Genomic portraits of methicillin-resistant staphylococci (MRS) from food fish unveiled the genes associated with staphylococcal food poisoning (SFP), virulence and antimicrobial resistance.

BACKGROUND: Characteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants. RESULTS: Three MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings. CONCLUSION: This study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread.

In vitro selection of DNA aptamers against staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.

Molecular Characteristics of Staphylococcus aureus Isolates form Food-Poisoning Outbreaks (2011-2022) in Sichuan, China.

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases in the world. This study aimed to investigate the molecular epidemiological characteristics of Staphylococcus aureus isolated from SFP. A total of 103 S. aureus isolates were obtained during 2011-2022 in Sichuan, southwest China. All isolates were tested for the genomic characteristics and phylogenetic analysis by performing whole-genome sequencing. Multilocus sequence typing analysis showed 17 multilocus sequence types (STs), ST7 (23.30%), ST5 (22.33%), and ST6 (16.50%) being the most common. A total of 45 virulence genes were detected, 22 of which were staphylococcal enterotoxin (SE) genes. Among the identified SE genes, selX exhibited the highest prevalence (86.4%). All isolates carried at least one SE gene. The results of the antimicrobial resistance (AMR) gene detection revealed 41 AMR genes of 12 classes. ß-lactam resistance genes (blal, blaR1, blaZ) and tetracycline resistance gene (tet(38)) exhibited a higher prevalence rate. Core genome single nucleotide polymorphism showed phylogenetic clustering of the isolates with the same region, year, and ST. The results indicated that the SFP isolates in southwest of China harbored multiple toxin and resistance genes, with a high prevalence of new SEs. Therefore, it is important to monitor the antimicrobial susceptibility and SE of S. aureus to reduce the potential risks to public health.

Epidemiological and molecular evidence of foodborne poisoning outbreak caused by enterotoxin gene cluster-harboring Staphylococcus aureus of new sequence type 7591.

OBJECTIVES: This paper presented a detailed analysis of the epidemiology and molecular characteristics of staphylococcal food poisoning (SFP) that occurred in a hotel in Hangzhou. METHODS: A total of 46 guests at the hotel underwent an epidemiological survey. Samples of stool from patients, vomit, swabs from the kitchen, leftover food items, and anal swabs from food handlers were taken and investigated for the presence of potential pathogenic bacteria. Molecular techniques and whole genome sequencing were performed to track the evolution of Staphylococcus aureus associated with the outbreak of SFP. RESULTS: Forty-six individuals displayed gastrointestinal symptoms. Seventeen isolates of S. aureus were discovered to carry the seg, sei, sem, sen, seo, and selu genes found in a specific enterotoxin gene cluster (egc) operon, but without the presence of classical enterotoxins such as SEA ∼ SEE. All egc-positive isolates shared identical pulsed-field gel electrophoresis profiles and were classified under new ST7591 (Clonal Complex 72) with identical spa typing t148. In addition, some isolates of S. aureus obtained from food sources sold in Hangzhou over the past 3 years and carrying egc genes were grouped under the ST72 lineage (CC72). Through whole genome sequencing, a strong genetic connection was revealed between these egc-positive isolates and clinical ST72 S. aureus found in China. CONCLUSIONS: S. aureus with non-classical egc enterotoxins was suggested to be a potential cause of SFP in humans.

Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay.

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases worldwide, resulting from the ingestion of staphylococcal enterotoxins (SEs), primarily SE type A (SEA), which is produced in food by enterotoxigenic strains of staphylococci, mainly S. aureus. Since newly identified SEs have been shown to have emetic properties and the genes encoding them have been found in food involved in poisoning outbreaks, it is necessary to have reliable tools to prove the presence of the toxins themselves, to clarify the role played by these non-classical SEs, and to precisely document SFP outbreaks. We have produced and characterized monoclonal antibodies directed specifically against SE type G, H or I (SEG, SEH or SEI respectively) or SEA. With these antibodies, we have developed, for each of these four targets, highly sensitive, specific, and reliable 3-h sandwich enzyme immunoassays that we evaluated for their suitability for SE detection in different matrices (bacterial cultures of S. aureus, contaminated food, human samples) for different purposes (strain characterization, food safety, biological threat detection, diagnosis). We also initiated and described for the first time the development of monoplex and quintuplex (SEA, SE type B (SEB), SEG, SEH, and SEI) lateral flow immunoassays for these new staphylococcal enterotoxins. The detection limits in buffer were under 10 pg/mL (0.4 pM) by enzyme immunoassays and at least 300 pg/mL (11 pM) by immunochromatography for all target toxins with no cross-reactivity observed. Spiking studies and/or bacterial supernatant analysis demonstrated the applicability of the developed methods, which could become reliable detection tools for the routine investigation of SEG, SEH, and SEI.

