ABSTRACT
Vancomycin (VAN) treatment in Clostridioides difficile infection (CDI) suffers from a relatively high rate of recurrence, with a variety of reasons behind this, including biofilm-induced recurrent infections. C. difficile can form monophyletic or symbiotic biofilms with other microbes in the gut, and these biofilms protect C. difficile from being killed by antibiotics. In this study, we analyzed the ecological relationship between Bacteroides thetaiotaomicron and C. difficile and their formation of symbiotic biofilm in the VAN environment. The production of symbiotic biofilm formed by C. difficile and B. thetaiotaomicron was higher than that of C. difficile and B. thetaiotaomicron alone in the VAN environment. In symbiotic biofilms, C. difficile was characterized by increased production of the toxin protein TcdA and TcdB, up-regulation of the expression levels of the virulence genes tcdA and tcdB, enhanced bacterial cell swimming motility and c-di-GMP content, and increased adhesion to Caco-2 cells. The scanning electron microscope (SEM) combined with confocal laser scanning microscopy (CLSM) results indicated that the symbiotic biofilm was elevated in thickness, dense, and had an increased amount of mixed bacteria, while the fluorescence in situ hybridization (FISH) probe and plate colony counting results further indicated that the symbiotic biofilm had a significant increase in the amount of C. difficile cells, and was able to better tolerate the killing of the simulated intestinal fluid. Taken together, C. difficile and B. thetaiotaomicron become collaborative in the VAN environment, and targeted deletion or attenuation of host gut B. thetaiotaomicron content may improve the actual efficacy of VAN in CDI treatment.
Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Bacteroides thetaiotaomicron , Biofilms , Clostridioides difficile , Symbiosis , Vancomycin , Biofilms/drug effects , Biofilms/growth & development , Clostridioides difficile/drug effects , Clostridioides difficile/physiology , Clostridioides difficile/genetics , Humans , Vancomycin/pharmacology , Anti-Bacterial Agents/pharmacology , Caco-2 Cells , Bacteroides thetaiotaomicron/drug effects , Bacteroides thetaiotaomicron/metabolism , Bacteroides thetaiotaomicron/physiology , Bacteroides thetaiotaomicron/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Bacterial Toxins/genetics , Enterotoxins/metabolism , Enterotoxins/genetics , Bacterial Adhesion/drug effectsABSTRACT
Bacillus cereus and Bacillus thuringiensis, which belong to the B. cereus group, are widely distributed in nature and can cause food poisoning symptoms. In this study, we collected 131 isolates belonging to the B. cereus group, comprising 124B. cereus and seven B. thuringiensis isolates, from fresh-cut lettuce production chain and investigated their potential risk by analyzing genotypic (enterotoxin and emetic toxin gene profiles) and phenotypic (antibiotic susceptibility, sporulation, and biofilm formation) characteristics. Enterotoxin genes were present only in B. cereus, whereas the emetic toxin gene was not detected in any of the B. cereus isolates. All isolates were susceptible to vancomycin, which is a last resort for treating B. cereus group infection symptoms, but generally resistant to ß-lactam antimicrobials, and had the ability to form spores (at an average sporulation rate of 24.6 %) and biofilms at 30 °C. Isolates that formed strong biofilms at 30 °C had a superior possibility of forming a dense biofilm by proliferating at 10 °C compared to other isolates. Additionally, confocal laser scanning microscopy (CLSM) images revealed a notable presence of spores within the submerged biofilm formed at 10 °C, and the strengthened attachment of biofilm inner cells to the substrate was further revealed through biofilm structure parameters analysis. Collectively, our study revealed the prevalence and contamination levels of B. cereus and B. thuringiensis at fresh-cut lettuce production chain and investigated their genotypic and phenotypic characteristics, aiming to provide valuable insights for the development of potential risk management strategies to ensure food safety, especially along the cold chain.
