Pyrogenic Toxin Superantigens Research Articles

Dynamics of antimicrobial resistance and virulence of staphylococcal species isolated from foods traded in the Cape Coast metropolitan and Elmina municipality of Ghana

The impact of staphylococci on food poisoning and infections could be higher than previously reported. In this study, we characterised the occurrence and coexistence of antimicrobial resistance and virulence genes of staphylococci isolates in foods. Staphylococci were isolated from 236 samples of selected street-vended foods and identified. The pattern of antimicrobial resistance and virulence genes in the staphylococci were assessed using disc diffusion, PCR and analysis of next-generation sequencing data. The food samples (70.76 %) showed a high prevalence of staphylococci and differed among the food categories. Forty-five Staphylococcus species were identified and comprised coagulase-negative and positive species. Staphylococcus sciuri (now Mammaliicoccus sciuri), S. aureus, S. kloosii, S. xylosus, S. saprophyticus, S. haemolyticus and S. succinus were the most abundant species. The staphylococcal isolates exhibited resistance to tetracycline, levofloxacin, ciprofloxacin, norfloxacin, gentamicin and amikacin and susceptibility to nitrofurantoin. Antimicrobial susceptibilities were also reported for cefoperazone, ceftriaxone, cefotaxime, nalidixic acid and piperacillin-tazobactam. The antimicrobial resistance and virulence genes commonly detected consisted of tet, arl, macB, van, gyr, nor, optrA, bcrA, blaZ, taeA and S. aureus lmrS. The isolates frequently exhibited multiple resistance (30.42 %) of up to eight antimicrobial drug classes. The isolates predominantly harboured genes that express efflux pump proteins (50.53 %) for antibiotic resistance compared with inactivation (10.05 %), target alteration (26.72 %), protection (7.67 %) and replacement (3.17 %). The virulence determinants comprised genes of pyrogenic toxin superantigens (eta, etb, tst), adhesions (clf, fnbA, fnbB, cna, map, ebp, spA, vWbp, coa) and genes that express exoproteins (nuclease, metalloprotease, γ-hemolysin, hyaluronate lyase). There was a statistically significant difference in the prevalence of staphylococci isolates and their antimicrobial resistance and virulence profile as revealed by the phenotypic, PCR and next-generation sequencing techniques. The findings suggest a higher health risk for consumers. We recommend a critical need for awareness and antimicrobial susceptibility and anti-virulence strategies to ensure food safety and counteract the spread of this clinically relevant genus.

Local Epidemiology of Nosocomial Staphylococcus aureus Infection in a Nigerian University Teaching Hospital.

Population-based studies of Staphylococcus aureus contribute to understanding the epidemiology of S. aureus infection. We enrolled surgical inpatients admitted to an African tertiary-care hospital in order to prospectively analyze the nosocomial impact of S. aureus. Data collection included an active sampling of the anterior nares and infectious foci within 48 h after admission and subsequently when clinically indicated. All S. aureus isolates were spa and agr genotyped. Possession of Panton-Valentine leukocidin (PVL) and other toxin genes was determined. We analyzed antibiotic susceptibility profiles by VITEK 2 systems and verified methicillin-resistant S. aureus (MRSA) by mecA/C PCR. Among 325 patients, 15.4% carried methicillin-susceptible S. aureus (MSSA) at admission, while 3.7% carried MRSA. The incidence densities of nosocomial infections due to MSSA and MRSA were 35.4 and 6.2 infections per 10,000 patient-days, respectively. Among all 47 nosocomial infections, skin and soft-tissue (40.4%) and bones or joints’ (25.5%) infections predominated. Six (12.7%) infection-related S. aureus isolates harbored PVL genes including two (4.2%) MRSA: overall, seventeen (36.2%) isolates carried pyrogenic toxin superantigens or other toxin genes. This study illustrates the considerable nosocomial impact of S. aureus in a Nigerian University hospital. Furthermore, they indicate a need for effective approaches to curtail nosocomial acquisition of multidrug-resistant S. aureus.

