact of tampon composition on S. aureus producing toxic shock syndrome toxin 1, but they are not recent and did not include menstrual cups. This study demonstrates that highly reproducible results for S. aureus growth and TSST-1 production can be obtained by using a simple protocol that reproduces the physiological conditions of tampon and cup usage as closely as possible, providing recommendations for tampon or cup use to both manufacturers and consumers. Notably, our results do not show that menstrual cups are safer than tampons and suggest that they require similar precautions., (Copyright © 2018 American Society for Microbiology.)

RelA is a global regulator for stationary phase development in the model bacterium Bacillus subtilis. The relA gene forms a bicistronic operon with the downstream dtd gene. In this study, we evaluated the significance of RelA and DTD proteins in spore formation and toxin production by an important gastrointestinal pathogen Clostridium perfringens. Our β-glucuronidase assay showed that in C. perfringens strain SM101, relA forms a bicistronic operon with its downstream dtd gene, and the relA promoter is expressed during both vegetative and sporulation conditions. By constructing double relA dtd and single dtd mutants in C. perfringens SM101, we found that: (1) RelA is required for maintaining the efficient growth capacity of SM101 cells during vegetative conditions; (2) both RelA and DTD are required for spore formation and enterotoxin (CPE) production by SM101; (3) RelA/DTD activate CodY, which is known to activate spore formation and CPE production in SM101 by activating a key sporulation-specific σ factor F; (4) as expected, RelA/DTD activate sporulation-specific σ factors (σ E , σ F , σ G and σ K ) by positively regulating Spo0A production; and finally (5) RelA, but not DTD, negatively regulates phospholipase C (PLC) production by repressing plc gene expression. Collectively, our results demonstrate that RelA modulates cellular physiology such as growth, spore formation and toxin production by C. perfringens type A strain SM101, although DTD also plays a role in these pleiotropic functions in coordination with RelA during sporulation. These findings have implications for the understanding of the mechanisms involved in the infectious cycle of C. perfringens.

Background/purpose: Among DNA-based techniques, polymerase chain reaction (PCR) is the most widely accepted molecular tool for the detection of pathogens. However, the technique involves several reagents and multiple pipetting steps that often lead to error-prone results. Additionally, the reagents entail a cold-chain facility to maintain their stability during storage and transportation. The main aim of the present study was to simplify the utility of a pre-optimized multiplex PCR format that was developed to detect toxigenic strains of Staphylococcus aureus by providing stable, pre-mixed, and ready-to-use master mix in a lyophilized formulation., Methods: Master mix containing all reagents except the template was lyophilized in the presence of an excipient lyoprotectant to achieve long-term stability without altering the sensitivity, specificity and PCR performance. Bromophenol blue was also included in the master mix to reduce the risk of external contamination during gel loading. The stability of lyophilized master mix was analyzed at different temperatures. The PCR performance was also examined after exposure of master mix to notable temperature fluctuations during transportation., Results: The shelf-life of lyophilized master mix was estimated to be 1.5 months at ambient temperature and 6 months at 4°C. Stability was unaffected by temperature fluctuations during transportation even in cold-chain-free conditions, thus reducing the cost required for cold storage., Conclusion: The sensitive, cost-effective, ready-to-use, and ambient temperature stable formulation could be implemented as a detection tool in food analysis and diagnostic laboratories and hospitals and for on-field application outside the laboratories, as well as for detection of toxigenic strains of S. aureus., (Copyright © 2016. Published by Elsevier B.V.)

Clostridium difficile , a major cause of nosocomial diarrhea and pseudomembranous colitis, still poses serious health-care challenges. The expression of its two main virulence factors, TcdA and TcdB, is reportedly repressed by cysteine, but molecular mechanism remains unclear. The cysteine desulfidase CdsB affects the virulence and infection progresses of some bacteria. The C. difficile strain 630 genome encodes a homolog of CdsB, and in the present study, we analyzed its role in C. difficile 630Δ erm by constructing an isogenic ClosTron-based cdsB mutant. When C. difficile was cultured in TY broth supplemented with cysteine, the cdsB gene was rapidly induced during the exponential growth phase. The inactivation of cdsB not only affected the resistance of C. difficile to cysteine, but also altered the expression levels of intracellular cysteine-degrading enzymes and the production of hydrogen sulfide. This suggests that C. difficile CdsB is a major inducible cysteine-degrading enzyme. The inactivation of the cdsB gene in C. difficile also removed the cysteine-dependent repression of toxin production, but failed to remove the Na 2 S-dependent repression, which supports that the cysteine-dependent repression of toxin production is probably attributable to the accumulation of cysteine by-products. We also mapped a δ 54 (SigL)-dependent promoter upstream from the cdsB gene, and cdsB expression was not induced in response to cysteine in the cdsR :: ermB or sigL :: ermB strain. Using a reporter gene fusion analysis, we identified the necessary promoter sequence for cysteine-dependent cdsB expression. Taken together, these results indicate that CdsB is a key inducible cysteine desulfidase in C. difficile which is regulated by δ 54 and CdsR in response to cysteine and that cysteine-dependent regulation of toxin production is closely associated with cysteine degradation.