[Behavior of Staphylococcus aureus and Staphylococcal Enterotoxins A and Q in Scrambled Eggs].

Staphylococcal food poisoning (SFP) is caused by Staphylococcus aureus, and its typical symptom of vomiting is evoked by staphylococcal enterotoxins (SEs). SEs are classified as classical and new types. SEQ is a new-type enterotoxin predicted to have a high potential risk for SFP. To elucidate the correlation between the number of S. aureus cells and the production of SEs as well as classical and new-type enterotoxins in the food environment, the numbers of S. aureus strain cells carrying sea and seq genes and the production of SEA and SEQ protein were examined under 3 pHs values (pH 6.0, 7.0 and 8.0) and 2 NaCl concentrations (0.5 and 1.0%) conditions. The experiments were performed at 25℃, resembling the setting of scrambled eggs at room temperature after cooking. By 24 hr after incubation, the cell number in the scrambled egg was ≥107/10 g, reaching 109/10 g by 48 hr under all conditions. The productions of both SEA and SEQ were detected in the scrambled egg under all conditions by 48 h. SEQ was detected from 24 hr at all 3 pH values in the egg containing 1.0% NaCl, whereas in the egg containing 0.5% NaCl, it was detected from 24 hr at pH 6.0 and from 48 hr at other pHs. The SEQ production was consistently 100-1,000 times less than that of SEA. These results suggest that the new-type enterotoxin SEQ has the potential to evoke symptoms related to SFP following the consumption of egg products cooked under relative lower pH and water activity.

[Staphylococcal Enterotoxin A Production and Inactivation in Bread during the Production Processes].

Staphylococcus aureus food poisoning is caused by the intoxication of staphylococcal enterotoxin (SE) produced in foods. Staphylococcal food poisoning is mostly due to staphylococcal enterotoxin type A (SEA) among SEs. There have been many studies on the growth and SEA production of S. aureus in various foods, but few studies in bread. Thus, the SEA production by S. aureus in dough during fermentation and the SEA inactivation in dough during baking were studied in the normal production processes of bread in this study. No growth of S. aureus or SEA production in dough, whose total weight was about 470 g, was observed during the fermentation at 25 and 35℃ for four hr, suggesting that the risk of SEA production in dough during fermentation under these conditions would be negligible. Any SEA injected at 6.0 and 0.56 ng/g in dough could not be detected after 20 and 10 min of baking at 200℃, respectively. These results showed that the baking process, which was completed in 25 min, was enough to inactivate SEA at those doses of SEA in the dough. The results on the production and inactivation of SEA in dough during the production processes in this study would be useful information on microbiological food safety of bread making.

A novel staphylococcal enterotoxin SE02 involved in a staphylococcal food poisoning outbreak that occurred in Tokyo in 2004.

Staphylococcal enterotoxins (SEs) are extracellular proteins, produced mainly by Staphylococcus aureus, which cause staphylococcal food poisoning (SFP) when ingested. Here, a novel SE was identified from two strains, which were identified as the causative microbes of the SFP outbreak that occurred in Tokyo in 2004. Both strains harbored the SEA gene, but its production was lower than that of other SEA-producing SFP isolates. Whole-genome sequencing analysis demonstrated that both strains harbored a SE-like gene besides sea. Phylogenetic analysis revealed that the amino acid sequence deduced from the SE-like gene belonged to the SEB group. Therefore, this gene was presumed to be a novel SE gene and termed "SE02." The stability of SE02 against heating and proteolytic digestions was a little different from that of SEA. SE02 has both superantigenic and emetic bioactivities. Namely, SE02 activated mouse splenocytes and exhibited emetic activity in the common marmoset. SE02 mRNA was highly expressed in both isolates during the exponential phase of cultivation. In addition, SE02 protein was produced at 20 °C and 25 °C, which reflects the actual situation of SFP. SE02 appears to be a novel emetic toxin that was likely the causative toxin in combination with SEA in the SFP outbreak.