Subject(s)
Bacillus cereus , Biofilms , Enterotoxins , Food Microbiology , Lactuca , Lactuca/microbiology , Biofilms/growth & development , Bacillus cereus/genetics , Bacillus cereus/metabolism , Bacillus cereus/isolation & purification , Bacillus cereus/physiology , Enterotoxins/genetics , Enterotoxins/metabolism , Bacillus thuringiensis/genetics , Bacillus thuringiensis/physiology , Spores, Bacterial/genetics , Anti-Bacterial Agents/pharmacology , Food Contamination/analysis , Microbial Sensitivity Tests , Foodborne Diseases/microbiology , GenotypeABSTRACT
Staphylococcal Enterotoxin Type B (SEB), produced by Staphylococcus aureus bacteria, is notorious for inducing severe food poisoning and toxic shock syndrome. While nanobody-based treatments hold promises for combating SEB-induced diseases, the lack of structural information between SEB and nanobodies has hindered the development of nanobody-based therapeutics. Here, we present crystal structures of SEB-Nb3, SEB-Nb6, SEB-Nb8, SEB-Nb11, and SEB-Nb20 at resolutions ranging from 1.59 Å to 2.33 Å. Crystallographic analysis revealed that Nb3, Nb8, Nb11, and Nb20 bind to SEB at the T-cell receptor (TCR) interface, while Nb6 binds at the major histocompatibility complex (MHC) interface, suggesting their potential to inhibit SEB function by disrupting interactions with TCR or MHC molecules. Molecular biological analyses confirmed the thermodynamic and kinetic parameters of Nb3, Nb5, Nb6, Nb8, Nb11, Nb15, Nb18, and Nb20 to SEB. The competitive inhibition was further confirmed by cell-based experiments demonstrating nanobody neutralization. These findings elucidate the structural basis for developing specific nanobodies to neutralize SEB threats, providing crucial insights into the underlying mechanisms and offering significant assistance for further optimization towards future therapeutic strategies.
Subject(s)
Enterotoxins , Protein Binding , Single-Domain Antibodies , Enterotoxins/chemistry , Enterotoxins/immunology , Enterotoxins/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunology , Humans , Models, Molecular , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/chemistry , Staphylococcus aureus/immunology , Crystallography, X-Ray , Thermodynamics , KineticsABSTRACT
Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells.
Subject(s)
Bacterial Toxins , Clostridioides difficile , Spores, Bacterial , Spores, Bacterial/metabolism , Spores, Bacterial/genetics , Clostridioides difficile/genetics , Clostridioides difficile/metabolism , Bacterial Toxins/metabolism , Bacterial Toxins/genetics , Humans , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Animals , Chlorocebus aethiops , Vero Cells , Enterotoxins/metabolism , Enterotoxins/geneticsABSTRACT
Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.
Subject(s)
Claudin-4 , Claudin-4/metabolism , Humans , Tight Junctions/metabolism , Cryoelectron Microscopy , Enterotoxins/metabolism , Enterotoxins/chemistry , Enterotoxins/immunology , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/metabolism , Protein Binding , Models, MolecularABSTRACT
Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting the growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed the production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets the virulence of S. aureus, and it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS.
Subject(s)
Bacterial Proteins , Bacterial Toxins , Enterotoxins , Staphylococcus aureus , Superantigens , Superantigens/metabolism , Superantigens/genetics , Enterotoxins/metabolism , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolism , Humans , Bacterial Toxins/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/antagonists & inhibitors , Female , Shock, Septic/drug therapy , Shock, Septic/metabolism , Shock, Septic/microbiology , Gene Expression Regulation, Bacterial/drug effects , Protein Kinases/metabolism , Protein Kinases/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Staphylococcal Infections/drug therapy , Staphylococcal Infections/metabolism , Staphylococcal Infections/microbiology , Virulence/drug effects , Lymphocytes/metabolism , Lymphocytes/drug effects , Menstrual Hygiene ProductsABSTRACT
The Klebsiella oxytoca species complex is part of the human microbiome, especially during infancy and childhood. K. oxytoca species complex strains can produce enterotoxins, namely, tilimycin and tilivalline, while also contributing to colonization resistance (CR). The relationship between these seemingly contradictory roles is not well understood. Here, by coupling ex vivo assays with CRISPR-mutagenesis and various mouse models, we show that K. oxytoca provides CR against Salmonella Typhimurium. In vitro, the antimicrobial activity against various Salmonella strains depended on tilimycin production and was induced by various simple carbohydrates. In vivo, CR against Salmonella depended on toxin production in germ-free mice, while it was largely toxin-independent in mice with residual microbiota. This was linked to the relative levels of toxin-inducing carbohydrates in vivo. Finally, dulcitol utilization was essential for toxin-independent CR in gnotobiotic mice. Together, this demonstrates that nutrient availability is key to both toxin-dependent and substrate-driven competition between K. oxytoca and Salmonella.
