Production of a new tetravalent vaccine targeting fimbriae and enterotoxin of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is an important type of pathogenic bacteria that causes diarrhea in pigs. The objective of this study was to prepare a novel tetravalent vaccine to effectively prevent piglet diarrhea caused by E. coli. In order to realize the production of K88ac-K99-ST1-LTB tetravalent inactivated vaccine, the biological characteristics, stability, preservation conditions, and safety of the recombinant strain BL21(DE3) (pXKKSL4) were studied, and the vaccine efficacy and minimum immune dose were measured. The results indicated that the biological characteristics, target protein expression, and immunogenicity of the 1st to 10th generations of the strain were stable. Therefore, the basic seed generation was preliminarily set as the 1st to 10th generations. The results of the efficacy tests showed that the immune protection rate could reach 90% with 1 minimum lethal dose (MLD) virulent strain attack in mice. The immunogenicity was stable, and the minimum immune dose was 0.1 mL per mouse. Our research showed that the genetically engineered vaccine developed in this way could prevent piglet diarrhea caused by enterotoxigenic E. coli through adhesin and enterotoxin. In order to realize industrial production of the vaccine as soon as possible, we conducted immunological tests and production process research on the constructed K88ac-K99-ST1-LTB tetravalent inactivated vaccine. The results of this study provide scientific experimental data for the commercial production of vaccines and lay a solid foundation for their industrial production.

Escherichia coli entérotoxinogènes (ETEC) est un type important de bactéries pathogènes qui cause de la diarrhée chez les porcs. L'objectif de l'étude était de préparer un nouveau vaccin tétravalent pour prévenir efficacement la diarrhée causée par E. coli chez les porcelets. Afin de réaliser la production du vaccin tétravalent inactivé K88ac-K99-ST1-LTB, les caractéristiques biologiques, la stabilité, les conditions de conservation, et la sécurité de la souche recombinante (BL21(DE3)(pXKKSL4) ont été étudiées et l'efficacité du vaccin et la dose immunitaire minimum ont été mesurées. Les résultats indiquent que les caractéristiques biologiques, l'expression des protéines cibles, et l'immunogénicité de la 1ère à la 10e génération de la souche étaient stables. Ainsi, la génération germinale de base a été établie de manière préliminaire comme étant de la 1ère à la 10e générations. Les résultats des tests d'efficacité ont démontré que le taux de protection immunitaire pouvait atteindre 90 % avec une attaque au moyen de 1 dose léthale minimale (MLD) d'une souche virulente chez les souris. L'immunogénicité était stable et la dose immunitaire minimum était de 0,1 mL par souris. Nos travaux ont démontré que le vaccin génétiquement élaboré développé de cette façon pourrait prévenir la diarrhée chez les porcelets causée par des E. coli entérotoxigénique via les adhésines et les entérotoxines. Afin d'atteindre la production industrielle de ce vaccin aussitôt que possible, nous avons mené des tests immunologiques et de la recherche sur le processus de production du vaccin tétravalent inactivé K88ac-K99-ST1-LTB. Les résultats de la présente étude fournissent des données scientifiques expérimentales pour la production commerciale de vaccins et jettent une base solide pour leur production industrielle.(Traduit par Docteur Serge Messier).

Enterotoxigenic Staphylococcus aureus in Brazilian artisanal cheeses: Occurrence, counts, phenotypic and genotypic profiles.