Staphylococcal Enterotoxin C-An Update on SEC Variants, Their Structure and Properties, and Their Role in Foodborne Intoxications.

Staphylococcal enterotoxins are the most common cause of foodborne intoxications (staphylococcal food poisoning) and cause a wide range of diseases. With at least six variants staphylococcal enterotoxin C (SEC) stands out as particularly diverse amongst the 25 known staphylococcal enterotoxins. Some variants present unique and even host-specific features. Here, we review the role of SEC in human and animal health with a particular focus on its role as a causative agent for foodborne intoxications. We highlight structural features unique to SEC and its variants, particularly, the emetic and superantigen activity, as well as the roles of SEC in mastitis and in dairy products. Information about the genetic organization as well as regulatory mechanisms including the accessory gene regulator and food-related stressors are provided.

Growth kinetics of Staphylococcus aureus and background microorganisms in camel milk.

Staphylococcus aureus is a common foodborne pathogen that is ubiquitous in nature. Consumption of contaminated foods, such as dairy products, can lead to food poisoning caused by heat-stable staphylococcal toxins that are not easily destroyed during pasteurization. The objective of this study was to investigate the growth kinetics of S. aureus and background microorganisms in camel milk stored at different temperatures between 8 and 43°C using one-step kinetic analysis to estimate the kinetic parameters from the observed growth curves. The growth of S. aureus showed apparent lag, exponential, and stationary phases, whereas no or negligible lag phase was observed for background microorganisms. Data analysis showed that the estimated minimum, optimum, and maximum growth temperatures were 5.9, 42.0, and 49.2°C for S. aureus, and 3.0, 38.6, and 49.2°C for the background microorganisms, respectively. The estimated optimum specific growth rate was higher for S. aureus (1.24 h-1) than for background microorganisms (0.995 h-1). This study found that camel milk may inhibit the growth of S. aureus, as it exhibits a lower specific growth rate than that in cow milk or cooked potato. It also has a longer lag phase than that in cow milk at comparable temperature ranges. This unique property is probably related to the presence of some antimicrobial compounds naturally occurring in camel milk. Validation of kinetic parameters and models showed that the root mean square error of prediction was only 0.5 log cfu/mL for S. aureus and background microorganisms, suggesting that the models are reasonably accurate. These models can be used for conducting risk assessments of S. aureus and predicting the general microbiological shelf life of camel milk to prevent foodborne staphylococcal poisoning.

Molecular detection of methicillin heat-resistant Staphylococcus aureus strains in pasteurized camel milk in Saudi Arabia.

Antibiotic- and heat-resistant bacteria in camel milk is a potential public health problem. Staphylococcus aureus (S. aureus) is an opportunistic pathogen in humans, dairy cattle and camels. We characterized the phenotype and genotype of methicillin-resistant staphylococcal strains recovered from pasteurized and raw camel milk (as control) distributed in the retail markets of Saudi Arabia. Of the 100 samples assessed between March and May 2016, 20 S. aureus isolates were recovered from pasteurized milk, 10 of which were resistant to cefoxitin, and as such, were methicillin-resistant. However, raw camel milk did not contain methicillin-resistant S. aureus (MRSA). Antimicrobial susceptibility tests showed that the resistance ratio for other antibiotics was 60%. We performed a polymerase chain reaction (PCR) assay using primers for the methicillin-resistant gene mecA and nucleotide sequencing to detect and verify the methicillin-resistant strains. Basic local alignment search tool (BLAST) analysis of the gene sequences showed a 96-100% similarity between the resistant isolates and the S. aureus CS100 strain's mecA gene. Ten of the methicillin-resistant isolates were heat-resistant and were stable at temperatures up to 85°C for 60 s, and three of these were resistant at 90°C for 60 or 90 s. The mean decimal reduction time (D85-value) was 111 s for the ten isolates. Sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis (PAGE) showed that there was no difference in the total protein profiles for the ten methicillin heat-resistant S. aureus (MHRSA) isolates and for S. aureus ATCC 29737. In conclusion, a relatively high percentage of the tested pasteurized camel milk samples contained S. aureus (20%) and MHRSA (10%).

