Classical Staphylococcal Enterotoxins Research Articles

Prevalence, Antibiotic Resistance, and Molecular Typing of Staphylococcus aureus Isolated from Ready-to-Eat Foods in Guangdong, South China.

The increasing global popularity of ready-to-eat (RTE) foods for their convenience simultaneously brings along a risk, as these products can be contaminated with various microorganisms, including potentially harmful pathogens. We aimed to investigate the food contamination of Staphylococcus aureus (S. aureus) in RTE foods in Guangdong, South China. All S. aureus isolates were subjected to characterization through antimicrobial susceptibility tests, multilocus sequence typing (MLST), and PCR analysis for detecting mec and blaZ genes. A total of 824 RTE food samples were collected from 2017 to 2022, of which 73 (8.9%) were found to be contaminated with S. aureus. Contamination levels were mostly in the range of 0.3-1.0 most probable number (MPN)/g, with 10 samples exceeding 110 MPN/g. Of the 73 S. aureus isolates, 10 were identified as methicillin-resistant S. aureus (MRSA). In MRSA, resistance was most frequently observed to penicillin (100%, 10/10), followed by erythromycin (80.0%, 8/10) and tetracycline (70%, 7/10). And in methicillin-sensitive S. aureus (MSSA), resistance was most frequently observed to penicillin (98.4%, 62/63), followed by tetracycline (30.2%, 19/63) and erythromycin (23.8%, 15/63). Overall, 98.6% (72/73) of the isolates demonstrated resistance to at least one antimicrobial agent, whereas 31.5% (23/73) were resistant to three or more antimicrobials. Fifty-seven S. aureus isolates harbored the penicillin-resistant gene blaZ, and 10 isolates carried the mec gene. In addition, 30.1% of the isolates harbored genes for classical staphylococcal enterotoxins (SEs), with seb being the most frequently detected SE gene. MLST revealed that the 73 isolates belonged to 14 different sequence types (STs), the most prevalent of which was ST7. In MRSA, the most common prevalent clone is ST6, and in MSSA, ST7 was the most common isolates. The prevalent multidrug resistance indicates that the resistance situation of foodborne S. aureus in Guangdong is severe, posing a potential threat to consumer safety and health.

Identification of the Enterotoxigenic Potential of Staphylococcus spp. from Raw Milk and Raw Milk Cheeses.

This study aimed to genotypic and phenotypic analyses of the enterotoxigenic potential of Staphylococcus spp. isolated from raw milk and raw milk cheeses. The presence of genes encoding staphylococcal enterotoxins (SEs), including the classical enterotoxins (sea-see), non-classical enterotoxins (seg-seu), exfoliative toxins (eta-etd) and toxic shock syndrome toxin-1 (tst-1) were investigated. Isolates positive for classical enterotoxin genes were then tested by SET-RPLA methods for toxin expression. Out of 75 Staphylococcus spp. (19 Staphylococcus aureus and 56 CoNS) isolates from raw milk (49/65.3%) and raw milk cheese samples (26/34.7%), the presence of enterotoxin genes was confirmed in 73 (97.3%) of them. Only one isolate from cheese sample (1.3%) was able to produce enterotoxin (SED). The presence of up to eight different genes encoding enterotoxins was determined simultaneously in the staphylococcal genome. The most common toxin gene combination was sek, eta present in fourteen isolates (18.7%). The tst-1 gene was present in each of the analyzed isolates from cheese samples (26/34.7%). Non-classical enterotoxins were much more frequently identified in the genome of staphylococcal isolates than classical SEs. The current research also showed that genes tagged in S. aureus were also identified in CoNS, and the total number of different genes detected in CoNS was seven times higher than in S. aureus. The obtained results indicate that, in many cases, the presence of a gene in Staphylococcus spp. is not synonymous with the ability of enterotoxins production. The differences in the number of isolates with genes encoding SEs and enterotoxin production may be mainly due to the limit of detection of the toxin production method used. This indicates the need to use high specificity and sensitivity methods for detecting enterotoxin in future studies.

Ready-to-Eat Foods: A Potential Vehicle for the Spread of Coagulase-Positive Staphylococci and Antimicrobial-Resistant Staphylococcus aureus in Buea Municipality, South West Cameroon.

