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.

Dicloxacilina en infecciones producidas por estafilococo productor de penicilinasa / Dicloxacillin in infections caused by penicillinase-producing staphylococcus

INTRODUCCIÓN: El amplio arsenal terapéutico destinado al tratamiento de las enfermedades infecciosas disponible en la actualidad hace imprescindible la realización de una selección rigurosa del agente antimicrobiano que se va a utilizar según una serie de criterios que permitan un uso adecuado de éstos. En primer lugar, habría que valorar la necesidad de instaurar un tratamiento antibiótico de acuerdo con los hallazgos clínicos del paciente. Una vez considerada esta necesidad, los criterios de selección del antimicrobiano más adecuado se deben hacer en función de los siguientes criterios: gravedad y estado general de base del paciente, consideración de los microorganismos más probables según el foco de la infección, el conocimiento del estado actual de resistencias de los posibles microorganismos implicados, la farmacocinética y la farmacodinámica de los antibióticos, los efectos secundarios y el coste de los antimicrobianos, la vía de administración y la duración del tratamiento. Las infecciones de piel y partes blandas están entre las infecciones más frecuentes en Pediatría. Los microorganismos implicados con mayor frecuencia son Staphylococcus aureus (S. aureus) y Streptococcus pyogenes (S. pyogenes). En ciertas situaciones deberá sospecharse que el S. aureus sea resistente a la meticilina (SARM). Las infecciones bacterianas de la piel pueden ocurrir a diferentes niveles de profundidad implicando la epidermis (impétigo), la dermis (ectima, erisipela) o el tejido celular subcutáneo (celulitis, abscesos subcutáneos), mientras que las infecciones de partes blandas se extenderán a mayor profundidad y afectarán a la fascia (fascitis necrotizante) o el músculo (piomiositis). El tratamiento antibiótico se debe iniciar de forma empírica cubriendo S. aureus y S. pyogenes y añadiendo un antibiótico con actividad inhibidora de la síntesis de toxinas cuando sea necesario. OBJETIVO: Evaluar rapidamente los parâmetros de eficácia, seguridade, costos y recomendaciones disponibles acerca del empleo de Dicloxacilina sódica para el tratamiento de personas con infecciones por gérmenes gram positivos, incluyendo estafilococos produtores de betalactamasa. METODOLOGÍA: Se realizo uma búsqueda bibliográfica en las principales bases de datos tales como PUBMED, LILACS, BRISA, COCHRANE, SCIELO, EMBASE, TRIPDATABASE como así también em sociedades científicas, agencias reguladoras, financiadores de salud y agencias de evaluación de tecnologias sanitárias. Se priorizó la inclusión de revisiones sistemáticas, ensayos clínicos controlados aleatorizados, evaluación de tecnologia sanitária y guias de práctica clínica. CONCLUSIONES: La Dicloxacilina, un antibiótico de la clase de las penicilinas, ha demostrado ser efectiva en el tratamiento de diversas infecciones bacterianas. Su eficacia radica en su capacidad para inhibir la síntesis de la pared celular bacteriana, lo que lleva a la muerte de las bacterias sensibles. Se utiliza comúnmente para tratar infecciones causadas por bacterias grampositivas, como Staphylococcus aureus, que son resistentes a la penicilina debido a la producción de la enzima beta-lactamasa. La Dicloxacilina es especialmente útil en el tratamiento de infecciones de la piel y tejidos blandos, como celulitis, forúnculos y abscesos, así como infecciones de heridas quirúrgicas y estafilococos en la piel. Alrededor del 5% de los pacientes tratados pueden esperar reacciones adversas. Los efectos adversos más comunes son náuseas, vómitos y diarrea. Como con otras penicilinas, se han producido reacciones de hipersensibilidad (inmediatas o tardías). La mayoría de estas son leves, sin embargo, se han reportado reacciones de hipersensibilidad graves, incluida la muerte (Aunque en extremo raras). Al costo aportado en solicitud, la tecnología tiene un impacto presupuestario mayor que los códigos disponibles actualmente en el Listado Oficial de Medicamentos (LOM). Actualmente con la concentración solicitada (250 mg/5 mL) existen 5 registros sanitarios activos, aunque su corta vida media obliga a administrarla con estrechos intervalos de dosificación (cada 6 horas) y el sabor de la solución oral es muy desagradable, lo que dificulta mucho el cumplimiento del tratamiento.[1] Las Guías Clínicas de Pediatria consideran tanto Cefadroxilo y Dicloxacilina como opciones para infecciones de la piel y tejidos blancos, y en otras alternativas terapéuticas se consideran Amoxicilina + Ácido clavulánico, Clindamicina y Claritromicina. Para el tratamiento de infecciones causadas por bacterias productoras de penicilinasa, neumococos del grupo A-beta, estreptococos hemolíticos y cepas de estafilococos resistentes a la penicilina G, se pueden considerar varias opciones terapéuticas. Algunas de estas opciones incluyen: Antibióticos antiestafilocócicos: Entre ellos se incluyen la oxacilina, la meticilina, la nafcilina y doxiciclina. Estos antibióticos son resistentes a la acción de la penicilinasa, una enzima producida por las bacterias estafilococos que inactiva la penicilina. Son eficaces contra infecciones causadas por estafilococos resistentes a la penicilina y a la meticilina. Cefalosporinas de primera generación, como la cefalexina y la cefadroxilo, pueden ser una opción de tratamiento para infecciones causadas por estafilococos productores de penicilinasa, neumococos del grupo A-beta, estreptococos hemolíticos y algunas cepas de estreptococos resistentes a la penicilina G. Estas cefalosporinas tienen actividad contra una amplia variedad de bacterias grampositivas y algunas gramnegativas, y son especialmente útiles en infecciones leves a moderadas de la piel y tejidos blandos, infecciones del tracto urinario y faringitis estreptocócica. Cefalosporinas de segunda generación: Las cefalosporinas como la cefuroxima, la cefaclor y la cefprozil pueden ser efectivas contra estreptococos y algunos tipos de estafilococos resistentes a la penicilina. Sin embargo, su eficacia contra cepas productoras de penicilinasa puede ser limitada. Macrólidos: Los macrólidos como la eritromicina, la claritromicina y la azitromicina pueden ser útiles en el tratamiento de infecciones causadas por estreptococos y neumococos. Sin embargo, algunos estafilococos pueden desarrollar resistencia a estos antibióticos, por lo que su uso puede ser limitado en infecciones estafilocócicas graves. Vancomicina: La vancomicina es un antibiótico que se utiliza para tratar infecciones causadas por estafilococos resistentes a la meticilina y otros microorganismos grampositivos. Es un agente de reserva que se reserva para infecciones graves o resistentes a otros antibióticos. En el LOM, se encuentra al menos un representante de casi todos los grupos de antibióticos mencionados, con formulaciones pediátricas disponibles en jarabe o suspensión (Cefadroxilo, Claritromicina, Amoxicilina + ácido clavulánico). En los casos en que no se dispone de estas formulaciones, como con la doxiciclina para niños mayores de 8 años o clindamicina, se pueden emplear las presentaciones en tabletas o cápsulas. Además, algunas formulaciones endovenosas, como la clindamicina, oxacilina e incluso la vancomicina, están disponibles para su uso en casos que lo requieran.

