Molecular epidemiology and identification of a Staphylococcus aureus clone causing food poisoning outbreaks in Japan.
ABSTRACT: Molecular characterization of isolates from staphylococcal food poisoning (SFP) outbreaks in Japan showed that the dominant lineage causing SFP outbreaks is clonal complex 81 (CC81), a single-locus variant of sequence type 1, coagulase type VII, positive for sea and/or seb, and positive for seh. Among various CC lineages producing staphylococcal enterotoxin A, CC81 showed the highest toxin productivity.
Project description:We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637-2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP.
Project description:The staphylococcal enterotoxins (SEs) are secreted by the bacteria Staphylococcus aureus and are the most common causative agent in staphylococcal food poisoning. The staphylococcal enterotoxin A (SEA) has been associated with large staphylococcal food poisoning outbreaks, but newer identified SEs, like staphylococcal enterotoxin H (SEH) has recently been shown to be present at similar levels as SEA in food poisoning outbreaks. Thus, we set out to investigate the thermo-stability of the three-dimensional structures of SEA, SEH and staphylococcal enterotoxin E (SEE), since heat inactivation is a common method to inactivate toxins during food processing. Interestingly, the investigated toxins behaved distinctly different upon heating. SEA and SEE were more stable at slightly acidic pH values, while SEH adopted an extremely stable structure at neutral pH, with almost no effects on secondary structural elements upon heating to 95°C, and with reversible formation of tertiary structure upon subsequent cooling to room temperature. Taken together, the data suggests that the family of staphylococcal enterotoxins have different ability to withstand heat, and thus the exact profile of heat inactivation for all SEs causing food poisoning needs to be considered to improve food safety.
Project description: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.
Project description:Staphylococcal food poisoning (SFP) caused by enterotoxigenic staphylococci is one of the main food-borne diseases. In contrast to Staphylococcus aureus, a systematic screening for the enterotoxins has not yet been performed on the genomic level for the coagulase-positive species S. intermedius. Therefore, the enterotoxigenic potential of 281 different veterinary (canine, n = 247; equine, n = 23; feline, n = 9; other, n = 2) and 11 human isolates of S. intermedius was tested by using a multiplex PCR DNA-enzyme immunoassay system targeting the staphylococcal enterotoxin genes sea, seb, sec, sed, and see. Molecular results were compared by in vitro testing of enterotoxin production by two immunoassays. A total of 33 (11.3%) S. intermedius isolates, including 31 (12.6%) canine isolates, 1 equine isolate, and 1 human isolate, tested positive for the sec gene. In vitro production of the respective enterotoxins was detected in 30 (90.9%) of these isolates by using immunological tests. In contrast, none of 65 veterinary specimen-derived isolates additionally tested and comprising 13 (sub)species of coagulase-negative staphylococci were found to be enterotoxigenic. This study shows on both molecular and immunological levels that a substantial number of S. intermedius isolates harbor the potential for enterotoxin production. Since evidence for noninvasive zoonotic transmission of S. intermedius from animal hosts to humans has been documented, an enterotoxigenic role of this microorganism in SFP via contamination of food products may be assumed.
Project description: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 <i>S. aureus</i>. 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 <i>S. aureus</i>, 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.
Project description:Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.
Project description:Staphylococcus aureus No. 10 is an isolate from a staphylococcal food poisoning outbreak in Japan, classified as clonal complex 81 subtype 1. It preferentially produces larger quantities of staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin H (SEH) in foods and media. Here, we report the complete annotated genome sequence of the chromosome and a plasmid.