Virulence, agr groups, antimicrobial resistance and epidemiology of Staphylococcus aureus isolated from bovine subclinical mastitis

Fifty-two Staphylococcus aureus recovered from papillary ostium and milk samples collected from cows with subclinical mastitis and milking environments in three small dairy herds located in southeastern Brazil were subjected to PCR identification based on the thermonuclease (nuc) gene. All the strains were submitted to in vitro antimicrobial susceptibility testing, and we investigated the sequence types (STs), agr groups (I-IV), virulence genes encoding for Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), biofilm-associated proteins, bi-component toxins, pyrogenic toxin superantigens, and enterotoxins. Screening for oxacillin resistance (2-6 μg/ml oxacillin), beta-lactamase activity assays, and PCR for the mecA/mecC genes detected 26 methicillin-susceptible S. aureus (MSSA) and 26 mec-independent oxacillin-nonsusceptible S. aureus (MIONSA). While MSSA isolates were found to be susceptible to all antimicrobial agents tested, or only resistant to penicillin and ampicillin, MIONSA isolates were multidrug-resistant. ST126-agr group II MSSA isolates were prevalent in milk (n=14) and carried a broad set of virulence genes (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla, and hlb), as well as the ST126-agr group II MIONSA isolated from milking liners (n=1), which also carried the eta gene. ST1-agr group III MIONSA isolates (n=4) were found in papillary ostium and milk, but most MIONSA isolates (n=21), which were identified in both papillary ostium and milking liners, were agr-negative and assigned to ST126. The agr-negative and agr group III lineages showed a low potential for virulence. Studies on the characterization of bovine-associated MSSA/MIONSA are essential to reduce S. aureus mastitis to prevent economic losses in dairy production and also to monitor the zoonotic potential of these pathogens associated with invasive infections and treatment failures in healthcare.

Device-Associated Menstrual Toxic Shock Syndrome.

In the 1980s, menstrual toxic shock syndrome (mTSS) became a household topic, particularly among mothers and their daughters. The research performed at the time, and for the first time, exposed the American public as well as the biomedical community, in a major way, to understanding disease progression and investigation. Those studies led to the identification of the cause, Staphylococcus aureus and the pyrogenic toxin superantigen TSS toxin 1 (TSST-1), and many of the risk factors, for example, tampon use. Those studies in turn led to TSS warning labels on the outside and inside of tampon boxes and, as important, uniform standards worldwide of tampon absorbency labeling. This review addresses our understanding of the development and conclusions related to mTSS and risk factors. We leave the final message that even though mTSS is not commonly in the news today, cases continue to occur. Additionally, S. aureus strains cycle in human populations in roughly 10-year intervals, possibly dependent on immune status. TSST-1-producing S. aureus bacteria appear to be reemerging, suggesting that physician awareness of this emergence and mTSS history should be heightened.

Leukotoxin and pyrogenic toxin Superantigen gene backgrounds in bloodstream and wound Staphylococcus aureus isolates from eastern region of China

BackgroundThe bicomponent leukotoxins and the pyrogenic toxin superantigens (PTSAgs) are important virulence factors of Staphylococcus aureus. It is necessary to survey the prevalence and expression of these toxin-encoding genes for understanding the possible pathogenic capacity of S. aureus to cause disease.MethodsFive leukotoxin genes and thirteen PTSAg determinants were detected for 177 S. aureus isolates from blood (n = 88) and wound (n = 89) infections by Polymerase Chain Reaction (PCR). The expression of leukotoxin ED (lukED) was determined by quantitative real-time PCR (qRT-PCR). The genetic backgrounds of isolates were analyzed by Staphylococcal Cassette Chromosome mec (SCCmec) typing (for methicillin-resistant S. aureus isolates), Pulsed-Field Gel Electrophoresis (PFGE), accessory gene regulator (agr) typing and Multilocus Sequence Typing (MLST, for representative isolates based on PFGE type) methods.Results99.4% (176/177) isolates contained at least one of leukotoxin genes. Among them, 94.9% (168/177), 81.4% (144/177) and 67.8% (120/177) isolates harbored hlgBC, lukED and lukAB, respectively. Compared to leukotoxin genes, there was a relatively lower overall prevalence of PTSAg genes [99.4% versus 72.9% (129/177), P < 0.001], and they were organized in 59 patterns, with the most common combination of the egc cluster with or without other PTSAg genes. Genetic analysis showed the distributions of certain toxin genes were associated with the genetic backgrounds of isolates. The egc cluster was a common feature of CC5 isolates, among which ST5 and ST764 isolates harbored more PTSAg genes. The lukED was not present in ST398 isolates, and its expression was quite different among isolates. No significant correlations were observed between the lukED expression levels of strains and the ST or agr types.ConclusionsThe present study elucidated the distribution of leukotoxin and PTSAg genes and the expression of lukED in blood and wound isolates, and analyzed the relationship between them with genetic characteristics of isolates. These data improve the current understanding of the possible pathogenicity of S. aureus.