Subject(s)
Klebsiella oxytoca , Salmonella Infections , Salmonella typhimurium , Klebsiella oxytoca/genetics , Klebsiella oxytoca/metabolism , Animals , Mice , Salmonella Infections/microbiology , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Salmonella typhimurium/growth & development , Salmonella typhimurium/drug effects , Humans , Disease Models, Animal , Enterotoxins/metabolism , Enterotoxins/genetics , Female , Mice, Inbred C57BL , Klebsiella Infections/microbiology , Microbiota , Gastrointestinal Microbiome , Antibiosis , BenzodiazepinonesABSTRACT
Clostridioides difficile infection (CDI) caused by toxigenic C. difficile is the leading cause of antimicrobial and healthcare-associated diarrhea. The pathogenicity of C. difficile relies on the synergistic effect of multiple virulence factors, including spores, flagella, type IV pili (T4P), toxins, and biofilm. Spores enable survival and transmission of C. difficile, while adhesion factors such as flagella and T4P allow C. difficile to colonize and persist in the host intestine. Subsequently, C. difficile produces the toxins TcdA and TcdB, causing pseudomembranous colitis and other C. difficile-associated diseases; adhesion factors bind to the extracellular matrix to form biofilm, allowing C. difficile to evade drug and immune system attack and cause recurrent infection. Cyclic diguanylate (c-di-GMP) is a near-ubiquitous second messenger that extensively regulates morphology, the expression of virulence factors, and multiple physiological processes in C. difficile. In this review, we summarize current knowledge of how c-di-GMP differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in C. difficile. We highlight that C. difficile spore formation and expression of toxin and flagella genes are inhibited at high intracellular levels of c-di-GMP, while T4P biosynthesis, cell aggregation, and biofilm formation are induced. Recent studies have enhanced our understanding of the c-di-GMP signaling networks in C. difficile and provided insights for the development of c-di-GMP-dependent strategies against CDI.
Subject(s)
Bacterial Proteins , Biofilms , Clostridioides difficile , Clostridium Infections , Cyclic GMP , Gene Expression Regulation, Bacterial , Phenotype , Virulence Factors , Clostridioides difficile/pathogenicity , Clostridioides difficile/genetics , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Virulence Factors/genetics , Virulence Factors/metabolism , Biofilms/growth & development , Humans , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Clostridium Infections/microbiology , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Spores, Bacterial/genetics , Flagella/genetics , Virulence , Enterotoxins/genetics , Enterotoxins/metabolism , AnimalsABSTRACT
Paeniclostridium sordellii hemorrhagic toxin (TcsH) and Clostridioides difficile toxin A (TcdA) are two major members of the large clostridial toxin (LCT) family. These two toxins share ~87% similarity and are known to cause severe hemorrhagic pathology in animals. Yet, the pathogenesis of their hemorrhagic toxicity has been mysterious for decades. Here, we examined the liver injury after systemic exposure to different LCTs and found that only TcsH and TcdA induce overt hepatic hemorrhage. By investigating the chimeric and truncated toxins, we demonstrated that the enzymatic domain of TcsH alone is not sufficient to determine its potent hepatic hemorrhagic toxicity in mice. Likewise, the combined repetitive oligopeptide (CROP) domain of TcsH/TcdA alone also failed to explain their strong hemorrhagic activity in mice. Lastly, we showed that disrupting the first two short repeats of CROPs in TcsH and TcdA impaired hemorrhagic toxicity without causing overt changes in cytotoxicity and lethality. These findings lead to a deeper understanding of toxin-induced hemorrhage and the pathogenesis of LCTs and could be insightful in developing therapeutic avenues against clostridial infections. IMPORTANCE: Paeniclostridium sordellii and Clostridioides difficile infections often cause hemorrhage in the affected tissues and organs, which is mainly attributed to their hemorrhagic toxins, TcsH and TcdA. In this study, we demonstrate that TcsH and TcdA, but not other related toxins. including Clostridioides difficile toxin B and TcsL, induce severe hepatic hemorrhage in mice. We further determine that a small region in TcsH and TcdA is critical for the hemorrhagic toxicity but not cytotoxicity or lethality of these toxins. Based on these results, we propose that the hemorrhagic toxicity of TcsH and TcdA is due to an uncharacterized mechanism, such as the presence of an unknown receptor, and future studies to identify the interactive host factors are warranted.