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.

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.

A comprehensive review on the detection of Staphylococcus aureus enterotoxins in food samples.

Staphylococcal enterotoxins (SEs), the major virulence factors of Staphylococcus aureus, cause a wide range of food poisoning and seriously threaten human health by infiltrating the food supply chain at different phases of manufacture, processes, distribution, and market. The significant prevalence of Staphylococcus aureus calls for efficient, fast, and sensitive methods for the early detection of SEs. Here, we provide a comprehensive review of the hazards of SEs in contaminated food, the characteristic and worldwide regulations of SEs, and various detection methods for SEs with extensive comparison and discussion of benefits and drawbacks, mainly including biological detection, genetic detection, and mass spectrometry detection and biosensors. We highlight the biosensors for the screening purpose of SEs, which are classified according to different recognition elements such as antibodies, aptamers, molecularly imprinted polymers, T-cell receptors, and transducers such as optical, electrochemical, and piezoelectric biosensors. We analyzed challenges of biosensors for the monitoring of SEs and conclude the trends for the development of novel biosensors should pay attention to improve samples pretreatment efficiency, employ innovative nanomaterials, and develop portable instruments. This review provides new information and insightful commentary, important to the development and innovation of further detection methods for SEs in food samples.

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.

Effect of environmental factors on expression of staphylococcal enterotoxin genes.