Despite increasing reports of skin and soft tissue infections caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Japan, the extent to which these strains cause nosocomial infections remains unknown, and this is especially true for bloodstream infections. In this study, we molecularly characterized MRSA isolates from Japanese blood samples. Among the 151 MRSA isolates collected from 53 medical facilities in 2011, 115 (76%) and 30 (20%) were classified as staphylococcal cassette chromosome mec (SCCmec) types II and IV, respectively, while the Panton-Valentine leukocidin (PVL) gene was detected in only two isolates. Among 66 MRSA isolates collected from Tokyo Medical University Hospital between 2012 and 2015, 43 (65%) and 20 (30%) were classifiable as SCCmec types II and IV, respectively. In 2015, highly virulent strains, such as the SCCmec type IV/PVL and SCCmec type IV/ toxic shock syndrome toxin-1 clonal types, increased in number. Therefore, the SCCmec type IV clone may cause invasive infections not only in community settings but also in healthcare settings in Japan.

Enterotoxigenic Escherichia coli (ETEC) is the main bacterial cause of dehydrating infant diarrhoea in less-developed countries. Labile toxin (LT) is the major virulent factor of ETEC. Easy diagnostic tests are necessary to reduce the number of cases. Immunological methods have some drawbacks and also have important limitations. For that reason, a Liquid Chromatography coupled to UV detector technique (LC-UV) has been optimize to a rapid identification and quantification of LT from bacteria cultures. It is also important to know optimal conditions for LT and with this purpose several enterotoxigenic E. coli strains have been studied to determine the influence of glucose concentration and different culture media on LT production. LC-UV technique demonstrated to be a good method for LT quantification showing values of 15 ng/mL and 45 ng/mL for limits of detection and quantification respectively. LT quantification revealed that toxin production is directly related to the concentration of glucose added in the broth medium. Tryptic Soy Broth is the most efficient culture medium for E. coli growth and enterotoxin production., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: To identify the cause on a foodborne breakout in a university of Hangzhou in 2014. Methods: Data on cases were gathered from the out-patient logs of the university affiliated or neighboring hospitals to describe the disease distribution and epidemiological curves. Case-control and field studies on hazard factors were conducted simultaneously. Results: The incubation period was 1.5-5.0 hours, of which the median was 3 during the outbreak. All the cases consumed food from a restaurant called Chen's Snacks nearby their university and suffered from the Staphylococcus aureus enterotoxin. Results from the Staphylococcus enterotoxin testing were positive in 3 stool and 6 food samples, out of the total 18 samples. Conclusion: This foodborne outbreak was caused through food poisoning by vermicelli which was contaminated with Staphylococcus aureus.

In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p < 0.05). TA, GN, BP, and RT significantly inhibited the expression of the sea gene in S. aureus C-29 (p < 0.05), while suppression attempts by PP and POP proved unsuccessful. After result analysis, it can be derived that TA, GN, BP, and RT inhibit the production of SEA. Of the six samples, each one significantly inhibited biofilm formation (p < 0.05). Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.

Staphylococcus aureus is a medically important pathogen with an abundance of virulence factors that are necessary for survival within a host, including the production of cytolytic toxins. The regulation of toxin production is mediated by the Agr quorum sensing system, and a poorly defined post-exponential growth phase signal independent of Agr. As part of a recent genome wide association study (GWAS) to identify novel loci that alter the expression of cytolytic toxins, a polymorphism in the cyoE gene, which encodes a protoheme IX farnesyltransferase, was identified. This enzyme is essential for processing heme into the electron transport chain for use as an electron acceptor. Interestingly, without this enzyme S. aureus were repressed in their ability to secrete cytolytic toxins, and this appears to be mediated through repression of the Agr quorum sensing system. We hypothesize that the loss of electron transport is inducing feedback inhibition of metabolic capabilities that suppress the TCA cycle, and that this coupled with decreased RNAIII transcription prevents synthesis of cytolytic toxins.