The consumption of ready-to-eat (RTE) foods contaminated with coagulase-positive staphylococci (CoPS) and especially Staphylococcus aureus puts consumers at a potential risk of food-borne disease or colonization and subsequent infection. This cross-sectional study determined the levels of CoPS and the presence of S. aureus in RTE foods sold in Buea municipality. A total of 420 RTE food samples, comprising 70 each of cake, bread, fruit salad, meat hot-pot, suya, and boiled rice were randomly purchased from February to August 2020. The CoPS counts were determined by culturing on Baird-Parker agar, and S. aureus was identified by amplification of the nuc gene using the polymerase chain reaction. All S. aureus isolates were screened for the presence of classical staphylococcal enterotoxin genes. To determine antimicrobial resistance profiles, each isolate was tested against 11 antimicrobials. Oxacillin-resistant S. aureus strains were analyzed for the presence of the mecA gene. Overall, 161 (38.3%) samples had detectable levels of CoPS ranging from 2.0 to 5.81 log10 CFU/g. Based on CoPS levels, 37 (8.81%) of the 420 RTE food samples-only fruit salad and meat hot-pot, had unsatisfactory microbiological quality. A total of 72 S. aureus isolates, comprising 52.78% from fruit salad, 16.67% from meat hot-pot, 12.5% from boiled rice, 9.72% from suya, 5.56% from bread, and 4.17% from cake, were recovered. None of the S. aureus isolates possessed any of the classical enterotoxin genes. All the isolates were susceptible to vancomycin and ofloxacin, while 68 (94.44%) and 66 (91.67%) were susceptible to oxacillin and ciprofloxacin, respectively. Resistance to penicillin (93.06%) was highest, followed by amoxicillin (91.67%) and erythromycin (79.17%). Four isolates were identified as methicillin-resistant S. aureus, all of which carried the mecA gene. A total of 24 antibiotypes were identified. Our findings showed that RTE foods sold in the Buea municipality are likely vehicles for the transmission of CoPS and antimicrobial-resistant S. aureus.

The Enterotoxin Gene Profiles and Enterotoxin Production of Staphylococcus aureus Strains Isolated from Artisanal Cheeses in Belgium.

A Staphyloccoccus aureus is one of the leading causes of food poisoning outbreaks (FPOs) worldwide. Staphylococcal food poisoning (SFP) is induced by the ingestion of food containing sufficient levels of staphylococcal enterotoxins (SEs). Currently, 33 SEs and SE-like toxins (SEls) have been described in the literature, but only five named "classical" enterotoxins are commonly investigated in FPOs due to lack of specific routine analytical techniques. The aims of this study were to (i) establish the genetic profile of strains in a variety of artisanal cheeses (n = 30) in Belgium, (ii) analyze the expression of the SE(l)s by these strains and (iii) compare the output derived from the different analytical tools. Forty-nine isolates of S. aureus were isolated from ten Belgian artisanal cheeses and were analyzed via microbiological, immunological, liquid chromatography mass spectrometry, molecular typing and genetic methods. The results indicated that classical SEs were not the dominant SEs in the Belgian artisanal cheeses that were analyzed in this study, and that all S. aureus isolates harbored at least one gene encoding a new SE(l). Among the new SE(l)s genes found, some of them code for enterotoxins with demonstrated emetic activity and ecg-enterotoxins. It is worth noting that the involvement of some of these new SEs has been demonstrated in SFP outbreaks. Thus, this study highlighted the importance of the development of specific techniques for the proper investigation of SFP outbreaks.

Effect of Temperature on the Expression of Classical Enterotoxin Genes among Staphylococci Associated with Bovine Mastitis.

Staphylococcal food poisoning (SFP), caused by the contamination of staphylococcal enterotoxins, is a common foodborne disease worldwide. The aims of this study were: (1) to investigate classical staphylococcal enterotoxin genes, sea, seb, sec, sed, and see, among Staphylococcus aureus and coagulase-negative staphylococci (CNS) associated with bovine mastitis; (2) to determine the effect of temperature on the expression of classical staphylococcal enterotoxin genes in staphylococci in milk. The detection of classical staphylococcal enterotoxin genes was performed using S. aureus (n = 51) and CNS (n = 47). The expression of classical enterotoxin genes, including sea, seb, sec, and see, was determined during the growth of staphylococci in milk subjected to ultra-high-temperature processing at two different temperatures: 8 °C and room temperature. Classical staphylococcal enterotoxin genes were expressed more frequently in S. aureus (35.30%) than in CNS (12.77%). The sec gene was most frequently detected in S. aureus (29.41%) and CNS (6.38%). Moreover, the expression of sea and sec was significantly higher at room temperature than at 8 °C after 16 h of incubation (p < 0.05). These results emphasize the importance of maintaining the storage temperature of milk below 8 °C to reduce the risk of SFP.