Subject(s)
Bacterial Toxins , Clostridioides difficile , Enterotoxins , Hemorrhage , Animals , Mice , Bacterial Toxins/toxicity , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Clostridioides difficile/genetics , Clostridioides difficile/pathogenicity , Enterotoxins/toxicity , Enterotoxins/genetics , Enterotoxins/metabolism , Liver/pathology , Clostridium Infections/microbiology , Humans , FemaleABSTRACT
Background: Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are well defined as food poisoning pathogens that are highly resistant and need continuous studies. Aim: The purpose of the work was to examine phenotypic and genotypic characteristics of both P. aeruginosa and S. aureus, and treatment trials with medicinal plants. Methods: Samples were examined for isolation of P. aeruginosa and S. aureus on selective media followed by biochemical confirmation, biofilm formation, genes detection, and expression of P. aeruginosa pslA biofilm gene was performed by quantitative real-time polymerase chain reaction after treatment with 0.312 mg/ml Moringa oleifera aqueous extract as a minimum inhibitory concentration. Results: The highest isolation rate of P. aeruginosa was 20% from both raw milk and Kariesh cheese, followed by 16% and 12% from ice cream and processed cheese, respectively, while the highest isolation rate of S. aureus was 36% from raw milk followed by 28% in ice cream and 16% in both Kariesh cheese and processed cheese. 30% of P. aeruginosa isolates were biofilm producers, while only 21% of S. aureus isolates were able to produce biofilm. The P. aeruginosa isolates harbor virulence-associated genes nan1, exoS, toxA, and pslA at 100%, 80%, 40%, and 40%, respectively. Staphylococcus aureus SEs genes were examined in S. aureus strains, where SEA and SEB genes were detected with 60%, but no isolate harbored SEC, SED, or SEE. The significant fold change of P. aeruginosa pslA expression was 0.40332 after treatment with M. oleifera aqueous extract. Conclusion: Pseudomonas aeruginosa and S. aureus harbor dangerous virulence genes that cause food poisoning, but M. oleifera extract could minimize their action.
Subject(s)
Foodborne Diseases , Moringa oleifera , Staphylococcal Infections , Animals , Staphylococcus aureus/genetics , Pseudomonas aeruginosa/genetics , Milk , Moringa oleifera/genetics , Enterotoxins/genetics , Enterotoxins/metabolism , Enterotoxins/pharmacology , Food Microbiology , Anti-Bacterial Agents/pharmacology , Staphylococcal Infections/microbiology , Staphylococcal Infections/veterinary , Biofilms , Foodborne Diseases/veterinary , Gene ExpressionABSTRACT
Microbial pigments are considered as one of the main sources of natural types, and the attention to them is increasing in the food and pharmaceutical industries. This study aimed to investigate the effects of pigments extracted from Micrococcus roseus (PEM) on the gene expression of a and b staphylococcal enterotoxins (sea and seb) and their acute toxicity. Real-time PCR was used to study the anti-enterotoxigenic activity of PEM against Staphylococcus aureus at sub-inhibitory concentrations. In addition, the acute toxicity of PEM was evaluated on albino mice through alkaline phosphatase (ALP), aspartate aminotransferas (AST), and alanine aminotransferase (ALT) of liver and its histopathological changes. Based on the results, the expression of sea and seb was decreased in the presence of PEM at sub-inhibitory concentrations. The 2-∆∆CT was measured 0.02 and 0.01 for the expression of sea and seb of S. aureus grown in the MHB containing 16 mg/ml PEM. The results showed that the expression of seb is more sensitive to PEM compared to the expression of sea. After treatment of mice with PEM for two weeks, the condition of mice was normal, and the results of liver enzymatic activities and histopathological changes showed insignificant difference compared to the control sample.