Staphylococcal enterotoxins (SEs) secreted by Staphylococcus aureus (S. aureus) can cause foodborne disease, nausea, vomiting and diarrhea, and even death. Regulation of SE expression is related to accessory gene regulators (Agr). It is important to reveal which environmental factors influence regulation of SE expression to prevent SE food poisoning outbreak. Hence, natural environmental factors which may have an impact on SE expression were selected, such as temperature, food types, strains, and competing strains. Seven strains of S. aureus carrying different SE genes were collected from the Chinese Academy of Inspection and Quarantine (CAIQ) strain bank for study. Strains were cultured with different conditions. Temperature was 8 °C, 22 °C, and 30 °C. Food type was milk powder and nutrient broth. Competing strains were Vibrio parahaemolyticus (V. parahaemolyticus), Escherichia coli (E. coli), and Bacillus cereus (B. cereus). The expression culture solution was pretreated by centrifugation, then determined by using SDS-PAGE, and distinguished SEs apart from each other by HPLC-ESI-TOF. There are 168 samples collected from SE expression culture; the result of SDS-PAGE suggests 23 samples were positive for SEs, and the other 145 samples were negative for SEs. The result of HPLC-ESI-TOF suggests that SEs with similar molecular weight can be distinguished in terms of m/z. The most important factor contributing to regulate expression of SEs was estimated by logistic regressive analysis. The result shows that McFadden R2 is 0.213; p value is 0.000 (p < 0.05); this result illustrates that the model is valid and meaningful. Strains, food types, temperature, and competing strands can explain the 21% change in SE expression. Temperature (z = 3.029, p = 0.002 < 0.01), strains (z = - 3.132, p = 0.002 < 0.01), and food types (z = - 2.415, p = 0.016 < 0.05) have significant impact on SE expression, and the competing strains (z = 1.230, p = 0.219 > 0.05) have no impact on the SE expression. More important impact on SE expression was estimated by OR value; the result shows that strength of temperature influencing on SE expression is bigger than strains and food types in terms of values of OR, temperature (OR = 2.862), strains (OR = 0.641), and food types (OR = 0.561); consequently, temperature is a key factor for stimulating SE expression and had high expression at 30 °C. Therefore, food easily contaminated with S. aureus should be monitored intensively at early and late summer, when proper temperature for expressing SEs may result in S. aureus food poisoning prevalence.

Stability and emetic activity of enterotoxin like X (SElX) with high carrier rate of food poisoning Staphylococcus aureus.

In order to analyze and clarify the thermal stability of food poisoning Staphylococcus aureus (S. aureus) enterotoxin-like X (SElX) and the biological characteristics of digestive enzymes, and to evaluate the risk of S. aureus carrying selx gene in food poisoning, the selx gene carrying rates of 165 strains isolated from 95 food poisoning events from 2006 to 2019 were first statistically analyzed. Subsequently, the purified recombinant SElX protein was digested and heated, and the superantigen activity was verified with mouse spleen cells and peripheral blood mononuclear cells of kittens. At the same time, the emetic activity and toxicity of SElX were evaluated using the kitten vomiting animal model, mice toxin model and in vitro cell models. The results showed the selx gene carrying rate of 165 food poisoning S. aureus strains was 90.30 %. SElX had significant resistance to heat treatment and pepsin digestion (pH = 4.0 and pH = 4.5), and had good superantigen activity and emetic activity. However, there is no significant lethal effect on mice and no significant toxicity to cells. Importantly, we found that SElX had an inhibitory effect on acidic mucus of goblet cells in various segments of the small intestine. The present study investigated the stability of SElX, and confirmed the emetic activity of SElX by establishing a kitten vomiting model for the first time, suggesting that SElX is a high risk toxin of food poisoning, which will provide new ideas for the prevention and control of S. aureus food poisoning.

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.

Stress Lowers Staphylococcal Enterotoxin C Production Independently of Agr, SarA, and SigB.