The present study aimed to assess the occurrence and counts of Staphylococcus aureus in Brazilian artisanal cheeses (BAC) produced in five regions of Brazil: Coalho and Manteiga (Northeast region); Colonial and Serrano (South); Caipira (Central-West); Marajó (North); and Minas Artisanal cheeses, from Araxá, Campos das Vertentes, Cerrado, Serro and Canastra microregions (Southeast). The resistance to chlorine-based sanitizers, ability to attach to stainless steel surfaces, and antibiogram profile of a large set of S. aureus strains (n = 585) were assessed. Further, a total of 42 isolates were evaluated for the presence of enterotoxigenic genes (sea, seb, sec, sed, see, seg, sei, sej, and ser) and submitted to typing using pulsed-field gel electrophoresis (PFGE). BAC presented high counts of S. aureus (3.4-6.4 log CFU/g), varying from 25 to 62.5%. From the S. aureus strains (n = 585) assessed, 16% could resist 200 ppm of sodium hypochlorite, whereas 87.6% produced strong ability to attach to stainless steel surfaces, corroborating with S. aureus ability to persist and spread in the environment. Furthermore, the relatively high frequency (80.5%) of multidrug-resistant S. aureus and the presence of enterotoxin genes in 92.6% of the strains is of utmost attention. It reveals the lurking threat of SFP that can survive when conditions are favorable. The presence of enterotoxigenic and antimicrobial-resistant strains of S. aureus in cheese constitutes a potential risk to public health. This result calls for better control of cheese contamination sources, and taking hygienic measures is necessary for food safety. More attention should be paid to animal welfare and hygiene practices in some dairy farms during manufacturing to enhance the microbiological quality of traditional cheese products.

Antibacterial effect of Moringa oleifera on Staphylococcus aureus and Pseudomonas aeruginosa isolated from raw milk and some dairy products with special reference to biofilm gene expression.

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.

Screening of antibiogram, virulence factors, and biofilm production of Staphylococcus aureus and the bio-control role of some probiotics as alternative antibiotics.

Background: Food safety is a serious challenge in the face of increasing population and diminishing resources. Staphylococcus aureus is a critical foodborne pathogen characterized by its capability to secret a diverse range of heat-resistant enterotoxins. Antibiotic usage in dairy herds resulted in the occurrence of antimicrobial resistance (AMR) patterns among bacterial species, which were consequently transmitted to humans via dairy products. Lactic acid bacteria (LAB) produce bacteriocins, which provide an excellent source of natural antimicrobials with the further advantage of being environmentally friendly and safe. Aim: Detection of multidrug resistance (MDR) S. aureus isolates in concerned samples, molecular characteristics, biofilm production, and the inhibitory role of LAB against it. Methods: Random samples of raw milk and other dairy products were analyzed for S. aureus isolation. Phenotypic and genotypic assessment of AMR was performed, in addition to detection of classical enterotoxin genes of S. aureus. Finally, evaluation of the antimicrobial action of some Lactobacillus strains against S. aureus. Results: Incidence rates of presumptive S. aureus in raw milk, Kariesh cheese, and yogurt samples were 50%, 40%, and 60%, respectively. The highest resistance of S. aureus was to Kanamycin (100%) and Nalidixic acid (89.3%), respectively. (78.66%) of S. aureus were MDR. 11.1% of S. aureus carried mecA gene. In concern with enterotoxins genes, PCR showed that examined isolates harbored sea with a percentage of (22.2%), while sed was found in (11.1%) of isolates. Regarding biofilm production, (88.88%) of S. aureus were biofilm producers. Finally, agar well diffusion showed that Lactobacillus acidophilus had the strongest antimicrobial action against S. aureus with inhibition zone diameter ranging from 18 to 22 mm. Conclusion: There is a widespread prevalence of MDR S. aureus in raw milk and dairy products. Production of staphylococcal enterotoxins, as well as biofilm production are responsible for public health risks. Therefore, installing proper hygienic routines and harsh food safety policies at food chain levels is substantial.

Epidemiology of enterotoxigenic Escherichia coli and impact on the growth of children in the first two years of life in Lima, Peru.