Biofilm formation and virulence factor analysis of Staphylococcus aureus isolates collected from ovine mastitis.

To perform a phenotypic and genotypic characterization of 258 Staphylococcus aureus isolates from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines. The potential for biofilm production was determined by phenotypic test of Congo Red Agar (CRA) and by PCR for the detection of icaA/D genes. Isolates were also screened by PCR for the presence of enterotoxins (sea, seb, sec, sed and see), toxic shock syndrome toxin (tsst), leukotoxins (lukD-E, lukM and lukPV83), haemolysins (hly-β and hly-γ), autolysin (atlA) genes and encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: clfA, clfB, fnbA, fnbB, bbp, cna, eno, fib, epbs, sdrC, sdrD and SdrE). None of the 258 isolates showed biofilm-forming ability on CRA and harboured icaA/D genes. The most frequent pyrogenic toxin superantigen genes amplified were sec plus tsst-1, which were found strictly in combination with 71·3% of the Staph.aureus isolates tested. None of the isolates harboured the genes encoding sea and see. Of the 258 isolates tested, 159 (61·6%) possessed all lukD-E/lukM/lukPV83 genes, 123 (47·7%) harboured both hly-β/hly-γ genes, whereas almost all (97·3%) were PCR positive for atlA gene. With respect to adhesion determinants, 179 (69·4%) isolates presented simultaneously four genes (fnbA, fib, clfA and clfB) for fibronectin- and fibrinogen-binding proteins. In this search, several putative virulence determinants have been identified in ovine Staph.aureus isolates collected in Sardinia. Some of the putative virulence determinants could be considered as components of a vaccine because of their role in ovine mastitis pathogenesis.

Molecular Characterization of Staphylococcus aureus Isolated from Bovine Mastitis and Close Human Contacts in South African Dairy Herds: Genetic Diversity and Inter-Species Host Transmission.

Staphylococcus aureus is one of the most common etiological agents of contagious bovine mastitis worldwide. The purpose of this study was to genetically characterize a collection of S. aureus isolates (bovine = 146, human = 12) recovered from cases of bovine mastitis and nasal swabs of close human contacts in the dairy environment. Isolates were screened for a combination of clinically significant antimicrobial and virulence gene markers whilst the molecular epidemiology of these isolates and possible inter-species host transmission was investigated using a combination of genotyping techniques. None of the isolates under evaluation tested positive for methicillin or vancomycin resistance encoding genes. Twenty seven percent of the bovine S. aureus isolates tested positive for one or more of the pyrogenic toxin superantigen (PTSAg) genes with the sec and sell genes predominating. Comparatively, 83% of the human S. aureus isolates tested positive for one or more PTSAg genes with a greater variety of genes being detected. Genomic DNA macrorestriction followed by pulsed-field gel electrophoresis (PFGE) of the bovine isolates generated 58 electrophoretic patterns which grouped into 10 pulsotypes at an 80% similarity level. The majority of the bovine isolates, 93.2% (136/146), clustered into four major pulsotypes. Seven sequence types (ST) were identified among the representative bovine S. aureus isolates genotyped, including: ST8 (CC8), ST97 (CC97), ST351 (CC705), ST352 (CC97), ST508 (CC45), ST2992 (CC97) and a novel sequence type, ST3538 (CC97). Based on PFGE analysis, greater genetic diversity was observed among the human S. aureus isolates. Bovine and human isolates from three sampling sites clustered together and were genotypically indistinguishable. Two of the isolates, ST97 and ST352 belong to the common bovine lineage CC97, and their isolation from close human contacts suggests zoonotic transfer. In the context of this study, the third isolate, ST8 (CC8), is believed to be a human clone which has transferred to a dairy cow and has subsequently caused mastitis. The detection of indistinguishable S. aureus isolates from bovine and human hosts at three of the sampling sites is suggestive of bacterial transmission and supports the need for vigilant monitoring of staphylococcal populations at the human-animal interface.

