Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China.
ABSTRACT: 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:Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd), PFGE types, accessory gene regulator (agr) groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1-7 (56 isolates) were methicillin resistant (MRSA) and 8-10 (36 isolates) were methicillin sensitive (MSSA). One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq), and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70%) but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant.
Project description:A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall, the genotyping results showed high genetic diversity of the strains regardless of their geographical distributions, and no strong correlation between genetic background and toxin genotypes of the strains. For genotyping S. aureus, PFGE appears to be more discriminatory than MLST. However, toxin gene typing combined with PFGE or MLST could increase the discriminatory power of genotyping S. aureus strains.
Project description:Staphylococcus aureus, a major food-poisoning pathogen, is a common contaminant in dairy industries worldwide, including in Brazil. We determined the occurrence of S. aureus in five dairies in Brazil over 8 months. Of 421 samples, 31 (7.4%) were positive for S. aureus and prevalence varied from 0 to 63.3% between dairies. Sixty-six isolates from the 31 samples were typed by Multi-Locus Sequence Typing to determine if these isolates were persistent or continuously reintroduced. Seven known sequence types (STs), ST1, ST5, ST30, ST97, ST126, ST188 and ST398, and four new ST were identified, ST3531, ST3540, ST3562 and ST3534. Clonal complex (CC) 1 (including the four new ST), known as an epidemic clone, was the dominant CC. However, there were no indications of persistence of particular ST. The resistance toward 11 antibiotic compounds was assessed. Twelve profiles were generated with 75.8% of strains being sensitive to all antibiotic classes and no Methicillin-resistant S. aureus (MRSA) strains were found. The enterotoxin-encoding genes involved in food-poisoning, e.g., sea, sed, see, and seg were targeted by PCR. The two toxin-encoding genes, sed and see, were not detected. Only three strains (4.5%) harbored seg and two of these also harbored sea. Despite the isolates being Methicillin-sensitive S. aureus (MSSA), the presence of CC1 clones in the processing environment, including some harboring enterotoxin encoding genes, is of concern and hygiene must have high priority to reduce contamination.
Project description:To investigate the distribution of staphylococcal enterotoxin (SE) A to I (SEA to SEI) genes (sea to sei) in Staphylococcus aureus, 146 isolates obtained in Japan from humans involved in and samples from food poisoning outbreaks, healthy humans, cows with mastitis, and bovine raw milk were analyzed by multiplex PCR. One hundred thirteen (77.4%) S. aureus isolates were found to be positive for one or more se genes. The se genotype was classified into 14 genotypes. seg and sei coexisted in the same S. aureus strain. The newly developed sandwich enzyme-linked immunosorbent assay showed that most seh-harboring S. aureus isolates were able to produce a significant amount of SEH. However, most of the S. aureus isolates harboring seg and about 60% of the isolates harboring sei did not produce a detectable level of SEG or SEI, while reverse transcription-PCR analysis proved that the mRNAs of SEG and SEI were transcribed in S. aureus strains harboring seg and sei genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning.
Project description:The impact of bacterial genetic characteristics on the outcome of patients with Staphylococcus aureus infections is uncertain. This investigation evaluated potential associations between bacterial genotype and clinical outcome using isolates collected as part of an international phase 2 clinical trial (FAST II) evaluating telavancin for the treatment of complicated skin and skin structure infections (cSSSI). Ninety S. aureus isolates from microbiologically evaluable patients with cSSSI enrolled in the FAST II trial from 11 sites in the United States (56 isolates, or 62%) and 7 sites in South Africa (34 isolates, or 38%) were examined for staphylococcal cassette chromosome mec, agr, and the presence of 31 virulence genes and subjected to pulsed-field gel electrophoresis (PFGE). South African methicillin-susceptible S. aureus (MSSA) isolates were more likely to carry certain virulence genes, including sdrD (P = 0.01), sea (P < 0.01), and pvl (P = 0.01). All 44 (49%) methicillin-resistant S. aureus (MRSA) isolates were from the United States; 37 (84%) were strain USA 300 by PFGE. In the United States, MRSA isolates were more likely than MSSA isolates to carry genes for sdrC (P = 0.03), map/eap (P = 0.05), fnbB (P = 0.11), tst (P = 0.02), sea (P = 0.04), sed (P = 0.04), seg (P = 0.11), sej (P = 0.11), agr (P = 0.09), V8 (P = 0.06), sdrD, sdrE, eta, etb, and see (P < 0.01 for all). MRSA isolates were more often clonal than MSSA isolates by PFGE. Isolates from patients who were cured were significantly more likely to contain the pvl gene than isolates from patients that failed or had indeterminate outcomes (79/84 [94%] versus 3/6 [50%]; P = 0.01). S. aureus strains from different geographic regions have different distributions of virulence genes.
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:A set of 84 Staphylococcus aureus isolates collected from the milk of cows with subclinical mastitis in Asturias (a cattle region of Spain) and six control strains were tested for sequences of genes encoding hemolysins (hla, hlb, hld, hlg, and hlg-2), leukotoxins (lukPV, lukM, and lukED), toxic shock syndrome toxin (tst), and enterotoxins (sea to see, seg to ser, and seu) by conventional and multiplex PCR. It was found that 84, 83, 11, and 39 isolates carried some type of hl, luk, tst, or se gene, respectively, which were arranged in 14 exotoxin genotypes. All of the isolates were negative for lukPV, hlg, sea, sed, see, sej, sek, sep, seq, and ser. Two gene groupings could be related with pathogenicity islands-[lukED, seg, sei, sem, sen, seo +/- seu] with Sabeta-1 and [tst, sec, sel] with SaPIbov, present in 45 and 13.1% of the isolates, respectively-while 11.9% of them carried both islands. Only one contained seb (together with upsilonSabeta-1), and another contained seh (together with lukED). The isolates were also analyzed by pulsed-field gel electrophoresis performed with SmaI. Thirty-nine SmaI profiles (similarity coefficient [S] = 0.94 to 0.21) were differentiated; 12, 1, and 10 of these, respectively, were generated by isolates presumptively carrying Sabeta-1, SaPIbov, or both. Five SmaI profiles (S > or = 0.8) formed a cluster, which contained 20 and 10 isolates carrying one (upsilonSabeta-1) or both islands. These data show the high frequency of genes encoding cytotoxins and pyrogenic toxin superantigens, their relationship with pathogenicity islands, and their distribution among a diversity of genetic types of S. aureus related to subclinical mastitis.