Background: Enterotoxigenic E. coli (ETEC) is a leading cause of diarrheal morbidity and mortality in children, although the data on disease burden, epidemiology, and impact on health at the community level are limited. Methods: In a longitudinal birth cohort study of 345 children followed until 24 months of age in Lima, Peru, we measured ETEC burden in diarrheal and non-diarrheal samples using quantitative PCR (LT, STh, and STp toxin genes), studied epidemiology and measured anthropometry in children. Results: About 70% of children suffered from one or more ETEC diarrhea episodes. Overall, the ETEC incidence rate (IR) was 73 per 100 child-years. ETEC infections began early after birth causing 10% (8.9-11.1) ETEC-attributable diarrheal burden at the population level (PAF) in neonates and most of the infections (58%) were attributed to ST-ETEC [PAF 7.9% (1.9-13.5)] and LT + ST-ETEC (29%) of which all the episodes were associated with diarrhea. ETEC infections increased with age, peaking at 17% PAF (4.6-27.7%; p = 0.026) at 21 to 24 months. ST-ETEC was the most prevalent type (IR 32.1) with frequent serial infections in a child. The common colonization factors in ETEC diarrhea cases were CFA/I, CS12, CS21, CS3, and CS6, while in asymptomatic ETEC cases were CS12, CS6 and CS21. Only few (5.7%) children had repeated infections with the same combination of ETEC toxin(s) and CFs, suggested genotype-specific immunity from each infection. For an average ETEC diarrhea episode of 5 days, reductions of 0.060 weight-for-length z-score (0.007 to 0.114; p = 0.027) and 0.061 weight-for-age z-score (0.015 to 0.108; p = 0.009) were noted in the following 30 days. Conclusion: This study showed that ETEC is a significant pathogen in Peruvian children who experience serial infections with multiple age-specific pathotypes, resulting in transitory growth impairment.

Targeting staphylococcal enterotoxin B binding to CD28 as a new strategy for dampening superantigen-mediated intestinal epithelial barrier dysfunctions.

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB116-132) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier.

The impact of indole and mucin on sporulation, biofilm formation, and enterotoxin production in foodborne Clostridium perfringens.

AIMS: Indole and mucin are compounds found in the host environment as they are produced by the host or by the host-associated microbiota. This study investigated whether indole and mucin impact Clostridium perfringens growth and sporulation, as well as enterotoxin production and biofilm formation. METHODS AND RESULTS: There was no impact on growth of Cl. perfringens for up to 400 µM indole and 240 mg/l mucin, and neither indole nor mucin affected sporulation. Reverse-transcriptase qPCR showed that mucin strongly upregulated the expression of Cl. perfringens enterotoxin (up to 121-fold increase), whereas indole had a much more modest effect (2-fold). This was also reflected in increased Cl. perfringens enterotoxin levels in mucin-treated Cl. perfringens (as assessed by a reversed passive latex agglutination assay). Finally, mucin and indole significantly increased biofilm formation of Cl. perfringens, although the effect size was relatively small (less than 1.5 fold). CONCLUSION: These results indicate that Cl. perfringens can sense its presence in a host environment by responding to mucin, and thereby markedly increased enterotoxin production.

A high-throughput gut-on-chip platform to study the epithelial responses to enterotoxins.

Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current methods lack high-throughput, robust, and translatable models capable of characterizing toxin-specific epithelial damage. Pressing concerns regarding enterotoxin contamination of foods and emerging interest in clinical applications of enterotoxins emphasize the need for new platforms. Here, we demonstrate how Caco-2 tubules can be used to study the effect of enterotoxins on the human intestinal epithelium, reflecting toxins' distinct pathogenic mechanisms. After exposure of the model to toxins nigericin, ochratoxin A, patulin and melittin, we observed dose-dependent reductions in barrier permeability as measured by TEER, which were detected with higher sensitivity than previous studies using conventional models. Combination of LDH release assays and DRAQ7 staining allowed comprehensive evaluation of toxin cytotoxicity, which was only observed after exposure to melittin and ochratoxin A. Furthermore, the study of actin cytoskeleton allowed to assess toxin-induced changes in cell morphology, which were only caused by nigericin. Altogether, our study highlights the potential of our Caco-2 tubular model in becoming a multi-parametric and high-throughput tool to bridge the gap between current enterotoxin research and translatable in vivo models of the human intestinal epithelium.

Rapid detection of major enterotoxin genes and antibiotic resistance of Staphylococcus aureus isolated from raw milk in the Yazd province, Iran.