Background: Staphylococcus aureus is the predominant bacterium responsible for various diseases in animals and humans. Preventive strategies could be better implemented by understanding the prevalence, genetic patterns, and the presence of enterotoxin and biofilm-producing genes along with the antibiotic susceptibility of this organism. This study was conducted in Rajasthan, the northwestern state of India, holding the largest population of cattle that makes it the second largest milk producer in India and no such prior information is available on these aspects., Methods: A total of 368 individual quarter bovine raw milk samples were collected from 13 districts of Rajasthan, and screened for the presence of S. aureus. Microbiological and molecular approaches were followed for bacterial identification. Genetic diversity was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of coagulase gene (coa), whereas enterotoxin and biofilm-producing genes were studied by PCR analysis. Antibiotic strips were employed to study the antibiotic resistance among strains., Results: In all, 73 S. aureus strains were obtained from 368 bovine raw milk samples out of that only 30 showed the presence of coa. Nine types of coa patterns ranging from 730 to 1130 bp were observed among these isolates. PCR-RFLP of coa distinguished the isolates into 15 genotypic patterns, of which patterns I, IV, V, and VI were predominant. Of the isolates, 30% were positive for sec, 10% for sea, and 3.3% for seb; these genes are responsible for enterotoxin production, whereas all isolates were found positive for icaAD and eno. The prevalence rates of other biofilm-producing genes fnbA, clfB, ebpS, sasG, fnbB, sasC, cna, bap, fib and, bbp were 97, 93, 90, 80, 80, 77, 53, 27, 10, and 6.6%, respectively. Twenty-seven (90%) strains were multidrug resistant, of which 15 were methicillin resistant. Maximum sensitivity was reported for kanamycin and it could be considered as a drug of choice for controlling S. aureus mediated cattle infections in the studied regions., Conclusions: Overall, these strains could cause several diseases to humans, insisting the need for developing a stricter hygiene program for improving milking practices and animal health.

Staphylococcal food poisoning is the result of consumption of food contaminated with staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus. To date, 23 SEs and SE-like enterotoxins (SEls) have been described in the literature. They are divided into classical SEs (SEA-SEE) and new SE/SEls (SEG-SElX). Some have proved to be foodborne-inducible, but others remain unidentified. In May 2016, at an elderly group home in Osaka city, Japan, an outbreak from foodborne pathogens occurred among lunch party participants. Within 2h 30min to 4h 40min, 15 of 53 participants presented gastrointestinal symptoms of vomiting, diarrhea, and nausea. A subsequent laboratory investigation detected S. aureus from most stool samples from patients, several left-over food items, a kitchen swab, and hand swabs from two food handlers. Classical SEs was not detected from S. aureus isolates or left-over food items. From examination for the presence of SE/SEl genes of 20 kinds by PCR, seg, sei, sem, sen, seo, and selu genes were detected in almost all isolates. These isolates exhibited identical or closely related types by coagulase type (type VII), Sma I digested pulsed-field gel electrophoresis analysis and multi-locus sequence typing (MLST-CC45 lineage). These results suggest that the foodborne outbreak was caused by S. aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical SEs. Additionally, some S. aureus isolates from human nasal swabs and healthy human feces harboring seg, sei, sem, sen, seo, and selu genes without production of classical SEs were classified into CC45 lineage using MLST. These findings suggest new SE/SEls as a potential cause of foodborne outbreaks., (Copyright © 2017. Published by Elsevier B.V.)

Escherichia coli heat-labile enterotoxin (LT) and its non-toxic mutant (LTm) are well-known powerful mucosal adjuvants and immunogens. However, the yields of these adjuvants from genetically engineered strains remain at extremely low levels, thereby hindering their extensive application in fundamental and clinical research. Therefore, efficient production of these adjuvant proteins from genetically engineered microbes is a huge challenge in the field of molecular biology. In order to explore the expression bottlenecks of LTm in E. coli , we constructed a series of recombinant plasmids based on various considerations and gene expression strategies. After comparing the protein expression among strains containing different recombinant plasmids, the signal sequence was found to be critical for the expression of LTm and its subunits. When the signal sequence was present, the strong hydrophobicity and instability of this amino acid sequence greatly restricted the generation of subunits. However, when the signal sequence was removed, abundantly expressed subunits formed inactive inclusion bodies that could not be assembled into the hexameric native form, although the inclusion body subunits could be refolded and the biological activity recovered in vitro. Therefore, the dilemma choice of signal sequence formed bottlenecks in the expression of LTm. These results reveal the expression bottlenecks of LTm, provide guidance for the preparation of LTm and its subunits, and certainly help to promote efficient preparation of this mucosal adjuvant protein.