Human scFvs That Counteract Bioactivities of Staphylococcus aureus TSST-1.

Some Staphylococcus aureus isolates produced toxic shock syndrome toxin-1 (TSST-1) which is a pyrogenic toxin superantigen (PTSAg). The toxin activates a large fraction of peripheral blood T lymphocytes causing the cells to proliferate and release massive amounts of pro-inflammatory cytokines leading to a life-threatening multisystem disorder: toxic shock syndrome (TSS). PTSAg-mediated-T cell stimulation circumvents the conventional antigenic peptide presentation to T cell receptor (TCR) by the antigen-presenting cell (APC). Instead, intact PTSAg binds directly to MHC-II molecule outside peptide binding cleft and simultaneously cross-links TCR-Vβ region. Currently, there is neither specific TSS treatment nor drug that directly inactivates TSST-1. In this study, human single chain antibodies (HuscFvs) that bound to and neutralized bioactivities of the TSST-1 were generated using phage display technology. Three E. coli clones transfected with TSST-1-bound phages fished-out from the human scFv library using recombinant TSST-1 as bait expressed TSST-1-bound-HuscFvs that inhibited the TSST-1-mediated T cell activation and pro-inflammatory cytokine gene expressions and productions.Computerized simulation, verified by mutations of the residues of HuscFv complementarity determining regions (CDRs),predicted to involve in target binding indicated that the HuscFvs formed interface contact with the toxin residues important for immunopathogenesis. The HuscFvs have high potential for future therapeutic application.

Staphylococcus aureus Toxic Shock Syndrome

Toxic shock syndrome (TSS), caused by toxic shock syndrome toxin-1 (TSST-1) and type B enterotoxins produced by Staphylococcus aureus [1,2]. The disease (TSS) was initially described in 1978 and came to public attention in 1980 with the occurrence of a series of menstrual-associated cases and at least 50% of non-menstrual cases. TSST-1 is a pyrogenic toxin superantigen produced by Staphylococcus aureus [3,4]. TSST-1 super antigen activate avast number of T cells in a T cell receptor Β chain Vβ-selective manner in direct association with major histocompatibility complex class II molecules on antigenpresenting cells especially Vβ2+ T cells [5-7].

Staphylococcus pseudintermedius can be misdiagnosed as Staphylococcus aureus in humans with dog bite wounds.

The purpose of this study was to investigate whether S. pseudintermedius is misdiagnosed as S. aureus by clinical laboratories when isolated fromhumans with dog bite wounds. In addition, we attempted to determine whether S. pseudintermedius isolates related to dog bite wounds share phenotypic and genotypic traits. S. pseudintermedius was identified by PCR targeting the nuc gene. Isolates were tested for antibiotic susceptibility using VetMIC GP-mo microdilution panels. The occurrence of genes encoding leukocidins, exfoliatins, pyrogenic toxin superantigens and enterotoxins was determined by PCR. The relatedness of S. pseudintermedius isolates was investigated using Multi Locus Sequence Typing (MLST). Out of 101 isolates defined as S. aureus by human clinical microbiology laboratories, 13 isolates were re-identified as S. pseudintermedius and one isolate was confirmed to carry the mecA gene, i.e. methicillin-resistant (MRSP). The MRSP isolate was also defined as multi-resistant. Two methicillin-susceptible S. pseudintermedius isolates were also multi-resistant and five were susceptible to all antibiotics tested. With the exception of three S. pseudintermedius isolates belonging to multi locus sequence type (MLST) 158, all the isolates belonged to unique STs. All isolates contained lukS/F-I, siet and se-int, and expA were identified in two isolates and expB and sec canine-sel in one isolate respectively. S. pseudintermedius is frequently misdiagnosed as S. aureus from humans with dog bite wounds showing that it can act as an opportunistic pathogen in humans. No common phenotypic and genotypic traits shared by the S. pseudintermedius isolates could be identified.

Staphylococcal superantigens interact with multiple host receptors to cause serious diseases.