Evaluation of Virulence Determinants Using Whole-Genome Sequencing and Phenotypic Biofilm Analysis of Outbreak-Linked Staphylococcus aureus Isolates.

Biofilms are a frequent cause of food contamination of potentially pathogenic bacteria, such as Staphylococcus aureus. Given its vast role in human disease, the possible impact of biofilm-producing S. aureus isolates in a food processing environment is evident. Sixty-nine S. aureus isolates collected from one firm following multiple staphylococcal food poisoning outbreak investigations were utilized for this analysis. Strain evaluations were performed to establish virulence determinants and the evolutionary relationships using data generated by shotgun whole-genome sequencing (WGS), along with end point polymerase chain reaction (PCR) and in vitro phenotypic assessments. S. aureus isolates were grouped into six well-supported clades in the phylogenetic tree, with the relationships within the clades indicating a strong degree of clonal structure. Our analysis identified four major sequence types 47.8% ST1, 31.9% ST45, 7.2% ST5, and 7.2% ST30 and two major spa types 47.8% t127 and 29.0% t3783. Extrapolated staphylococcal enterotoxin (SE) analysis found that all isolates were positive for at least 1 of the 23 SEs and/or SE-like toxin genes. Enterotoxigenic assessments found that 93% of the isolates expressed a classical SE(A–E). SE gene concurrence was observed at 96.2%, based on PCR and WGS results. In total, 46 gene targets were distinguished. This included genes that encode for adhesion and biofilm synthesis such as clfA, clfB, bbp, ebpS, ica, bap and agr. Our evaluation found agr group III to be the most prevalent at 55%, followed by 35% for agr group I. All isolates harbored the complete intercellular adhesion operon that is recognized to contain genes responsible for the adhesion step of biofilm formation by encoding proteins involved in the syntheses of the biofilm matrix. Phenotypic characterization of biofilm formation was evaluated three times, with each test completed in triplicate and accomplished utilizing the microtiter plate method and Congo red agar (CRA). The microtiter plate results indicated moderate to high biofilm formation for 96% of the isolates, with 4% exhibiting weak to no biofilm development. CRA results yielded all positive to intermediate results. The potential to inadvertently transfer pathogenic bacteria from the environment into food products creates challenges to any firm and may result in adulterated food.

Prevalence of classical Staphylococcal enterotoxin genes of Staphylococcus aureus isolated from ready-to-eat food in Ho Chi Minh City, Vietnam

Contamination of Staphylococcus aureus in ready-to-eat (RTE) food is a leading cause of foodborne illness in Vietnam. The aim of this study was to analyze the frequency of classical Staphylococcal enterotoxin genes (sea, seb, sec, sed, and see genes) in S. aureus strains isolated from ready-to-eat food in Ho Chi Minh, Vietnam (76 strains of&amp;nbsp; S. aureus were isolated from 200 random samples of ready - to - eat (RTE) food, which include pate, pork roll, barbecue pork, roasted pork and roasted duck, 40 samples of each), all the samples were collected from different fast food services on street in district 5, 6, 7 and 8 in Ho Chi Minh City, Vietnam. All the trains of S. aureus were stored at -70oC for analyzing Staphylococcal enterotoxin genes). The obtained results in this study indicated that out of 76 S. aureus strains, there are 6 strains (7.9%) carried SE genes: &amp;nbsp;sea gene (1.32%) was detected in one pate sample (Dist. 8), sec gene was detected (3.95%) in three samples, which are two pork roll samples (Dist. 7 and 8) and one roasted duck sample (Dist. 8), and finally see gene was detected in one pate sample (Dist. 8)and in one pork roll sample (Dist.&amp;nbsp; 8). This could be a serious public health risk and highlight the need to implement good hygiene practices.