Project description:Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases in the world. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and spa typing methods were used to characterize Staphylococcus aureus isolates from food surveillance during 2013-2015 in southwest China, and Staphylococcal cassette chromosome mec (SCCmec) typing was used for methicillin-resistant S. aureus (MRSA). Isolates were also examined for their antibiotic resistance and carriage of virulence genes.Isolation rate of S. aureus was 2.60% during the three years' surveillance and 29.50% of them were MRSA. All the S. aureus had hla genes (100%), 14.34% of the strains had tst, and 16.73% had PVL. 163 PFGE-SmaI patterns, 41 ST types and 36 spa types were obtained for all the S. aureus. Among them, ST6-t701 (13.15%), ST7-t091 (12.75%), ST59-t437 (9.96%) and ST5-t002 (7.57%) were the prevalent genotypes. Most of MRSA in this study belonged to SCCmec IV and V, accounted for 74.32% and 20.27% respectively. ST6-SCCmec IV-t701 (36.50%) was the most prevalent clone among isolates from food, followed by ST59-SCCmec V-t437 (20.30%), ST5-SCCmec IV-t002 (12.20%) and ST59-SCCmec IV-t437 (12.20%). Some strains had the identical PFGE patterns, ST and spa types with isolates from patients.S. aureus isolated from food in southwest China displayed heterogeneity. Isolates had the same genotype profiles with isolates from patients, indicating high homology.
Project description:We report the complete nucleotide sequence and analysis of pETBTY825, a Staphylococcus aureus TY825 plasmid encoding exfoliative toxin B (ETB). S. aureus TY825 is a clinical isolate obtained from an impetigo patient in 2002. The size of pETBTY825, 60.6 kbp, was unexpectedly larger than that of the archetype pETBTY4 (∼30 kbp). Genomic comparison of the plasmids shows that pETBTY825 has the archetype pETBTY4 as the backbone and has a single large extra DNA region of 22.4 kbp. The extra DNA region contains genes for resistance to aminoglycoside [aac(6')/aph(2″)], macrolide (msrA), and penicillin (blaZ). A plasmid deletion experiment indicated that these three resistance elements were functionally active. We retrospectively examined the resistance profile of the clinical ETB-producing S. aureus strains isolated in 1977 to 2007 using a MIC determination with gentamicin (GM), arbekacin (ABK), and erythromycin (EM) and by PCR analyses for aac(6')/aph(2″) and msrA using purified plasmid preparations. The ETB-producing S. aureus strains began to display high resistance to GM, which was parallel with the detection of aac(6')/aph(2″) and mecA, after 1990. Conversely, there was no significant change in the ABK MIC during the testing period, although it had a tendency to slightly increase. After 2001, isolates resistant to EM significantly increased; however, msrA was hardly detected in ETB-producing S. aureus strains, and only five isolates were positive for both aac(6')/aph(2″) and msrA. In this study, we report the emergence of a fusion plasmid carrying the toxin gene etb and drug resistance genes. Prevalence of the pETBTY825 carrier may further increase the clinical threat, since ETB-producing S. aureus is closely related to more severe impetigo or staphylococcal scalded-skin syndrome (SSSS), which requires a general antimicrobial treatment.
Project description:Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA) is an important issue associated with significant morbidity and mortality and well known as a predominant pathogen causing bloodstream infection (BSIs) globally. To estimate the antibiotic resistance and molecular characteristics of S. aureus causing BSIs in Shanghai, 120 S. aureus isolates (20 isolates each year) from the patients with S. aureus BSIs from 2013 to 2018 were randomly selected and enrolled in this study. Fifty-three (44.2%) MRSA isolates were determined, and no isolate was found resistant to vancomycin, daptomycin, synercid, linezolid and ceftaroline. The toxin genes tst, sec, seg and sei were found more frequently among MRSA isolates compared with MSSA isolates (all P?<?0.0001). Twenty-nine sequence types (STs) were identified, and ST5 (23.3%) was the most common ST, followed by ST398 (11.7%) and ST764 (10.0%). SCCmec II (73.6%) was the most frequent SCCmec type among MRSA isolates. The dominant clonal complexes (CCs) were CC5 (ST5, ST764, ST965 and ST3066; 36.7%) and the livestock-associated clone CC398 (ST398, 11.7%). MRSA-CC5 was the predominant CC among MRSA isolates (37/53, 69.8%), and CC5-II MRSA was found in 34 isolates accounting for 91.9% (34/37) among CC5 MRSA isolates. In addition, all 29 tst-positive MRSA isolates were CC5-MRSA as well. Our study provided the properties and genotypes of S. aureus causing BSIs at Ruijin Hospital in Shanghai from 2013 to 2018, and might suggest of value clues for the further study insights into pathogenic mechanisms intrinsically referring to the development of human-adapted S. aureus clones and their diffusions.