Klebsiella oxytoca enterotoxins tilimycin and tilivalline have distinct host DNA-damaging and microtubule-stabilizing activities.

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.

Porcine Enterotoxigenic Escherichia coli Strains Differ in Their Capacity To Secrete Enterotoxins through Varying YghG Levels.

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.

The effect of protective cultures on Staphylococcus aureus growth and enterotoxin production.

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.

Identification of bacterial hazards in the production of artisan fresh cheese in Cuba.

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.

Interleukin-10 (IL-10) Produced by Mutant Toxic Shock Syndrome Toxin 1 Vaccine-Induced Memory T Cells Downregulates IL-17 Production and Abrogates the Protective Effect against Staphylococcus aureus Infection.

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.

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile.

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, σTcdR , 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 σTcdR 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, σD and CodY, revealed neither is required for bimodal toxin gene expression. However, σD 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 σTcdR , 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.

Enterotoxin gene profile and molecular epidemiology of Aeromonas species from fish and diverse water sources.

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.

Suppressive effect of Lactobacillus fermentum Lim2 on Clostridioides difficile 027 toxin production.

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 log10 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.

Short communication: Characterization of enterotoxin-producing Staphylococcus aureus isolated from mastitic cows.

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.

Molecular Characterization of Enterotoxigenic Staphylococcus aureus Isolated from Raw Cow Milk in Poland.

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.

NanR Regulates Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain F4969.

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.

RelA/DTD-mediated regulation of spore formation and toxin production by Clostridium perfringens type A strain SM101.

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 ß-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 (σE, σF, σG and σK) 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.

CdtR Regulates TcdA and TcdB Production in Clostridium difficile.

Clostridium difficile is a global health burden and the leading cause of antibiotic-associated diarrhoea worldwide, causing severe gastrointestinal disease and death. Three well characterised toxins are encoded by this bacterium in two genetic loci, specifically, TcdB (toxin B) and TcdA (toxin A) in the Pathogenicity Locus (PaLoc) and binary toxin (CDT) in the genomically distinct CDT locus (CdtLoc). Toxin production is controlled by regulators specific to each locus. The orphan response regulator, CdtR, encoded within the CdtLoc, up-regulates CDT production. Until now there has been no suggestion that CdtR influences TcdA and TcdB production since it is not carried by all PaLoc-containing strains and CdtLoc is not linked genetically to PaLoc. Here we show that, in addition to CDT, CdtR regulates TcdA and TcdB production but that this effect is strain dependent. Of clinical relevance, CdtR increased the production of TcdA, TcdB and CDT in two epidemic ribotype 027 human strains, modulating their virulence in a mouse infection model. Strains traditionally from animal lineages, notably ribotype 078 strains, are increasingly being isolated from humans and their genetic and phenotypic analysis is critical for future studies on this important pathogen. Here we show that CdtR-mediated toxin regulation did not occur in other strain backgrounds, including a ribotype 078 animal strain. The finding that toxin gene regulation is strain dependent highlights the regulatory diversity between C. difficile isolates and the importance of studying virulence regulation in diverse lineages and clinically relevant strains. Our work provides the first evidence that TcdA, TcdB and CDT production is linked by a common regulatory mechanism and that CdtR may act as a global regulator of virulence in epidemic 027 strains.

Impact of Currently Marketed Tampons and Menstrual Cups on Staphylococcus aureus Growth and Toxic Shock Syndrome Toxin 1 Production In Vitro.

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.

Epidemiology of toxic shock syndrome toxin-1 harboring Staphylococcus aureus obtained from clinical samples in Iran: A Systematic Review and Meta-analysis.

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.

CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101.

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.

Genetic and genomic diversity of NheABC locus from Bacillus strains.

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.

Coagulase gene polymorphism, enterotoxigenecity, biofilm production, and antibiotic resistance in Staphylococcus aureus isolated from bovine raw milk in North West India.

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.

Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by 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.

Degradation of staphylococcal enterotoxin A by a Pseudomonas aeruginosa isolate from raw milk.

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.