Subject(s)
Enterotoxins , Liver , Pigments, Biological , Staphylococcus aureus , Animals , Mice , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Liver/pathology , Liver/drug effects , Enterotoxins/genetics , Enterotoxins/toxicity , Enterotoxins/metabolism , Micrococcus/drug effects , Micrococcus/genetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/toxicity , Staphylococcal Infections/microbiology , Male , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Microbial Sensitivity Tests , Alanine Transaminase/metabolism , Alanine Transaminase/bloodABSTRACT
Production of soluble proteins is essential for structure/function studies; however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens emerged as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine-binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA, and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein perdeuteration using deuterated minimal media with deuterium oxide as solvent and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news, since isotopic labeling is expensive and often ineffective but represents a necessary prerequisite for some structural biology techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein perdeuteration in amounts suitable for structural biology studies.
Subject(s)
Escherichia coli , Vibrio , Escherichia coli/genetics , Escherichia coli/metabolism , Enterotoxins/metabolism , Cholera Toxin/metabolismABSTRACT
Staphylococcus aureus (S. aureus) enterotoxins have aroused great concern to food safety owing to its increased risk of food poisoning. The current research aimed to investigate the anti-virulence mechanisms of phloretin against S. aureus in terms of toxin activity and gene expression. The results indicated that phloretin could effectively inhibit the production of hemolysins and enterotoxins, and its anti-virulence effect was exerted in a concentration-dependent manner. Transcriptome results indicated that phloretin could downregulate the transcription level of majority virulence factors related genes (68 %) of S. aureus, including the quorum sensing-related genes (agrB, agrC, agrA, sspA, splF, splD and others) and bacterial secretion system-related genes (secDF, secY2, and yidC). In addition, it was speculated that phloretin was most likely to bind to the AgrA DNA binding domain, thereby affecting the expression of downstream virulence genes (hla, seb, spa, rot, geh, etc) based on molecular docking. Finally, the application in cooked chicken indicated that phloretin could effectively decrease the content of enterotoxins and improve the storage quality of cooked chicken. These findings not only evidenced the feasible anti-virulence activity of phloretin, but also provided a new strategy to prevent S. aureus food poisoning in cooked meat preservation.
Subject(s)
Foodborne Diseases , Staphylococcal Infections , Animals , Staphylococcus aureus , Virulence/genetics , Chickens/microbiology , Molecular Docking Simulation , Phloretin/pharmacology , Phloretin/metabolism , Enterotoxins/genetics , Enterotoxins/metabolism , Staphylococcal Infections/microbiology , Gene Expression Profiling , Anti-Bacterial Agents/pharmacologyABSTRACT
Menstrual toxic shock syndrome (mTSS) is a rare but life-threatening disease associated with the use of high-absorbency tampons. The production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) superantigen is involved in nearly all cases of mTSS and is tightly controlled by regulators responding to the environment. In the prototypic mTSS strain S. aureus MN8, the major repressor of TSST-1 is the carbon catabolite protein A (CcpA), which responds to glucose concentrations in the vaginal tract. Healthy vaginal Lactobacillus species also depend on glucose for both growth and acidification of the vaginal environment through lactic acid production. We hypothesized that interactions between the vaginal microbiota [herein referred to as community state types (CSTs)] and S. aureus MN8 depend on environmental cues and that these interactions subsequently affect TSST-1 production. Using S. aureus MN8 ΔccpA growing in various glucose concentrations, we demonstrate that the supernatants from different CSTs grown in vaginally defined medium (VDM) could significantly decrease tst expression. When co-culturing CST species with MN8 ∆ccpA, we show that Lactobacillus jensenii completely inhibits TSST-1 production in conditions mimicking healthy menstruation or mTSS. Finally, we show that growing S. aureus in "unhealthy" or "transitional" CST supernatants results in higher interleukin 2 (IL-2) production from T cells. These findings suggest that dysbiotic CSTs may encourage TSST-1 production in the vaginal tract and further indicate that the CSTs are likely important for the protection from mTSS.IMPORTANCEIn this study, we investigate the impact of the vaginal microbiota against Staphylococcus aureus in conditions mimicking the vaginal environment at various stages of the menstrual cycle. We demonstrate that Lactobacillus jensenii can inhibit toxic shock syndrome toxin-1 (TSST-1) production, suggesting the potential for probiotic activity in treating and preventing menstrual toxic shock syndrome (mTSS). On the other side of the spectrum, "unhealthy" or "transient" bacteria such as Gardnerella vaginalis and Lactobacillus iners support more TSST-1 production by S. aureus, suggesting that community state types are important in the development of mTSS. This study sets forward a model for examining contact-independent interactions between pathogenic bacteria and the vaginal microbiota. It also demonstrates the necessity of replicating the environment when studying one as dynamic as the vagina.