Pathogenic Potential and Antimicrobial Resistance Profile of Staphylococcus aureus in Milk and Beef from the Northwest and Southwest Regions of Cameroon.

Staphylococcus aureus is a major foodborne pathogen and commensal of the skin and mucous membranes of animals and humans. Its virulence relies on the production of a variety of toxins resistant to denaturing conditions. Increasing reports of S. aureus food poisoning and contamination of foods of animal origin elsewhere necessitates the investigation of these foods in Cameroon, to implement safety measures. This cross-sectional study evaluated S. aureus contamination in milk and beef in the Northwest and Southwest Regions of Cameroon, where cow milk is usually not pasteurized before consumption, and beef is the main source of protein. The distribution of antibiotic-resistant isolates and those with enterotoxin-producing potential was also investigated to provide data of public health and food safety benefit. S. aureus was isolated from 39 raw milk and 250 beef samples by standard methods. Confirmation of isolates was by PCR to detect the nuc gene. S. aureus was investigated for classical staphylococcal enterotoxin (SE) genes (sea, seb, sec, sed, and see) by PCR. Their susceptibility to 9 antibiotics was tested by the disk diffusion method. The chi-square test was used to compare the contamination of samples, antibiotic resistance, and the distribution of SE genes. S. aureus was isolated from 11.1% of samples. Contamination was higher in milk (48%) than in beef (5.2%) (P < 0.001). The sea was the most frequently (90%) harboured gene. A large proportion of isolates (88%) harboured more than one virulence gene. Isolates were generally resistant to erythromycin (82%), vancomycin (80%), tetracycline (76%), and oxacillin (74%). Multidrug resistance (MDR) was common (92%). Milk and beef samples in study area were contaminated with MDR enterotoxigenic S. aureus strains and may constitute a potential hazard to consumers. Thus, the need for implementation of proper hygienic measures when handling these products and pasteurization of milk cannot be overemphasized.

Characterization of Oxacillin-Susceptible mecA-Positive Staphylococcus aureus from Food Poisoning Outbreaks and Retail Foods in China.

In this study, we explored the prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) in staphylococcal food poisoning outbreak isolates and foodborne isolates, and then investigated their molecular characteristics, classical staphylococcal enterotoxins (SEs), and drug resistance. Eight (2.9%) of 275 isolates from food poisoning outbreaks and 7 (3.8%) of 184 isolates from retail foods were identified as OS-MRSA isolates. Among the 15 OS-MRSA isolates, the most frequently detected toxin genes were hld (100%), hla (93.3%), pvl (80.0%), and hlb (46.7%) followed by seg and seq (33.3%, each), hlg (26.7%), seb and hlgv (20.0%, each), sec, seh, sel, sep, and tst (13.3%, each), and sei, sem, sen, and seo (6.7%, each). None of isolates carried other tested virulence genes. The most frequently detected classical SEs were SEB and SEC (26.7%, each), followed by SEA and SEE (20.0%, each), and SED (6.7%). Resistance was most frequently observed in ampicillin, penicillin, and cefoxitin (100%, each), followed by trimethoprim/sulfamethoxazole (93.3%), erythromycin (73.3%), amoxicillin/clavulanic acid (46.7%), tetracyclines (26.7%), and ciprofloxacin (6.7%). All isolates were susceptible to other tested antibiotics. A dominant molecular type belonged to ST398-IVa-t034 (26.7%), followed by ST59-IVa-t437 (20.0%), ST88-III-t14340 and ST1-IVa-t114 (13.3%, each), and ST5-II-t002, ST630-t4549, ST5-II, and ST4495-t10738 (6.7%, each). Our findings indicated that OS-MRSA strains had a low prevalence rate among outbreak strains and foodborne strains, which frequently harbored SCCmec IVa, and carried a variety of toxin genes, and also expressed numerous classical SEs. In addition, all OS-MRSA isolates were susceptible to the majority of antibacterial agents except β-lactam. Our study is the first to report that OS-MRSA isolates are associated with food poisoning outbreaks worldwide.

NAuRA: Genomic Tool to Identify Staphylococcal Enterotoxins in Staphylococcus aureus Strains Responsible for FoodBorne Outbreaks.