Virulence factors and antimicrobial resistance of Staphylococcus aureus isolated from the production process of Minas artisanal cheese from the region of Campo das Vertentes, Brazil.

Staphylococcus aureus is one of the main pathogens found in cheeses produced with raw milk, including Minas artisanal cheese from Brazil. However, information about S. aureus isolated from artisanal cheeses and its sources of production in small-scale dairies is very limited. We aimed to characterize the virulence factors of S. aureus isolated from raw milk, endogenous starter culture, Minas artisanal cheese, and cheese handlers from the region of Campo das Vertentes, Minas Gerais, Brazil. We identified the staphylococcal isolates by MALDI-TOF mass spectrometry. We evaluated biofilm production on Congo red agar and polystyrene plates. We used PCR to detect icaA, icaB, icaC, sea, seb, sec, sed, see, tsst-1, agr, and mecA. We evaluated the expression of staphylococcal toxin genes in PCR-positive staphylococcal isolates using quantitative reverse-transcription PCR, and we evaluated the production of these toxins and their hemolytic activity in vitro. We also evaluated the antimicrobial resistance profile of the staphylococcal isolates. For statistical analysis, we used cluster analysis, χ2 tests, and correspondence tests. We analyzed 76 staphylococcal isolates. According to PCR, 18.42, 18.42, 2.63, and 77.63% were positive for sea, tsst-1, sec, and agr, respectively. We found low expression of staphylococcal toxin genes according to quantitative reverse-transcription PCR, and only 2 staphylococcal isolates produced toxic shock syndrome toxins. A total of 43 staphylococcal isolates (56.58%) had hemolytic activity; 53 were biofilm-forming on Congo red agar (69.73%), and 62 on polystyrene plates (81.58%). None of the staphylococcal isolates expressed the mecA gene, and none presented a multi-drug resistance pattern. The highest resistance was observed for penicillin G (67.11%) in 51 isolates and for tetracycline (27.63%) in 21 isolates. The staphylococcal isolates we evaluated had toxigenic potential, with a higher prevalence of sea and tsst-1. Biofilm production was the main virulence factor of the studied bacteria. Six clusters were formed whose distribution frequencies differed for hemolytic activity, biofilm formation (qualitative and quantitative analyses), and resistance to penicillin, tetracycline, and erythromycin. These findings emphasize the need for effective measures to prevent staphylococcal food poisoning by limiting S. aureus growth and enterotoxin formation throughout the food production chain and the final product.

Genetic Diversity of Composite Enterotoxigenic Staphylococcus epidermidis Pathogenicity Islands.

The only known elements encoding enterotoxins in coagulase-negative staphylococci are composite Staphylococcus epidermidis pathogenicity islands (SePIs), including SePI and S. epidermidis composite insertion (SeCI) regions. We investigated 1545 Staphylococcus spp. genomes using whole-genome MLST, and queried them for genes of staphylococcal enterotoxin family and for 29 ORFs identified in prototype SePI from S. epidermidis FRI909. Enterotoxin-encoding genes were identified in 97% of Staphylococcus aureus genomes, in one Staphylococcus argenteus genome and in nine S. epidermidis genomes. All enterotoxigenic S. epidermidis strains carried composite SePI, encoding sec and sel enterotoxin genes, and were assigned to a discrete wgMLST cluster also containing genomes with incomplete islands located in the same region as complete SePI in enterotoxigenic strains. Staphylococcus epidermidis strains without SeCI and SePI genes, and strains with complete SeCI and no SePI genes were identified but no strains were found to carry only SePI and not SeCI genes. The systematic differences between SePI and SeCI regions imply a lineage-specific pattern of inheritance and support independent acquisition of the two elements in S. epidermidis. We provided evidence of reticulate evolution of mobile elements that contain elements with different putative ancestry, including composite SePI that contains genes found in other coagulase-negative staphylococci (SeCI), as well as in S. aureus (SePI-like elements). We conclude that SePI-associated elements present in nonenterotoxigenic S. epidermidis represent a scaffold associated with acquisition of virulence-associated genes. Gene exchange between S. aureus and S. epidermidis may promote emergence of new pathogenic S. epidermidis clones.