Project description:Ingestion of staphylococcal enterotoxins preformed by Staphylococcus aureus in food leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. There are five major staphylococcal enterotoxins: SEA, SEB, SEC, SED, and SEE. While variants of these toxins have been described and were linked to specific hosts or levels or enterotoxin production, data on sequence variation is still limited. In this study, we aim to extend the knowledge on promoter and gene variants of the major enterotoxins SEB, SEC, and SED. To this end, we determined seb, sec, and sed promoter and gene sequences of a well-characterized set of enterotoxigenic Staphylococcus aureus strains originating from foodborne outbreaks, human infections, human nasal colonization, rabbits, and cattle. New nucleotide sequence variants were detected for all three enterotoxins and a novel amino acid sequence variant of SED was detected in a strain associated with human nasal colonization. While the seb promoter and gene sequences exhibited a high degree of variability, the sec and sed promoter and gene were more conserved. Interestingly, a truncated variant of sed was detected in all tested sed harboring rabbit strains. The generated data represents a further step towards improved understanding of strain-specific differences in enterotoxin expression and host-specific variation in enterotoxin sequences.
Project description:Staphylococcal food poisoning, one of the most common food-borne diseases, results from ingestion of one or more staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus in foods. In the present study, 64 S. aureus isolates recovered from foods and food handlers, associated or not associated with food-poisoning outbreaks in Spain, were investigated. They were assigned to 31 strains by spa typing, multilocus sequence typing (MLST), exotoxin gene content, and antimicrobial resistance. The strains belonged to 10 clonal complexes (CCs): CC5 (29.0%), CC30 (25.8%), CC45 (16.1%), CC8, CC15 (two strains each), CC1, CC22, CC25, CC59, and CC121 (one strain each). They contained hemolysin genes (90.3%); lukED (77.4%); exfoliatin genes eta, etd (6.5% each), and etb (3.2%); tst (25.8%); and the following enterotoxin or enterotoxin-like genes or clusters: sea (38.7%), seb (12.9%), sec (16.1%), sed-selj with or without ser (22.9%), selk-selq (6.5%), seh, sell, selp (9.7% each), egc1 (32.3%), and egc2 (48.4%). The number of se and sel genes ranged from zero to 12. All isolates carrying tst, and most isolates with genes encoding classical enterotoxins (SEA, SEB, SEC, and SED), expressed the corresponding toxin(s). Two CC5 isolates from hamburgers (spa type t002, sequence type 5 [ST5]; spa type t2173, ST5) were methicillin resistant and harbored staphylococcal cassette chromosome mec (SCCmec) IVd. Six (19.4%) were mupirocin resistant, and one (spa type t120, ST15) from a food handler carried mupA (MIC, 1,250 ?g/ml). Resistance to ampicillin (blaZ) (61.3%), erythromycin (ermA-ermC or ermC) (25.8%), clindamycin (msrA-msrB or msrB) (16.1%), tetracycline (tetK) (3.2%), and amikacin-gentamicin-kanamycin-tobramycin (aphA with aacA plus aphD or aadD) (6.5%) was also observed. The presence of S. aureus strains with an important repertoire of virulence and resistance determinants in the food chain represents a potential health hazard for consumers and merits further observation.
Project description:Although coagulase-positive staphylococci are considered to be the main factor responsible for food poisoning, an increasing role for the coagulase-negative staphylococci in the production of enterotoxins has been observed in recent years. This study was conducted to assess the occurrence of genes responsible for the production of staphylococcal enterotoxins (SE), enterotoxin-like toxins (SEI) and toxic shock syndrome toxin-1 (TSST-1) in coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food from bars and restaurants. One hundred and eighteen CoNS strains were tested using polymerase chain reaction (PCR) to five superantigenic toxin genes, including five different types of classical enterotoxins (sea, seb, sec, sed and see) and the toxic shock syndrome toxin-1 (tsst-1) as well as to supertoxin-like genes. PCR-positive isolates were then tested using immunoenzymatic methods (SET-RPLA, Vidas SET 2) for toxin expression. Out of 118 CoNS strains, the presence of staphylococcal enterotoxins was confirmed in 72% of them. The most frequently found enterotoxin-like genotype was ser, selu. Two of the tested strains had up to ten different enterotoxin genes in the genome at the same time. Although no production of enterotoxins was detected in the CoNS, which means that their possible role in the epidemiology of food-borne diseases is minimal, the data demonstrated that the toxigenic capacity of the CoNS should not be ignored, and that this group of microorganisms should be continuously monitored in food.