INTRODUCTION: Raw milk is a nutrient-rich food, but it may harbour harmful bacteria, such as enterotoxigenic Staphylococcus aureus (S. aureus), which can cause staphylococcal food poisoning. Antibiotic resistance of S. aureus in raw milk can increase the risk of such infections, particularly among susceptible individuals. OBJECTIVE: This study aimed to investigate the prevalence of enterotoxin genes a, d, g, i and j and the antibiotic resistance of S. aureus isolated from raw milk samples. METHODS: During a 6-month sampling period, 60 raw milk specimens were obtained from diverse locations in Yazd province, Iran. Antibiogram profiling was conducted via the disc diffusion method. In addition, staphylococcal enterotoxin (SE) genes a, d, g, i, and j were detected through real-time PCR analysis. RESULTS: Bacteriological assays confirmed the presence of S. aureus in 11 samples (18.3%). All isolates demonstrated 100% resistance to penicillin G but exhibited sensitivity to vancomycin, while resistance to other antibiotics ranged from 36.4% to 45.5%. The prevalence of enterotoxin genes in these strains showed variable distribution, with sea being the predominant SE (45.5%), followed by sed (36.4%), seg (18.2), sej and sei (9.1% each). CONCLUSIONS: This study discovered the presence of multiple enterotoxins in S. aureus strains obtained from raw milk samples. These strains also demonstrated resistance to a variety of antibiotics. Since enterotoxigenic S. aureus is known to cause human food poisoning, monitoring food hygiene practices, especially during raw milk production, is critical.

Meat and meat products as potential sources of emerging MDR Bacillus cereus: groEL gene sequencing, toxigenic and antimicrobial resistance.

BACKGROUND: Bacillus cereus is implicated in severe foodborne infection in humans. This study intended to assess the occurrence, groEL gene sequencing, biofilm production, and resistance profiles of emerged multidrug resistant (MDR) B. cereus in meat and meat product samples. Moreover, this work highlights the virulence and toxigenic genes (hblABCD complex, nheABC complex, cytK, ces, and pc-plc) and antimicrobial resistance genes (bla1, tetA, bla2, tetB, and ermA). METHODS: Consequently, 200 samples (sausage, minced meat, luncheon, beef meat, and liver; n = 40 for each) were indiscriminately collected from commercial supermarkets in Port Said Province, Egypt, from March to May 2021. Subsequently, food samples were bacteriologically examined. The obtained isolates were tested for groEL gene sequence analysis, antibiotic susceptibility, biofilm production, and PCR screening of toxigenic and resistance genes. RESULTS: The overall prevalence of B. cereus among the inspected food samples was 21%, where the highest predominance was detected in minced meat (42.5%), followed by beef meat (30%). The phylogenetic analysis of the groEL gene exposed that the examined B. cereus strain disclosed a notable genetic identity with other strains from the USA and China. Moreover, the obtained B. cereus strains revealed ß-hemolytic activity, and 88.1% of the recovered strains tested positive for biofilm production. PCR evidenced that the obtained B. cereus strains usually inherited the nhe complex genes (nheA and nheC: 100%, and nheB: 83.3%), followed by cytK (76.2%), hbl complex (hblC and hblD: 59.5%, hblB: 16.6%, and hblA: 11.9%), ces (54.7%), and pc-plc (30.9%) virulence genes. Likewise, 42.9% of the examined B. cereus strains were MDR to six antimicrobial classes and encoded bla1, bla2, ermA, and tetA genes. CONCLUSION: In summary, this study highlights the presence of MDR B. cereus in meat and meat products, posing a significant public health risk. The contamination by B. cereus is common in minced meat and beef meat. The molecular assay is a reliable fundamental tool for screening emerging MDR B. cereus strains in meat and meat products.

Vaginal community state types (CSTs) alter environmental cues and production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1).

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.

Immunogenicity and safety in rabbits of a Clostridioides difficile vaccine combining novel toxoids and a novel adjuvant.