Staphylococcal enterotoxin C (SEC) can cause staphylococcal food poisoning, one of the most prevalent foodborne intoxications. It is produced by Staphylococcus aureus during growth in the food matrix. While the surrounding bacteria in food matrices usually repress the growth of S.aureus, the organism possesses a remarkable growth advantage under stressful conditions encountered in many foods. Examples for such food matrices are pastry and bakery products with their high sugar content that lowers water availability. While S. aureus can still grow in these challenging environments, it remains unclear how these conditions affect SEC expression. Here, the influence of 30% glucose on sec mRNA in a qPCR assay and SEC protein expression was investigated for the first time in an ELISA. In addition, regulatory knockout mutants Δagr, ΔsarA, and ΔsigB were generated to investigate regulatory gene elements in glucose stress. In five out of seven strains, glucose stress led to a pronounced decrease in sec mRNA transcription and SEC protein levels were substantially lower under glucose stress. It could be shown that key regulatory elements Δagr, ΔsarA, and ΔsigB in strain SAI48 did not contribute to the pronounced downregulation under glucose stress. Based on these findings, glucose effectively lowers SEC synthesis in the food matrix. However, the mechanism by which it acts on toxin expression and regulatory elements in S. aureus remains unclear. Future studies on other regulatory elements and transcriptomics may shed light on the mechanisms.

Investigation of a Staphylococcus aureus sequence type 72 food poisoning outbreak associated with food-handler contamination in Italy.

On August 2019 a staphylococcal food poisoning outbreak occurred in an elderly home in Piedmont, Italy. The epidemiological investigation performed among the persons that consumed the meal identified chicken salad as the most likely source of the outbreak. Staphylococcus aureus was isolated from a total of seven samples, namely one vomit sample from a guest of the nursing home, two food samples (chicken salad with and without mayonnaise) and nasal swabs collected from a total of four persons working in the kitchen of the nursing home. The maximum likelihood tree obtained using single nucleotide polymorphisms analysis revealed that the isolates from the aforementioned samples clustered together. Multilocus sequence typing revealed that they belonged to Sequence Type 72. Fourier transform infrared spectroscopy (FTIR) was used in parallel to single nucleotide polymorphisms and whole genome sequencing for the determination of the degree of relatedness of the isolates. The results of the FTIR showed the same clustering obtained with single nucleotide polymorphisms and whole genome sequencing and revealed the source of infection. This study underlines the importance of both laboratory evidence and epidemiological data for outbreak investigation and further confirms that FTIR is a suitable support for the short-term epidemiological investigation on source attribution in case of a S. aureus infection.

First report of enterotoxigenic Staphylococcus argenteus as a foodborne pathogen.

Staphylococcal enterotoxins preformed in food are the causative agents of staphylococcal food poisoning outbreaks (SFPO). In this study we characterised in depth two coagulase-positive non-pigmented staphylococci involved in two independent outbreaks that occurred in France. While indistinguishable from Staphylococcus aureus using PCR methods and growth phenotype comparisons, both isolates were identified as Staphylococcus argenteus by whole genome sequencing. The genomes were analysed for the presence of enterotoxin genes, whose expression was determined in laboratory medium and, for the first time, in artificially-contaminated milk samples by using liquid chromatography-mass spectrometry and ELISA methods. The concentration measured for the SEB toxin in milk (0.67 ng/ml) was comparable to concentrations reported for other types of enterotoxins behind SFPO. From a collection of publicly available genomes, we performed an unprecedented systematic investigation of the enterotoxin gene set of S. argenteus, including variants and pseudogenes. The most prevalent genes were sex, followed by sel26, sel27 and sey. The egc cluster was less frequent and most of the time carried a dysfunctional seg gene. Our results shed light on the enterotoxigenic properties of S. argenteus, and emphasize the importance in monitoring of S. argenteus as an emerging foodborne pathogen.

Prevalence of staphylococcal toxin in food contaminated by Staphylococcus spp.: Protocol for a systematic review with meta-analysis.