Food contamination by staphylococcal enterotoxins (SEs) is responsible for many food poisoning outbreaks (FPOs) each year, and they represent the third leading cause of FPOs in Europe. SEs constitute a protein family with 27 proteins. However, enzyme immunoassays can only detect directly in food the five classical SEs (SEA-SEE). Thus, molecular characterization methods of strains found in food are now used for FPO investigations. Here, we describe the development and implementation of a genomic analysis tool called NAuRA (Nice automatic Research of alleles) that can detect the presence of 27 SEs genes in just one analysis- and create a database of allelic data and protein variants for harmonizing analyses. This tool uses genome assembly data and the 27 protein sequences of SEs. To include the different divergence levels between SE-coding genes, parameters of coverage and identity were generated from 10,000 simulations and a dataset of 244 assembled genomes from strains responsible for outbreaks in Europe as well as the RefSeq reference database. Based on phylogenetic inference performed using maximum-likelihood on the core genomes of the strains in this collection, we demonstrated that strains responsible for FPOs are distributed throughout the phylogenetic tree. Moreover, 71 toxin profiles were obtained using the NAuRA pipeline and these profiles do not follow the evolutionary history of strains. This study presents a pioneering method to investigate strains isolated from food at the genomic level and to analyze the diversity of all 27 SE-coding genes together.

Microbial safety status of Serro artisanal cheese produced in Brazil

Considering the growing consumption of artisanal foods worldwide, we aimed to evaluate the microbial safety of Serro artisanal cheese (SAC), produced in Minas Gerais State, Brazil. This cheese is produced with raw milk using 1 of 2 natural starter cultures: "pingo" and "rala." A total of 53 SAC samples (pingo = 8; rala = 45) were obtained from different farmers and subjected to conventional and molecular assays to detect and enumerate Listeria monocytogenes, Salmonella spp., coagulase-positive staphylococci (CPS), diarrheagenic Escherichia coli, Mycobacterium tuberculosis, and Brucella abortus. The SAC samples were also subjected to an ELISA to detect classical staphylococcal enterotoxins (CSE: SEA, SEB, SEC, SED, SEE) and to PCR assays to detect staphylococcal enterotoxin-related genes (sea, seb, sec, sed, see). Coagulase-positive staphylococci isolates were obtained and tested by the same assays to detect their potential in CSE production and presence of CSE-related genes. None of the SAC samples showed any of the screened food-borne pathogens and zoonotic agents, and none showed the presence of CSE by phenotypic and genotypic approaches. Despite the absence of microbial hazards, mean counts of CPS in SAC samples were 5.2 log cfu/g (pingo starter) and 4.6 log cfu/g (rala starter), indicating poor hygiene practices during production. None of the tested CPS isolates (n = 116) produced CSE or presented CSE-related genes. Despite the relative microbial safety, hygienic conditions during SAC production must be improved to meet official guidelines established in Brazil.

Impact of co-cultivation with Enterococcus faecalis over growth, enterotoxin production and gene expression of Staphylococcus aureus in broth and fresh cheeses

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

The T cell activating properties and antitumour activity of Staphylococcal Enterotoxin-like Q.

Staphylococcal enterotoxins (SEs), as typical superantigens, exhibit promising antitumour activity in the clinic, but their unavoidable side effects related to fever and emesis seriously limit their application for the treatment of malignant tumours. Fortunately, the identification of Staphylococcal enterotoxin-like toxins (SEls), which possess amino acid sequences similar to those of classical SEs but exhibit no or low emetic activity, has provided a set of potential immunomodulatory candidates for cancer therapy. The aim of this study was to examine the effect of SElQ on lymphocyte activation and to further demonstrate its antitumour activity both in vitro and in vivo. High-purity SElQ was successfully harvested, and in vitro results confirmed that SElQ can significantly activate mouse- and human-derived lymphocytes in a dose-dependent manner, particularly CD4+ and CD8+ T cells, which showed significant increases in both percentage and absolute number. Further examination revealed that in addition to the originally recognized TCR Vβ5 and 21, TCR Vβ14, 17 and 18 were activated in SElQ-induced human PBMCs. Moreover, the expression of IL-2 and IFN-γ was significantly upregulated in vitro and in vivo after SElQ treatment. Based on the findings that SElQ induces lymphocyte activation and cytokine release, we then confirmed its antitumour activity both in vitro and in vivo. The data showed that treatment with a low concentration of SElQ (30µg/mouse) could inhibit the growth of tumours by approximately 30% and no significant toxicity was observed. Taken together, our results demonstrated that SElQ can significantly induce T cell activation and cytokine release and further elicit substantial antitumour activity and thus provide support for the potential application of SElQ in cancer immunotherapy.