Genotypes, Enterotoxin Gene Profiles, and Antimicrobial Resistance of Staphylococcus aureus Associated with Foodborne Outbreaks in Hangzhou, China.

Staphylococcal food poisoning is an illness caused by the consumption of food that contains sufficient amounts of one or more enterotoxins. In the present study, a total of 37 S. aureus isolates were recovered from leftover food, swabs from a kitchen environment, and patient feces associated with four foodborne outbreaks that occurred in Hangzhou, southeast China, and were characterized by multilocus sequence typing (MLST), spa typing, pulse-field gel electrophoresis (PFGE), and antimicrobial susceptibility. Classical enterotoxin and enterotoxin-like genes were profiled by PCR analysis. ST6-t304 was the most common clone (40.54%), followed by ST2315-t11687 (32.43%). Six clusters (A to F) were divided based on PFGE patterns, and Clusters A and C were the most common types, constituting 86.49% of all isolates. Moreover, sea was the most frequently identified enterotoxin gene (81.08%), followed by the combination of seg-sei-selm-seln-sleo-selu and sec-sell (each 48.65%). Five isolates also harbored the exotoxin cluster sed-selj-ser. In addition, resistance to penicillin (97.30%), erythromycin (37.85), tetracycline (32.43%), clindamycin, gentamicin, and sulfamethoxazole (each 10.81%) was observed. Our research demonstrated the link between leftover foods and patients by molecular typing and detecting the profiles of enterotoxin or enterotoxin-like genes in human and food isolates. S. aureus maintains an extensive repertoire of enterotoxins and drug resistance genes that could cause potential health threats to consumers.

The occurrence of Staphylococcus aureus, enterotoxigenic and methicillin-resistant strains in Iranian food resources: a systematic review and meta-analysis.

INTRODUCTION: Staphylococcus aureus, in particular its enterotoxigenic strains, is a well-known pathogen in food microbiology as one of the most common causes of food poisoning. This study aimed to investigate the prevalence of S. aureus enterotoxigenic strains in Iranian food resources by using a systematic review and meta-analysis. METHODS: A systematic search was performed using electronic databases from papers that were published by Iranian authors to the end of July 2017. Then, 34 publications which met our inclusion criteria were selected for data extraction and analysis. RESULTS: The average pooled prevalence of S. aureus in food resources was 15.5% (95% CI: 12.7%-18.9%) ranging from 1.1% to 52%. According to the examined publications the estimated average prevalence of enterotoxigenic strains was estimated at 53.7% (95% CI: 41.4%-65.6%) ranging from 13% to 83.3%. The average prevalence of methicillin-resistant S. aureus in contaminated resources was estimated at 15.5% (95% CI: 11.1%-21.4%) ranging from 0% to 32%. The results showed that SEA and SEG were the most prevalent classic and new staphylococcal enterotoxins' (SEs) types. The results of antibiotic resistance pattern showed the highest resistance rates to ß-lactams, tetracycline and erythromycin. CONCLUSIONS: The present analysis showed a significant presence of enterotoxigenic strains of S. aureus and a high rate of staphylococcal enterotoxins among food resources in Iran. However, also the prevalence of MRSA strains is remarkable and requires an improvement of the hygienic conditions to reduce the risk of MRSA dissemination in community and hospitals. Furthermore, the rational use of antibiotics in the environments related to food resources is recommended.

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

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

Effects of Temperature Abuse on the Growth and Staphylococcal Enterotoxin A Gene (sea) Expression of Staphylococcus aureus in Milk.