Staphylococcal enterotoxin A (SEA) was the major virulence factor of Staphylococcus aureus and a biomarker of S. aureus. To establish a fast, low cost, high accuracy, reliable, and simple method for detecting S. aureus, SEA was analyzed by HPLC-ESI-TOF. SEA was not yet commercially available in universal, so SEA was prepared before it was analyzed by HPLC-ESI-TOF. The result showed that high purified SEA was successfully prepared and SEA has normal distribution in mass spectra. A large amount of recombinant SEA (rSEA) was obtained by engineering technology and was purified by Ni affinity chromatography column, and the expression and purity of rSEA and SEA were analyzed by SDS-PAGE. The factors effected on ionization of SEA were studied, and the qualitative analysis of SEA by HPLC-ESI-TOF. The result showed that large amount of SEs expressed within a short time at 28 °C or thereabouts, and there was no impurity bands in electrophorogram after rSEA was purified by Ni affinity chromatography column. In addition, the SEA which had homologous AA sequence with wild SEA was made by rSEA. The retention of SEA in column and ionization of SEA in ESI-TOF were studied for qualitative analysis of S. aureus. The result showed that the content of formic acid in mobile phase was an important factor for ionization of SEs in ESI-TOF. And the result provided theoretical foundation for qualitative detection of S. aureus. [SEs + nH + + mNH 4 + ] n+m+ was shown on ESI-TOF spectra when SEA was detected by ESI-TOF in positive ion mode, and the numerical value of n+m was less than or equal to the number of basic amino acids in SEs. This method was applied to determine SEA in water samples preliminarily, and the detection limit of SEA in spiked water sample was 3 mg/kg. The limit of detection of 3 mg/kg was low sensitivity for low molecular weight matters, but it was high sensitivity for SEA which had a high molecular weight of 27 kDa. Of SEA, 3 mg/kg was equivalent to 10 -4  mmol/kg of SEA. This study can provide evidence for establishing method to determine SEA in real samples.

Non-hemolytic enterotoxin (NHE), a tri-partite, proteinaceous toxin encoded by contiguous nheA, nheB and nheC genes of Bacillus cereus sensu lato (B. cereus s.l.), is considered to be associated with the foodborne diarrheic syndrome. However, B. cereus s.l. includes a number of closely related strains, and the occurrence of NHE among them, and other members of Bacillus is unclear. Consequently, we aimed to determine the distribution and evolution of NHE within Bacillus by confirming the presence of the nheA, B and C sequences and variation within them using published data, and to analyze the genomic and genetic diversity. The phylogenetic tree of NHE proteins (NheA, NheB and NheC) from 81 different B. cereus s.l. strains was constructed. And on the genetic determinants of the NHE toxin did not bring any obvious link between the nheABC genes sequence of a strain and its virulence in the diarrhoeal pathogenesis. Analysis of the genomic diversity of the nheA, B and C loci revealed that their upstream regions were more conserved than the downstream sequences. Multilocus sequence typing schemes (MLST) based on seven concatenated housekeeping genes and nheA, B and C genes of the 75 strains were developed. The neighbor joining phylogenetic tree based on seven housekeeping genes together with nheA, B and C genes was similiar to published Bacillus phylogenetic trees. And on the genetic determinants of the NHE toxin did not bring any obvious link between the nheABC genes sequence of a strain and its virulence in the diarrhoeal pathogenesis.The results indicate that nheA, B and C genes do not affect the diversity of housekeeping genes, and this specific NHE protein does not participate in the diarrheic syndrome.