Histamine release from intestinal mast cells induced by staphylococcal enterotoxin A (SEA) evokes vomiting reflex in common marmoset.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are known as causative agents of emetic food poisoning. We previously demonstrated that SEA binds with submucosal mast cells and evokes mast cell degranulation in a small emetic house musk shrew model. Notably, primates have been recognized as the standard model for emetic assays and analysis of SE emetic activity. However, the mechanism involved in SEA-induced vomiting in primates has not yet been elucidated. In the present study, we established common marmosets as an emetic animal model. Common marmosets were administered classical SEs, including SEA, SEB and SEC, and exhibited multiple vomiting responses. However, a non-emetic staphylococcal superantigen, toxic shock syndrome toxin-1, did not induce emesis in these monkeys. These results indicated that the common marmoset is a useful animal model for assessing the emesis-inducing activity of SEs. Furthermore, histological analysis uncovered that SEA bound with submucosal mast cells and induced mast cell degranulation. Additionally, ex vivo and in vivo pharmacological results showed that SEA-induced histamine release plays a critical role in the vomiting response in common marmosets. The present results suggested that 5-hydroxytryptamine also plays an important role in the transmission of emetic stimulation on the afferent vagus nerve or central nervous system. We conclude that SEA induces histamine release from submucosal mast cells in the gastrointestinal tract and that histamine contributes to the SEA-induced vomiting reflex via the serotonergic nerve and/or other vagus nerve.

Prevalence and distribuion of staphylococcal enterotoxin genes among Staphylococcus aureus isolates from chicken and turkey carcasses in Algeria

This study is aimed to determine the prevalence of staphylococcus aureus (S.aureus) by biochemical tests in poultry carcasses. It is also intend to detect the distribution of genes for classical staphylococcal enterotoxins A, B, C, D and E (sea, seb, sec, sed and see) and for gene femA, specific for S.aureus species, using multiplex PCR. A total of 385 samples of neck skins from fresh poultry carcasses were collected during the period 2012-2013 from 16 different slaughterhouses located in the region of Algiers, Algeria. The overall prevalence of S.aureus in freshly slaughtered poultry carcasses was 41.56%, with an individual prevalence of 40.63% and 45.71% for chicken and turkey respectively. From the 95 strains of S.aureus identified by biochemical tests, 82 (86.32%) isolates were femA positive using multiplex PCR. The investigation has also revealed the presence of both enterotoxins B and D, with a predominance of seb (13.33%) followed by sed (1.67%), in the chicken carcasses while in turkey only sed was detected (4.55%) It has been found that strains of S.aureus of poultry origin can be enterotoxigenic with the predominance of genes encoding for enterotoxins seb in chicken and sed in turkey. As enterotoxins can be produced in adequate amounts to induce foodborne illnesses, these potential dangers must be considered in terms of a real risk to public health.

Prevalence of Enterotoxin Genes inStaphylococcus aureusIsolates from Pork Production

In this study, 130 Staphylococcus aureus isolates from samples associated with pork production were tested for prevalence of 18 staphylococcal enterotoxin (SE) genes. Approximately 94.6% (123/130) of isolates from different stages of pork production harbored one or more SE genes forming 37 different enterotoxin gene profiles. Seb was present in 60.0% of the S. aureus isolates, the highest among the genes tested. The genes, sed, sej, seo, sep, ser, and seu, were not found. The five classical SE genes (including sea, seb, sec, sed, see) had lower prevalence than the egc gene cluster (seg, sei, sem, sen, seo, or seu). Notably, ∼6.9% (9/130) isolates harbored five SE genes. Classical SE genes were relatively higher in raw meat isolates than swine farm isolates, suggesting that raw meat isolates have a greater potential for classical staphylococcal food poisoning. Incomplete egc clusters were mainly distributed in swine farm isolates, and some of them coexisted with other classical SE genes (seb, sec), showing that swine farms could be potential sources of enterogenic S. aureus of food safety concern. Characterizing the distributions of enterotoxin genes among S. aureus may provide epidemiological information for the benefit of public health and food safety.