Staphylococcus aureus strains that cause human diseases produce a large family of pyrogenic toxin superantigens (SAgs). These include toxic shock syndrome toxin-1 (TSST-1), the staphylococcal enterotoxins (SEs), and the SE-like proteins; to date, 23 staphylococcal SAgs have been described. Among the SAgs, three have been highly associated with human diseases (TSST-1, SEB, and SEC), likely because they are produced in high concentrations compared to other SAgs. Another major family of exotoxins produced by S. aureus is the cytolysins, particularly α-, β-, γ-, and δ-toxins, phenol soluble modulins, and leukocidins. This review discusses the association of SAgs with human diseases and particularly the "outside-in" signaling mechanism that leads to SAg-associated diseases. We discuss SAg interactions with three host immune cell receptors, including variable regions of the β-chain of the T cell receptor, MHC II α- and/or β-chains, and an epithelial/endothelial cell receptor that may include CD40. To a lesser extent, we discuss the role of cytolysins in facilitating disease production by SAgs.

Characterization of Colonizing Staphylococcus aureus Isolated from Surgical Wards' Patients in a Nigerian University Hospital

In contrast to developed countries, only limited data on the prevalence, resistance and clonal structure of Staphylococcus aureus are available for African countries. Since S. aureus carriage is a risk factor for postoperative wound infection, patients who had been hospitalized in surgical wards in a Nigerian University Teaching Hospital were screened for S. aureus carriage. All S. aureus isolates were genotyped (spa, agr) and assigned to multilocus sequence types (MLST). Species affiliation, methicillin-resistance, and the possession of pyrogenic toxin superantigens (PTSAg), exfoliative toxins (ETs) and Panton-Valentine Leukocidin (PVL) were analyzed. Of 192 patients screened, the S. aureus carrier rate was 31.8 % (n = 61). Of these isolates, 7 (11.5%) were methicillin-resistant (MRSA). The isolates comprised 24 spa types. The most frequent spa types were t064, t084, t311, and t1931, while the most prevalent MLST clonal complexes were CC5 and CC15. The most frequent PTSAg genes detected were seg/sei (41.0%) followed by seb (29.5%), sea (19.7%), seh (14.7%) and sec (11.5). The difference between the possession of classical and newly described PTSAg genes was not significant (63.9% versus 59.0% respectively; P = 0.602). PVL encoding genes were found in 39.3% isolates. All MRSA isolates were PVL negative, SCCmec types I and VI in MLST CC 5 and CC 30, respectively. Typing of the accessory gene regulator (agr) showed the following distribution: agr group 1 (n = 20), group II (n = 17), group III (n = 14) and group IV (n = 10). Compared to European data, enterotoxin gene seb and PVL-encoding genes were more prevalent in Nigerian methicillin-susceptible S. aureus isolates, which may therefore act as potential reservoir for PVL and PTSAg genes.

Exotoxin gene backgrounds in bloodstream and wound Staphylococcus aureus isolates from geriatric patients attending a long-term care Spanish hospital

The exotoxin gene content was established for 62 Staphylococcus aureus isolates causing bloodstream (n = 31) and wound (n = 31) infections in geriatric patients attending a long-term care Spanish hospital from 1996 to 2006. Content was determined based on PCR screening of genes encoding five haemolysins, three exfoliatins, three leukotoxins and 21 pyrogenic toxin superantigens (PTSAgs), in addition to markers of genomic (νSaβ) and pathogenicity (SaPIs) islands. Exotoxin genes were abundant in both bloodstream (11-23 genes) and wound (8-19 genes) isolates, and they were arranged in 55 combinations with only two represented in both groups. All isolates were positive for genes encoding haemolysins (hl; 3-5) and PTSAgs (tst, se and sel; 5-14), whereas exfoliatin (et) and leukotoxin (luk) genes appeared in 98.4 and 51.6 % of isolates, respectively. The hlg, lukPV, tst, sec and selu genes were found significantly more frequently in bloodstream than in wound isolates, whereas hlg-variant, sea, seb, see and selk-selq were more frequent in wound isolates (P<0.05). Distinctive exotoxin gene combinations could potentially be associated with specific mobile genetic elements, including genomic islands [lukED, egc1 (seg, seln, sei, selm, selo) and egc2 (seg, seln, selu, sei, selm, selo)]; pathogenicity islands (etd, seb, sec, sell, selq, selk and tst); bacteriophages (eta, lukPV, sea, selp, selk, selq and see); and plasmids (sed, selj, ser, ses and set). The abundance of exotoxin genes and variety of arrangements shown by S. aureus from geriatric patients could play a role in the adaptation of the pathogen.