Objectives: To establish risk factors for Clostridium difficile colonization among hospitalized patients in England., Methods: Patients admitted to elderly medicine wards at three acute hospitals in England were recruited to a prospective observational study. Participants were asked to provide a stool sample as soon as possible after enrolment and then weekly during their hospital stay. Samples were cultured for C. difficile before ribotyping and toxin detection by PCR. A multivariable logistic regression model of risk factors for C. difficile colonization was fitted from univariable risk factors significant at the p < 0.05 level., Results: 410/727 participants submitted ≥1 stool sample and 40 (9.8%) carried toxigenic C. difficile in the first sample taken. Ribotype 106 was identified three times and seven other ribotypes twice. No ribotype 027 strains were identified. Independent predictors of colonization were previous C. difficile infection (OR 4.53 (95% C.I. 1.33-15.48) and malnutrition (MUST score ≥2) (OR 3.29 (95% C.I. 1.47-7.35)). Although C. difficile colonised patients experienced higher 90-day mortality, colonization was not an independent risk for death., Conclusions: In a non-epidemic setting patients who have previously had CDI and have a MUST score of ≥2 are at increased risk of C. difficile colonization and could be targeted for active surveillance to prevent C. difficile transmission., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recently, we found that staphylococcal enterotoxin A (SEA)-producing Staphylococcus aureus strains produced SEA in raw milk with microbial contaminants at high temperatures like 40 °C only. Moreover, the concentration of SEA produced in raw milk gradually decreased after the peak. The reason(s) for SEA degradation in raw milk was studied in this study. Degradation of SEA spiked in raw milk was observed at 40 °C, but not at 25 °C. A Pseudomonas aeruginosa isolate from raw milk degraded SEA spiked in broth at 40 °C. A sample partially purified with a chromatographic method from culture supernatant of the isolate degraded SEA. Two main proteolytic bands were observed in the sample by zymographic analysis with casein. These results suggested that the SEA in raw milk might be degraded by a protease(s) produced by the P. aeruginosa isolate. This finding might be the first report on SEA degradation by a proteolytic enzyme(s) derived from Pseudomonas bacteria to our knowledge.

Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY -null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY -null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY -null mutant strain but significantly increased in the SM101 codY -null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation., (Copyright © 2017 American Society for Microbiology.)

Emetic toxin-producing Bacillus cereus (emetic B. cereus) is the third member of B. cereus group whose toxins are encoded by megaplasmids, beside anthrax and insecticidal toxins of B. anthracis and B. thuringiensis, respectively. A total of 18 emetic isolates collected from food poisoning events, clinical and non-random food samples in Zhejiang province of China, were analyzed by plasmid screening, pulse field gel electrophoresis, multilocus sequence typing, and toxic gene identification to investigate their genotypic diversity. In this study, 13 plasmid profile types, 14 pulse types and 6 different STs from emetic isolates were detected, in which ST 1035,1038,1053,1054 and 1065 were first assigned and reported. The toxic gene ces existed on its own, or coexisted with other toxic genes bceT, cytk, entFM and nhe, but never with hbl in emetic isolates. The results demonstrated that the emetic B. cereus strains from China were heterologous at genotypic level., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The aim of this study was to identify and characterise Bacillus cereus from a unique national collection of 564 strains associated with 140 strong-evidence food-borne outbreaks (FBOs) occurring in France during 2007 to 2014. Starchy food and vegetables were the most frequent food vehicles identified; 747 of 911 human cases occurred in institutional catering contexts. Incubation period was significantly shorter for emetic strains compared with diarrhoeal strains A sub-panel of 149 strains strictly associated to 74 FBOs and selected on Coliphage M13-PCR pattern, was studied for detection of the genes encoding cereulide, diarrhoeic toxins (Nhe, Hbl, CytK1 and CytK2) and haemolysin (HlyII), as well as panC phylogenetic classification. This clustered the strains into 12 genetic signatures (GSs) highlighting the virulence potential of each strain. GS1 (nhe genes only) and GS2 (nhe, hbl and cytK2), were the most prevalent GS and may have a large impact on human health as they were present in 28% and 31% of FBOs, respectively. Our study provides a convenient molecular scheme for characterisation of B. cereus strains responsible for FBOs in order to improve the monitoring and investigation of B. cereus-induced FBOs, assess emerging clusters and diversity of strains., (This article is copyright of The Authors, 2016.)

Clostridium difficile is the leading cause of antibiotic-associated diarrhea and colitis in hospitalized humans. Recently, C. difficile infection (CDI) has been increasingly recognized as a cause of neonatal enteritis in food animals such as pigs, resulting in stunted growth, delays in weaning, and mortality, as well as colitis in large birds such as ostriches. C. difficile is a strictly anaerobic spore-forming bacterium, which produces two toxins A (TcdA) and B (TcdB) as its main virulence factors. The majority of strains isolated from animals produce an additional binary toxin (C. difficile transferase) that is associated with increased virulence. C. difficile is ubiquitous in the environment and has a wide host range. This review summarizes the epidemiology, clinical presentations, risk factors, and laboratory diagnosis of CDI in animals. Increased awareness by veterinarians and animal owners of the significance of clinical disease caused by C. difficile in livestock and avians is needed. Finally, this review provides an overview on methods for controlling environmental contamination and potential therapeutics available.