BACKGROUND: Food contamination by Staphylococcus spp. enterotoxigenic strains is quite common and despite underreporting caused by the short duration of clinical symptoms and lack of medical care, staphylococcal food poisoning is one of the most common Foodborne Diseases (FBD) in the world. This study describes a systematic review protocol with meta-analysis on the prevalence and types of staphylococcal enterotoxins in food, and the profile of contaminated foods. METHODS: The research will be conducted through the selection of studies reporting the analysis of staphylococcal enterotoxins in food contaminated by Staphylococcus spp. Searches will happen on the following databases: Medline (OVID), GALE, Science Direct, CAB Direct (CABI), Google Scholar, in addition to manual search in the list of references of articles, directory of theses and dissertations, and countries' health agencies. Reports will be imported into the application Rayyan. Two researchers will independently select studies and extract data, and a third reviewer will solve conflicting decisions. The primary outcome will be the identification of staphylococcal enterotoxins in food, and the secondary outcomes will include staphylococcal enterotoxin types and foods involved. To assess the risk of bias in the studies, the tool developed by the Joanna Briggs Institute (JBI) will be used. For data synthesis, a meta-analysis will be performed. However, in case that is not possible, a narrative synthesis of the most relevant results will be carried out. DISCUSSION: This protocol will serve as the basis for a systematic review that aims to relate the results of existing studies on the staphylococcal enterotoxin prevalence and types in food, and the profile of the contaminated foods. The results will broaden the perception of food safety risks, highlight existing literature gaps, contribute to the study of the epidemiological profile and may guide the allocation of health resources for the development of preventive measures related. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration number: CRD42021258223.

Microcalorimetric Investigations of Reversible Staphylococcal Enterotoxin Unfolding.

Staphylococcal food poisoning (SFP) is a common food-borne illness often associated with contamination during food handling. The genes for Staphylococcal enterotoxin (SE) isoforms SEA and SEB are frequently detected in human nasal Staphylococcus aureus isolates and these toxins are commonly associated with SFP. Past studies described the resistance of preformed SE proteins to heat inactivation and their reactivation upon cooling in foods. Full thermodynamic analyses for these processes have not been reported, however. The thermal stabilities of SEA, SEB, and SEH and reversibility of unfolding in simple buffers were investigated at pH 4.5 and pH 6.8 using differential scanning calorimetry (DSC). SEA and SEB unfolding was irreversible at pH 6.8 and at least partially reversible at pH 4.5 while SEH unfolding was irreversible at pH 4.5 and reversible at pH 6.8. Additional studies showed maximum refolding for SEB at pH 3.5-4.0 and diminished refolding at pH 4.5 with increasing ionic strength. SE-stimulated secretion of interferon-gamma by human peripheral blood mononuclear cells was used to assess residual SE biological activity following heat treatments using conditions matching those used for DSC studies. The biological activities of SEB and SEH exhibited greater resistance to heat inactivation than that of SEA. The residual activities of heat-treated SEB and SEH were measurable but diminished further in the presence of reconstituted nonfat dry milk adjusted to pH 4.5 or pH 6.8. To different extents, the pH and ionic strengths typical for foods influenced the thermal stabilities of SEA, SEB, and SEH and their potentials to renature spontaneously after heat treatments.

Multiplex Detection of 24 Staphylococcal Enterotoxins in Culture Supernatant Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Staphylococcal food poisoning outbreaks are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SEs). Among the 27 SEs described in the literature to date, only a few can be detected using immuno-enzymatic-based methods that are strongly dependent on the availability of antibodies. Liquid chromatography, coupled to high-resolution mass spectrometry (LC-HRMS), has, therefore, been put forward as a relevant complementary method, but only for the detection of a limited number of enterotoxins. In this work, LC-HRMS was developed for the detection and quantification of 24 SEs. A database of 93 specific signature peptides and LC-HRMS parameters was optimized using sequences from 24 SEs, including their 162 variants. A label-free quantification protocol was established to overcome the absence of calibration standards. The LC-HRMS method showed high performance in terms of specificity, sensitivity, and accuracy when applied to 49 enterotoxin-producing strains. SE concentrations measured depended on both SE type and the coagulase-positive staphylococci (CPS) strain. This study indicates that LC-MS is a relevant alternative and complementary tool to ELISA methods. The advantages of LC-MS clearly lie in both the multiplex analysis of a large number of SEs, and the automated analysis of a high number of samples.

Molecular markers for the detection of pathogenic and food poisoning potential of methicillin resistant Staphylococcus aureus isolated from wounds of hospitalized patients.