Structurally Refined β‐Lactones as Potent Inhibitors of Devastating Bacterial Virulence Factors

Disarmed forces: Inhibition of the central virulence regulator ClpP by structurally refined beta-lactones resulted in dramatically reduced production of devastating virulence factors, including pyrogenic toxin superantigens derived from pathogenic multiresistant Staphylococcus aureus strains. Targeting of this virulence regulator could present an attractive strategy for neutralizing the harmful effects of bacterial pathogens, and help the host immune response to eliminate the disarmed bacteria.

Multiplex PCRs for assignment of Staphylocoagulase types and subtypes of type VI Staphylocoagulase

Staphylocoagulases (SCs) have been classified by the differences in antigenicity using a serological method. We have developed a system to classify them based on the nucleotide differences in SC genes ( coa). The system was composed of three multiplex PCRs (M-PCRs): M-PCR:A, identifying types III, IV, VII, and VIII; M-PCR:B, identifying types I, II, V, and VI; M-PCR:C, identifying three subtypes of type VI. In this study, we found that coa genes of the serotype VI were not identical, but classified into three subtypes based on the nucleotide differences, especially in D2 and the central region: VIa, the coa gene carried by stp12 from human; and VIb and VIc, the coa genes carried by strains IFH556 and IFH514 isolated from bovine raw milk. The primer pair used in M-PCR:B was designed to identify all three subtypes of type VI coa. The results showed that coa types of 154 out of 155 Staphylococcus aureus strains from various origins assigned by M-PCR:A and B were identical to those obtained by serological methods, leaving a serotype IV strain unclassifiable. All 73 type VI strains were classified into one of three subtypes by M-PCR:C. Furthermore, we found that type VIa and VIb strains carried characteristic pyrogenic toxin superantigen genes, while no toxin genes were identified in type VIc strains, suggesting the correlation between the subtype of type VI coa gene and the carriage of genomic islands. Our results showed that these M-PCRs are convenient methods for SC typing that might be useful for epidemiological studies.

Virulence genes of bovineStaphylococcus aureusfrom persistent and nonpersistent intramammary infections with different clinical characteristics

To screen putative virulence genes in Staphylococcus aureus causing persistent and nonpersistent bovine intramammary infections (IMI) with different clinical characteristics. To examine, whether a possible relationship exists between genetic profile and infection persistence, clinical signs of infection, clonal type determined by pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance. One-hundred and sixty-one S. aureus isolates derived from bovine IMI, consisting of 17 different PFGE types, were screened by conventional and multiplex-polymerase chain reaction (PCR) for 24 virulence genes for haemolysins (hla-hlg), leukocidins (lukED, lukM), exfoliative toxins (eta, etb), enterotoxins (sea-seo, seu), toxic-shock syndrome toxin (tst), and genes encoding penicillin (blaZ) and methicillin resistance (mecA). The majority of S. aureus isolated at the onset of mastitis carried haemolysin genes (76.7-97.4%), lukED (96.6%), and at least one gene for pyrogenic toxin superantigen (PTSAg) (69.0%). Strains carrying PTSAg-encoding genes were more common among predominant PFGE types and in persistent IMI. Strains concomitantly possessing sed, sej, and blaZ, putatively plasmid-encoded, were typically found in connection with persistent IMI. Our results suggest that certain genetic elements are over-representative in S. aureus isolates especially from persistent bovine mastitis. This phenomenon seems to be in connection with clonal type and is often concomitant with penicillin resistance. This is the first study to investigate associations between a large number of bacterial factors and outcome of S. aureus mastitis. The finding that widespread clonal types of S. aureus causing bovine mastitis of low treatment response may harbour characteristic genes could be improved for strain-specific diagnostic purposes.