Clostridioides difficile infection (CDI) is a serious healthcare-associated disease, causing symptoms such as diarrhea and pseudomembranous colitis. The major virulence factors responsible for the disease symptoms are two secreted cytotoxic proteins, TcdA and TcdB. A parenteral vaccine based on formaldehyde-inactivated TcdA and TcdB supplemented with alum adjuvant, has previously been investigated in humans but resulted in an insufficient immune response. In search for an improved response, we investigated a novel toxin inactivation method and a novel, potent adjuvant. Inactivation of toxins by metal-catalyzed oxidation (MCO) was previously shown to preserve neutralizing epitopes and to annihilate reversion to toxicity. The immunogenicity and safety of TcdA and TcdB inactivated by MCO and combined with a novel carbohydrate fatty acid monosulphate ester-based (CMS) adjuvant were investigated in rabbits. Two or three intramuscular immunizations generated high serum IgG and neutralizing antibody titers against both toxins. The CMS adjuvant increased antibody responses to both toxins while an alum adjuvant control was effective only against TcdA. Systemic safety was evaluated by monitoring body weight, body temperature, and analysis of red and white blood cell counts shortly after immunization. Local safety was assessed by histopathologic examination of the injection site at the end of the study. Body weight gain was constant in all groups. Body temperature increased up to 1 ˚C one day after the first immunization but less after the second or third immunization. White blood cell counts, and percentage of neutrophils increased one day after immunization with CMS-adjuvanted vaccines, but not with alum. Histopathology of the injection sites 42 days after the last injection did not reveal any abnormal tissue reactions. From this study, we conclude that TcdA and TcdB inactivated by MCO and combined with CMS adjuvant demonstrated promising immunogenicity and safety in rabbits and could be a candidate for a vaccine against CDI.

Transcriptome analysis reveals the inhibitory mechanism of phloretin on virulence expression of Staphylococcus aureus and its application in cooked chicken.

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.

Outlining the skin-homing and circulating CLA+NK cells in patients with severe atopic dermatitis.

Atopic dermatitis (AD) is a complex, multifactorial skin disease, characterized by pruritus and predominant Th2 inflammation. Innate immune cells may play a role in AD development and are composed of granulocytes, macrophages, innate-like T cells, and innate lymphoid cells. This study investigates the phenotypic and functional profile of circulating CLA+ natural killer (NK) cells and its role in the skin-homing to NK cells infiltrated in adults' skin with AD. We selected 44 AD patients and 27 non-AD volunteers for the study. The results showed increased frequencies of both CLA+CD56bright and CLA+CD56dim NK cell populations in the peripheral blood, mainly in severe AD patients. Upon SEB stimulation, we observed an augmented percentage of CLA+CD56dim NK cells expressing CD107a, IFN-γ, IL-10, and TNF, reinforcing the role of staphylococcal enterotoxins in AD pathogenesis. Additionally, we demonstrated increased dermal expression of both NK cell markers NCAM-1/CD56 and pan-granzyme, corroborating the skin-homing, mostly in severe AD. Further studies are necessary to elucidate the potential role of NK cells in the chronification of the inflammatory process in AD skin, as well as their possible relationship with staphylococcal enterotoxins, and as practicable therapeutic targets.

Coordination of prophage and global regulator leads to high enterotoxin production in staphylococcal food poisoning-associated lineage.

Staphylococcus species in food produce Staphylococcal enterotoxins (SEs) that cause Staphylococcal food poisoning (SFP). More than 20 SE types have been reported, among which Staphylococcal enterotoxin A (SEA) has been recognized as one of the most important SEs associated with SFP. However, the regulatory mechanisms underlying its production remain unclear. Previously, we identified a major SFP clone in Japan, CC81 subtype-1, which exhibits high SEA production. In this study, we attempted to identify the factors contributing to this phenomenon. Thus, we demonstrated that the attenuation of the activity of endogenous regulator, Staphylococcal accessory regulator S (SarS), and the lysogenization of a high SEA-producing phage contributed to this phenomenon in CC81 subtype-1. Furthermore, our results indicated that SarS could directly bind to the promoter upstream of the sea gene and suppress SEA expression; this low SarS repression activity was identified as one of the reasons for the high SEA production observed. Therefore, we revealed that both exogenous and endogenous factors may probably contribute to the high SEA production. Our results confirmed that SE production is a fundamental and critical factor in SFP and clarified the associated production mechanism while enhancing our understanding as to why a specific clone frequently causes SFP. IMPORTANCE: The importance of this study lies in its unveiling of a molecular regulatory mechanism associated with the most important food poisoning toxin and the evolution of Staphylococcal food poisoning (SFP)-associated clone. SFP is primarily caused by Staphylococcus aureus, with Staphylococcal enterotoxin A (SEA) being commonly involved in many cases. Thus, SEA has been recognized as a major toxin type. However, despite almost a century since its discovery, the complete mechanism of SEA production is as yet unknown. In this study, we analyzed an SEA-producing SFP clone isolated in East Asia and discovered that this strain, besides acquiring the high SEA-producing phage, exhibits remarkably high SEA production due to the low activity of SarS, an intrinsic regulatory factor. This is the first report documenting the evolution of the SFP clone through the coordinated action of exogenous mobile genetic factors and endogenous regulators on this notorious toxin.

Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration.

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.

Survival, Growth, and Toxin Production of Bacillus cereus During Cooking and Storage of Fresh Rice Noodles.

Retail stores maintain fresh rice noodles (FRNs) at room temperature because refrigeration negatively impacts FRNs' texture. The room temperature and high water activity of FRNs help spore-forming Bacillus cereus to grow and produce toxins. In this study, the effect of steam cooking on survival and different storage temperatures on the growth and enterotoxins production of B. cereus in FRNs were investigated. White rice flour was used to make FRNs. Three treatments of FRNs were used in this study; uninoculated, inoculated (with 4.0 log CFU/ml of B. cereus spores), and autoclaved as a negative control. A slurry of rice flour, cornstarch, and water was steam cooked for 4 min at 90°C and incubated for 168 h at 4°C, and for 72 h at 22 and 32°C. Incubated FRNs were tested for pH, B. cereus growth, and enterotoxins production. Steam cooking reduced the total number of B. cereus spores by 0.7 ± 0.3 log CFU/g. Surviving B. cereus spores in inoculated and uninoculated FRNs germinated over 72 h of storage. No B. cereus was detected in negative controls. An interaction was observed across storage temperatures and time (p < 0.05). The B. cereus population in uninoculated FRNs increased by more than 7.0 log CFU/g at 22 and 32°C over 72 h, while inoculated FRNs showed a 5.0 log bacterial increase at these storage temperatures. No growth was observed at 4°C in both inoculated and uninoculated FRNs. The pH of inoculated FRNs was reduced from 6.9 ± 0.1 to 5.7 ± 0.0 at 32°C and to 6.2 ± 0.1 at 22°C, and the pH of uninoculated FRNs was reduced from 7.0 ± 0.1 to 5.8 ± 0.2 at 32°C and to 6.5 ± 0.0 at 22°C, indicative of FRNs spoilage. B. cereus in inoculated FRNs produced enterotoxins after 12 h of storage at 32°C, and over 24 h of storage at 22°C, while no toxin was detected at 4°C. Our findings show that storing FRNs at room temperature for 24 h leads to enterotoxin production, emphasizing the importance of proper FRN storage and their potential risk to consumers. Nevertheless, further research should investigate the effect of other foodborne pathogens on these products.

Isofagomine Inhibits Multiple TcdB Variants and Protects Mice from Clostridioides difficile-Induced Mortality.

Clostridioides difficile causes life-threatening diarrhea and is one of the leading causes of nosocomial infections. During infection, C. difficile releases two gut-damaging toxins, TcdA and TcdB, which are the primary determinants of disease pathogenesis and are important therapeutic targets. Once in the cytosol of mammalian cells, TcdA and TcdB use UDP-glucose to glucosylate host Rho GTPases, which leads to cytoskeletal changes that result in a loss of intestinal integrity. Isofagomine inhibits TcdA and TcdB as a mimic of the glucocation transition state of the glucosyltransferase reaction. However, sequence variants of TcdA and TcdB across the clades of infective C. difficile continue to be identified, and therefore, evaluation of isofagomine inhibition against multiple toxin variants is required. Here, we show that isofagomine inhibits the glucosyltransferase domain of multiple TcdB variants and protects TcdB-induced cell rounding of the most common full-length toxin variants. Furthermore, we demonstrate that isofagomine protects against C. difficile-induced mortality in two murine models of C. difficile infection. Isofagomine treatment of mouse C. difficile infection also permitted the recovery of the gastrointestinal microbiota, an important barrier to preventing recurring C. difficile infection. The broad specificity of isofagomine supports its potential as a prophylactic to protect against C. difficile-induced morbidity and mortality.