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China.

The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST) and polymerase chain reaction (PCR) analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE) genes (sea, seb, sec, sed, see, seg, seh, sei, sej), the exfoliative toxin genes (eta and etb), the toxic shock syndrome toxin-1 gene (tst), and the Panton-Valentine leucocidin-encoding gene (pvl). The isolates encompassed 26 different sequence types (STs), including four new STs (ST3482, ST3484, ST3485, ST3504), clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7%) detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%), sea (33.3%), and seg (33.3%). The classical SE genes (sea–see), which contribute significantly to staphylococcal food poisoning (SFP), were detected in 72.5% of the S. aureus isolates. In addition, pvl, eta, etb, and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies.

Staphylococcus aureus undergoes major transcriptional reorganization during growth with Enterococcus faecalis in milk

Previous studies have demonstrated the antagonistic potential of lactic acid bacteria (LAB) present in raw milk microbiota over Staphylococcus aureus, albeit the molecular mechanisms underlying this inhibitory effect are not fully understood. In this study, we compared the behavior of S. aureus ATCC 29213 alone and in the presence of a cheese-isolated LAB strain, Enterococcus faecalis 41FL1 in skimmed milk at 30 °C for 24 h using phenotypical and molecular approaches. Phenotypic analysis showed the absence of classical staphylococcal enterotoxins in co-culture with a 1.2-log decrease in S. aureus final population compared to single culture. Transcriptional activity of several exotoxins and global regulators, including agr, was negatively impacted in co-culture, contrasting with the accumulation of transcripts coding for surface proteins. After 24 h, the number of transcripts coding for several metabolite responsive elements, as well as enzymes involved in glycolysis and acetoin metabolism was increased in co-culture. The present study discusses the complexity of the transcriptomic mechanisms possibly leading to S. aureus attenuated virulence in the presence of E. faecalis and provides insights into this interspecies interaction in a simulated food context.

Development and Evaluation of Simple Dot-Blot Assays for Rapid Detection of Staphylococcal Enterotoxin-A in Food.

The present study was aimed to develop and evaluate dot-blot assays for rapid detection of staphylococcal enterotoxin-A (SEA) in food. Dot blots were developed in two formats, indirect and sandwich utilizing mouse monoclonal anti-SEA and rabbit polyclonal anti-SEA antibodies. In indirect dot-blot format, recombinant SEA was directly coated on NCM dot-blot strip and detection was carried out by anti-SEA antibodies. In sandwich dot-blot format, SEA was trapped between anti-SEA capture and detection antibodies. Both the dot-blot assays exhibited a sensitivity of ~48ngml-1 when tested in different food matrices. The developed assays were highly specific as no cross-reactivity was detected with other classical staphylococcal enterotoxins, toxigenic bacteria and foodborne pathogens. Sensitivity and specificity of developed indirect and sandwich dot-blot assays with respect to PCR was found to be 100 and 99%, respectively. The results shows that the developed dot-blot assays can be used as rapid preliminary screening tests for detection of SEA in food or determining the toxigenic potential of staphylococci, especially in resource-limited settings.

Characterization of Staphylococcus xylosus LQ3 and its application in dried cured sausage

The aim of this study was to characterize the food safety aspects and enzymatic profile of Staphylococcus xylosus LQ3 in vitro and its influence in the curing process and antioxidant capacity in dried cured sausage. S. xylosus LQ3 isolate was susceptible to all antimicrobials tested and did not show any antimicrobial resistance genes. LQ3 was negative for hemolytic and DNase activities. Moreover, no genes encoding for alpha and beta hemolysis or for classical staphylococcal enterotoxins were identified. Enzymatic tests showed that S. xylosus LQ3 presented lipolytic, proteolytic, nitrate reductase (25 °C and 37 °C), and superoxide dismutase activities. The sausage with S. xylosus LQ3 (T1) was lighter and redder in color than the control without S. xylosus (C1). LQ3 treated sausage (T1) presented lower lipid oxidation in comparison to C1. S. xylosus LQ3 demonstrated potential to be used as starter culture for the production of fermented sausage, because it showed microbiological and toxicological safety and a relevant enzymatic profile. Moreover, it contributed to the formation of the typical coloration of cured products and limited lipid oxidation in situ.