Pathogenic strains of Staphylococcus aureus are mostly resistant to methicillin and they can cause severe infections. The current study was planned to assess the food poisoning potential of pathogenic, methicillin resistant Staphylococcus aureus by molecular detection of enterotoxin A (Eta) gene. A total of 100 septic wound samples from patients admitted in surgical ward (n=50) and burn unit (n=50) of Mayo Hospital Lahore were collected aseptically. These samples were processed primarily for bacterial growth on nutrient agar and purified on mannitol salt agar where twenty (20) samples showed pin-point colonies with yellow discoloration of media. Moreover, isolates were further characterized on the basis of microscopic appearance and biochemical assays where fourteen (14) isolates were declared Staphylococcus. DNA of these isolates were subjected to 16S rRNA gene amplification and sequences of S. aureus were submitted to NCBI GenBank viz., MW344063.1, MW341438.1, MW344064.1, MW344065.1, MW341439.1, MW341440.1, MW345971.1, MW345972.1, MW345973.1, MW716458.1. All the isolates (n=10) demonstrated molecular confirmation of pathogenicity and methicillin resistance by amplification of Coa and mecA gene. Out of these ten isolates, three amplified enterotoxin A (Eta) gene were confirmed. It is concluded that enterotoxin A of S. aureus which causes food poisoning is present in pathogenic, methicillin resistant S. aureus isolated from various wounds infections.

Ex Vivo and In Vitro Methods for Detection of Bioactive Staphylococcal Enterotoxins.

Staphylococcus aureus is a major bacterial cause of clinical infections and foodborne illnesses.Through the synthesis of a group of Staphylococcal enterotoxins (SEs), gastroenteritis occurs and the SEs function as superantigens to massively activate T cells. The ability to rapidly detect and quantify SEs is imperative in order to learn the causes of staphylococcal outbreaks and to stop similar outbreaks in the future. Also, the ability to discern active toxin is essential for development of food treatment and processing methods. Here, we discuss the various methodologies for detection and analysis of SEs.

Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey / Caracterização genômica perfis de resistência antimicrobiana, produção de enterotoxinas e biofilme Staphylococcus aureus resistente à meticilina isolado de origem clínica e de produtos de origem animal no leste da Turquia

Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

High production of egc2-related staphylococcal enterotoxins caused a food poisoning outbreak.

Staphylococcal enterotoxins (SEs) produced by enterotoxigenic Staphylococcus aureus in food cause staphylococcal food poisoning. We recently reported a foodborne outbreak due to S. aureus harboring new SE/SE-like (SEl) genes (seg, sei, sem, sen, seo, and selu) related to enterotoxin gene cluster (egc) 2 as with other research groups. However, the pathogenicity of SEs production remains unclear. Therefore, we herein investigated egc2-related SEs production from S. aureus isolates and leftover food items during a foodborne outbreak using a sandwich enzyme-linked immunosorbent assay suitable for the quantification of SEs. S. aureus isolates produced markedly high levels of egc2-related SEs, and the leftover food item "Sushi" contained SEs over the toxin dose that causes food poisoning symptoms. A representative isolate was subjected to whole-genome sequencing. The isolate was homologous with previously reported ST45 strains, particularly the unique genomic island νSaß structure mostly consisting of egc2. The present study indicates that egc2-related SEs are food poisoning causative agents based on high SE production levels within an actual foodborne outbreak.

Investigation of Staphylococcus aureus positive for Staphylococcal enterotoxin S and T genes.

Staphylococcus aureus produces staphylococcal enterotoxins (SEs) and causes food poisoning. It is known that almost all SE-encoding genes are present on various types of mobile genetic elements and can mobilize among S. aureus populations. Further, plasmids comprise one of SE gene carriers. Previously, we reported novel SEs, SES and SET, harbored by the plasmid pF5 from Fukuoka5. In the present study, we analyzed the distribution of these SEs in various S. aureus isolates in Japan. We used 526 S. aureus strains and found 311 strains positive for at least one SE/SE-like toxin gene, but only two strains (Fukuoka5 and Hiroshima3) were positive for ses and set among the specimens. We analyzed two plasmids (pF5 and pH3) from these strains and found that they were different. Whereas these plasmids partially shared similar sequences involved in the ser/selj/set/ses gene cluster, other sequences were different. A comparison of these plasmids with those deposited in the NCBI database revealed that only one plasmid had the ser/selj/set/ses cluster with a stop mutation in set similar to that in pH3. In addition, the chromosomes of Fukuoka5 and Hiroshima3, positive for ses and set, were classified into different genotypes. Despite the low rate of gene positivity for these SEs, it is suggested that there is diversity in plasmids and strains carrying these two SEs. Consequently, regarding the entire feature of SE prevalence, we improved the multiplex PCR detection method for the SE superfamily to obtain further insight.