The aim of this study was to determine the effects of different temperatures and storage time on Staphylococcus aureus growth, sea gene expression, and synthesis of staphylococcal enterotoxin A (SEA) in the pasteurized and UHT-pasteurized milk. Pasteurized and UHT-pasteurized milk were inoculated with 3.98 log10 CFU/mL of S. aureus (ATCC 13565). Inoculated milk samples were stored at 8°C, 15°C, and 22°C for 24, 48, and 72 h, respectively. SEA synthesis was detected with a fully automated miniVIDAS instrument using the Enzyme-Linked Fluorescent Assay (ELFA) technology. The patterns of gene regulation were detected by quantitative reverse transcriptase PCR. The 2-ΔΔCT method has been used as a relative quantification strategy for gene expression responses data analysis. The results indicated that growth rate, sea gene expression, and SEA synthesis were influenced by type of milk, storage time, and temperature. Incubation of milk at different temperatures (15°C and 22°C) and times was used to simulate inadequate transport and storage conditions. Storage of pasteurized milk at 22°C for 24 h significantly upregulated the expression of sea gene compared with milk stored at 8°C, which coincides with the achieved S. aureus number of 105 CFU/mL and detected amount of SEA. In addition, storage of UHT-pasteurized milk at 22°C for 24 h and at 15°C for 48 h significantly upregulated the sea gene expression compared with milk stored at 8°C, which coincides with the detected amount of SEA and the dynamics of S. aureus number change. It can, therefore, be concluded that implementing good hygiene practices to avoid pre- and post-heat treatment milk contamination and maintaining the cold chain at temperature <8°C throughout the entire dairy production chain are of paramount importance to decrease the risk of staphylococcal food poisoning.

Prevalence of Enterotoxin Genes in Staphylococcus aureus Isolates 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.

The Validation of the Dai Nippon Medi·Ca SA Method for AOAC Research Institute Performance Tested MethodsSM Certification.

A ready-made dry medium method for Staphylococcus aureus count, the Medi·Ca SA method incubated at 35 or 37°C, was compared with the Baird-Parker method (AOAC Official MethodSM 975.55) for 11 food matrices: raw beef, raw ground beef, raw lamb, cooked ham, raw salmon, frozen prawn, fresh chilled pasta, pasteurized milk, natural cheese, cream puff, and potato salad. The mean difference between the two methods at each contamination level for each matrix was <0.5 log10, and the 95% confidence intervals on the mean differences fell within the range of -0.50 to 0.50. Standard deviation of repeatability and RSDr values of the Medi·Ca SA method were generally the same level as those of the Baird-Parker method, and r2 ranged from 0.98 to 1.00. Product consistency and stability studies showed little variability between productions lots and a shelf-life of 16 months. Incubation time within the range of 22-26 h and variations to the sample volume did not adversely affect the results. These results showed that the Medi·Ca SA method is a reasonable alternative to the reference method for selected food matrices and makes it possible to simultaneously detect and enumerate S. aureus in only 24 h.

Analysis of the VIDAS® Staph Enterotoxin III (SET3) for Detection of Staphylococcal Enterotoxins G, H, and I in Foods.

BACKGROUND: Staphylococcal food poisoning (SFP) frequently causes illnesses worldwide. SFP occurs from the ingestion of staphylococcal enterotoxins (SEs) preformed in foods by enterotoxigenic strains of Staphylococcus species, primarily S. aureus. SEG, SEH, and SEI induce emesis and have been implicated in outbreaks. Immunological-based methods are deemed the most practical methods for the routine analysis of SEs in foods given their ease of use, sensitivity, specificity, and commercial availability. These kits are routinely used to test for SEA-SEE. However, only recently has a kit been developed to detect SEG, SEH, and SEI. OBJECTIVE: Our research examined the performance of the novel VIDAS® Staph Enterotoxin III (SET3) for the detection of staphylococcal enterotoxins SEG, SEH, and SEI in foods. METHODS: Here we assess the sensitivity and specificity of SET3 using duplicate test portions of six foods at varying concentrations of inclusivity and exclusivity inocula: pure SEG, SEH, SEI, S. aureus strain extracts positive for seg, seh, and sei, as well as SEA, SEB, SEC, SED, and SEE. RESULTS: The overall detection limit was less than 2.09 ng/mL for foods inoculated with SEG, SEH, and SEI, with no cross reactivity observed. HIGHLIGHTS: Integrating concurrent testing to detect the presence of SEA-SEE and SEG-SEI utilizing the SET3 along with the VIDAS SET2, Ridascreen® SET total, or other comparable kits will be instrumental for the future food assessments in our laboratory and may become the new standard for SE analysis of foods.