Subject(s)
Bacterial Toxins , Lactobacillus , Shock, Septic , Staphylococcal Infections , Female , Humans , Staphylococcus aureus/metabolism , Shock, Septic/microbiology , Cues , Enterotoxins/metabolism , Superantigens/metabolism , Vagina/microbiology , Bacteria/metabolism , Staphylococcal Infections/microbiology , Glucose/metabolismABSTRACT
Foodborne diseases caused by Staphylococcus aureus contamination on meat and meat products has gained increasing attention in recent years, while the pathogenicity of S. aureus is mainly attributed to its virulence factors production, which is primarily regulated by quorum sensing (QS) system. Herein, we aimed to uncover the inhibitory effects and mechanisms of citral (CIT) on virulence factors production by S. aureus, and further explore its potential application in pork preservation. Susceptibility test confirmed the antibacterial properties of CIT against S. aureus, the minimal inhibitory concentration (MIC) was 0.25 mg/mL. Treatment with sub-MICs of CIT reduced the hemolytic activity by inhibiting the production of α-hemolysin, and staphylococcal enterotoxins (SEs) production was significantly inhibited by CIT in both culture medium and pork without affecting bacterial growth. Transcriptomic analysis indicated that the differentially expression genes encoding α-hemolysin, SEs, and other virulence factors were down-regulated after treatment with 1/2MIC CIT. Moreover, the genes related to QS including agrA and agrC were also down-regulated, while the global transcriptional regulator sarA was up-regulated. Data here demonstrated that CIT could inhibited S. aureus virulence factors production through disturbing QS systems. In a challenge test, the addition of CIT caused a remarkable inhibition of S. aureus population and delay in lipid oxidation and color change on pork after 15 days incubation at 4 °C. These findings demonstrated that CIT could not only efficiently restrain the production of S. aureus virulence factors by disturbing QS, but also exhibit the potential application on the preservation of meat products.
Subject(s)
Acyclic Monoterpenes , Staphylococcal Infections , Staphylococcus aureus , Humans , Virulence Factors/genetics , Virulence Factors/metabolism , Hemolysin Proteins , Bacterial Proteins/metabolism , Enterotoxins/metabolism , Meat , Staphylococcal Infections/microbiology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolismABSTRACT
Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.
Subject(s)
Bacillus cereus , Bacterial Proteins , Cattle , Animals , Mice , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacillus cereus/genetics , Bacillus cereus/metabolism , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Virulence Factors/metabolism , Enterotoxins/metabolismABSTRACT
The present reserach aimed to detect and isolate the genes involved in the staphylococcal enterotoxins (SEs) production in strains isolated from unprocessed cow's milk and to examine the impact of Bifidobacterium bifidum probiotic cell-free supernatant (CFS) on their expression. Standard techniques were used for isolation and identification of Staphylococci strains in unprocessed milk. The PCR was used to identify strains carrying enterotoxin genes. The B. bifidum CFS was applied to strains containing the target genes, and the genes expression levels were quantified using Real-time PCR. Using 16SrDNA sequencing, the phylogenic relationship of the isolated strains was determined. Analysis revealed that bacteria such as Staphylococcus species were found in the 72% of the samples. The PCR test showed the presence of various SE superantigens, including SEA (16.7%), SEC (11.7%), SED (8.3%), SEE (6.7%), and SEB (1.7%) in isolated strains. The B. bifidum CFS had obvious antimicrobial activity against strains 24, 51, 54, and 35 of Staphylococcus species, and the minimum inhibitory concentration and minimum bactericidal concentration values for these strains treated with B. bifidum CFS were in the range of 31.25-125 µg/ml. Strains 51 and 24 were clustered with S.aureus ATCC 25923, and strains 54 and 35 were clustered with S.aureus ATCC 12600, respectively. The RT-PCR exhibited that probiotics CFS suppressed the expression of SEA, SEB, SEC, and SEE genes (P<0.05). The average fold change for SEA, SEB, SEC, and SED genes was -1.681, -1.28, -1.52, and -0.84, respectively. The research demonstrated that probiotic bacteria can lower enterotoxin production by downregulating the expression of SEs genes.