Novel multiplex PCR for the detection of the Staphylococcus aureus superantigen and its application to raw meat isolates in Korea

A multiplex PCR assay that allows for the rapid screening of the 19 genes that encode staphylococcal enterotoxins (SEs) ( sea to see, and seg to sei), SE-like (SEl) toxins ( sej to ser, and seu), and toxic shock syndrome toxin-1 (TSST-1) ( tst) was developed in this study. These toxins are included in the pyrogenic toxin superantigen (PTSAg) family and are responsible for many diseases such as staphylococcal food poisoning (SFP) and TSS. The primers were designed based on dual priming oligonucleotide (DPO) technology to detect all of the 19 SAg genes in three sets of PCR. The developed multiplex PCR was applied to 143 Staphylococcus aureus strains isolated from pork and chicken meat in Korea. Almost 50% of the strains possessed at least one of the 19 SAg genes. The most frequently found genes were seg, sei, sem, and sen (53 isolates, 37%), which were often found simultaneously in the same isolate. In those isolates, the seo (39 isolates, 27%) or seu (6 isolates, 4%) genes were frequently found together and this combination ( seg, sei, sem, sen, and seo or seu) was considered to be a part of the enterotoxin gene cluster ( egc). The sea gene (10 isolates, 7%) was the gene most frequently detected out of all the classical SE genes ( sea to see). Although these classical SEs are considered to be major etiological factors in SFP, newly described SE or SEl genes ( seg to ser, and seu) were more frequently detected than the classical SE genes in this study. There was no isolate detected containing the seb, sec, sek, sel, or seq genes. S. aureus possessing mobile genetic elements known to encode these SAg genes, such as egc, were presumed to be widely distributed among pork and chicken meats in Korea. The multiplex PCR developed in this study could be applied to the investigation of SAg genes in S. aureus strains isolated from various sources.

A quantitative real-time immuno-PCR approach for detection of staphylococcal enterotoxins

Bacterial intoxications represent a substantial public health concern with enterotoxins produced by Staphylococcus aureus among the most common causes of food poisoning. In addition to their role in the pathogenicity of food poisoning, staphylococcal enterotoxins have profound effects on the immune system as members of the family of pyrogenic toxin superantigens. As the classical diagnostic bioassays as well as the routinely used immunological methods are hampered by several drawbacks regarding sensitivity, specificity, and practicability, there is a need for the timely identification of toxins by highly sensitive and specific methods. To combine the versatility of an enzyme immunoassay (EIA) with the amplification power of the PCR, a quantitative real-time immuno-PCR (qRT-iPCR) was developed for the detection of staphylococcal enterotoxins A and B and compared to a commercially available EIA. A broadly applicable tool for signal amplification of pre-formed immunocomplexes was established by covalent binding of a reporter DNA to secondary detection antibodies. Therefore, the amino-modified reporter DNA was coupled successfully to N-succinimidyl-S-actyl-thioacetate-activated secondary detection antibodies. The qRT-iPCR was able to detect highly reproducibly as low as approximately 0.6 to 6 pg (4 to 40 amol/microl) of staphylococcal enterotoxin B and staphylococcal enterotoxin A, respectively. In conclusion, the qRT-iPCR approach was shown to overcome clearly the sensitivity limit of traditional immunological detection procedures for bacterial toxins, as demonstrated in this study for staphylococcal enterotoxins. The development of a stable antibody-DNA conjugate providing a universal signal amplification offers a versatile as well as a highly sensitive and specific tool for diagnostic and research purposes generally applicable for pre-formed antibody-antigen complexes.

Cytotoxin and pyrogenic toxin superantigen gene profiles of Staphylococcus aureus associated with subclinical mastitis in dairy cows and relationships with macrorestriction genomic profiles.

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.

Relationships between toxin gene content and genetic background in nasal carried isolates of Staphylococcus aureus from Asturias, Spain

Staphylococcus aureus recovered from nasal carriers, producers and non-producers (43 isolates each) of classical pyrogenic toxin superantigens (PTSAgs), were screened for 17 additional PTSAg-genes by PCR. Percentages of 88.4 and 65.1 were positive for some new enterotoxin-gene, and 76.7 and 55.8 for enterotoxin-gene-clusters ( egc-like), respectively. The 86 isolates belonged to 17 toxin-genotypes (all eta-, etb-, etd-, see- and sep-negative), and generated 40 SmaI-genomic profiles that in a dendrogram of similarity ( S⩾0.7) clustered into nine lineages and 11 non-clustered branches. Correlations between classical PTSAgs and SmaI-lineages were established and egc-like groupings appeared dispersed in six lineages.