Prevalence and genomic characterization of the Bacillus cereus group strains contamination in food products in Southern China.

The Bacillus cereus group, as one of the important opportunistic foodborne pathogens, is considered a risk to public health due to foodborne diseases and an important cause of economic losses to food industries. This study aimed to gain essential information on the prevalence, phenotype, and genotype of B. cereus group strains isolated from various food products in China. A total of 890 strains of B. cereus group bacteria from 1181 food samples from 2020 to 2023 were identified using the standardized detection method. These strains were found to be prevalent in various food types, with the highest contamination rates observed in cereal flour (55.8 %) and wheat/rice noodles (45.7 %). The tested strains exhibited high resistance rates against penicillin (98.5 %) and ampicillin (98.9 %). Strains isolated from cereal flour had the highest rate of meropenem resistance (7.8 %), while strains from sausages were most resistant to vancomycin (16.8 %). A total of 234 out of the 891 B. cereus group strains were randomly selected for WGS analysis, 18.4 % of which displayed multidrug resistance. The species identification by WGS analysis revealed the presence of 10 distinct species within the B. cereus group, with B. cereus species being the most prevalent. The highest level of species diversity was observed in sausages. Notably, B. anthracis strains lacking the anthrax toxin genes were detected in flour-based food products and sausages. A total of 20 antibiotic resistance genes have been identified, with ß-lactam resistance genes (bla1, bla2, BcI, BcII, and blaTEM-116) being the most common. The B. tropicus strains exhibit the highest average number of virulence genes (23.4). The diarrheal virulence genes nheABC, hblACD, and cytK were found in numerous strains. Only 4 of the 234 (1.7 %) sequenced strains contain the ces gene cluster linked to emetic symptoms. These data offer valuable insights for public health policymakers on addressing foodborne B. cereus group infections and ensuring food safety.

Prevalence of virulence- and antibiotic resistance-associated genotypes and phenotypes in Staphylococcus aureus strains from the food sector compared to clinical and cow mastitis isolates.

Background: Infections by the pathogen Staphylococcus aureus currently represent one of the most serious threats to human health worldwide, especially due to the production of enterotoxins and the ability to form biofilms. These structures and the acquisition of antibiotic resistance limit the action of antibiotics and disinfectants used to combat this microorganism in the industry and the clinic. Methods: This work reports a comparative phenotypic and genotypic study of 18 S. aureus strains from different origins: clinical samples, milk from mastitic cows and food industry surfaces, most of which were isolated in Northern Spain. Results: Genetically, the strains were very diverse but, in most cases, a closer proximity was observed for those from the same source. Notably, the average number of virulence genes was not significantly different in strains from the food sector. Of the 18 strains, 10 coded for at least one enterotoxin, and four of them carried 6 or 7 enterotoxin genes. The latter were all veterinary or clinical isolates. Most strains carried prophages, plasmids and/or pathogenicity islands. Regarding antibiotic resistance, although phenotypically all strains showed resistance to at least one antibiotic, resistance genes were only identified in 44.5% of strains, being mastitis isolates those with the lowest prevalence. Virulence-related phenotypic properties such as haemolytic activity, staphyloxanthin production, biofilm-forming capacity and spreading ability were widely distributed amongst the isolates. Conclusions: Our results indicate that production of virulence factors, antibiotic resistance and biofilm formation can be found in S. aureus isolates from diverse environments, including the food industry, although some of these traits are more prevalent in strains isolated from infections in cows or humans. This emphasizes on the importance of monitoring the spread of these determinants not only in samples from the clinical environment, but also along the food chain, a strategy that falls under the prism of a one-health approach.