Subject(s)
Bifidobacterium bifidum , Enterotoxins , Milk , Probiotics , Staphylococcus aureus , Enterotoxins/genetics , Enterotoxins/metabolism , Animals , Probiotics/pharmacology , Milk/microbiology , Cattle , Staphylococcus aureus/drug effects , Staphylococcus aureus/physiology , Bifidobacterium bifidum/physiology , Gene ExpressionABSTRACT
Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.
Subject(s)
Cattle , Animals , Mice , Bacterial Proteins/metabolism , Bacillus cereus/metabolism , Hemolysin Proteins/metabolism , Virulence Factors/metabolism , Enterotoxins/metabolismABSTRACT
Enterotoxigenic
Subject(s)
Adaptation, Physiological/physiology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/physiology , Oils, Volatile/pharmacology , Origanum/metabolism , Staphylococcal Food Poisoning/prevention & control , Staphylococcus aureus/metabolism , Acetic Acid/pharmacology , Enterotoxins/metabolism , Food Microbiology , Lactic Acid/pharmacology , Microbial Sensitivity Tests , Potassium Chloride/pharmacology , Rosmarinus/metabolism , Sodium Chloride/pharmacology , Staphylococcal Food Poisoning/microbiology , Staphylococcus aureus/pathogenicityABSTRACT
Many forms of diarrhoeal disease, particularly so called "secretory" diarrhoealdisease are thought to arise by the active secretion of chloride ion from the enterocytes,creating an osmotic gradient for fluid movement into the small intestinallumen. This model implies that normally occurring intestinal secretion is catastrophicallyenhanced by bacterial enterotoxins. This review advocates that neither normalnor abnormal intestinal secretion from the enterocytes occurs and that no competentproof for chloride secretion exists. Prior to 1970, the physiological evidence failed tosupport the concept of the formation of intestinal juice as a normal intestinal event.The concept was later revived to explain the high rate of fluid entry into the lumenafter exposure to cholera toxin. Much evidence has been advanced for the chloridesecretion hypothesis, the dominant secretory paradigm after 1974, but is the evidencesufficiently compelling for it to be regarded as proving the chloride secretory model?. The evidence falls into four categories and a fifth conjectural argument that proposes that an abnormal chloride ion channel in cystic fibrotic sufferers confers a natural selective advantage by preventing diarrhoeal disease. Secretion is putatively demonstrated by 1) showing that mass transfer of fluid is into the lumen (secretion) and not merely a failure to transport out of the lumen (failed absorption). Support is offered by 2) chloride ion flux measurements in vitro in Ussing chambers and by 3) shortcircuit current measurements that are consistent with and purport to show chloride ion movement into the lumen. In addition, 4) pharmacological agents are identified that affect short-circuit current and these are assumed to be anti-secretory, consistent with the biochemical mechanism for secretion, confirmed wherever possible by mouse knock-out models. Finally, the proxy methods used to study water movement such as elevated short-circuit current measurements show these to be absent in cystic fibrotic patients. The enterocyte secretion hypothesis is challenged here on the basis of an examination of the methods used to show secretion, particularly after exposing the small intestine to heat stable enterotoxin (STa) from E.coli. STa is thought to be secretory because fluid entry into the lumen is claimed, enhanced isotopic flux of chloride ion towards the lumen occurs, an increase in short-circuit current is found, preventable by various drugs that are deemed likely to be anti-secretory and also because the short-circuit current changes after STa are not seen in cystic fibrotic patients. Using volume recovery in vivo, STa is found not to be secretory but only anti-absorptive. Hence, other techniques used to show secretion are not fit for that purpose. If STa is identified as secretory and yet no secretion occurs, how reliable is the evidence for other toxins being secretory when these methods are used? This review concludes that chloride ion secretion is unproven. A review of the literature indicates that secretion occurs not because epithelial cells actively pump water but by interdiction of fluid absorption, increased conductivity through tight junctions and an increased hydrostatic driving force through elevated capillary pressure.The exclusive focus on chloride secretion may explain the failure to develop antisecretory drugs over the last three decades (AU)
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