Screening of Staphylococcus aureus nasal strains isolated from medical students for toxin genes.

Three hundred twenty-one students (156 students with no clinical exposure and 165 students with clinical exposure) were screened for nasal colonization by Staphylococcus aureus; 20.9% of students were S. aureus nasal carriers, and 40.3% of S. aureus isolates harbored toxin genes. The most prevalent genes were tst (15.0 %) and sec (13.4 %). Isolates with multiple genes were only found among clinical students (p = 0.045). Six of 11 PFGE clones were positive for toxin genes. Methicillin-resistant (MRSA) isolates were only detected in the clinical students (4.5 %). The exposure of students to the hospital environment neither radically increased S. aureus nasal carriage, nor the frequency of clinically important toxin gene presence, but it could have influenced the positive selection of toxigenic MRSA strains.

Prevalence of staphylococcal enterotoxins, toxin genes and genetic-relatedness of foodborne Staphylococcus aureus strains isolated in the Marmara Region of Turkey.

Staphylococcus aureus is a major foodborne pathogen and it has the ability to produce a number of extracellular toxins. We analyzed 1070 food samples obtained from retail markets and dairy farms in the Marmara Region of Turkey for the presence of S. aureus. Out of 147 isolates, 92 (62.6%) were enterotoxigenic. PCR was used to investigate the presence of staphylococcal enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu), exfoliative toxin genes (eta and etb) and the toxic-shock syndrome toxin gene (tst). The PCR results showed that 53.3% of the isolates contained staphylococcal enterotoxin-like (SEl) toxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu) which were more frequent than classical enterotoxin genes (sea to see). Furthermore, seo, sei, sem, seg, seu and sec were found in 37.0, 32.7, 30.4, 29.3, 29.3 and 27.2% of the isolates, respectively. The tst gene was detected and confirmed by DNA sequencing in 9 isolates. The presence of eta and etb were not found in the isolates. Enterotoxigenic capabilities of isolates with SEA-SEE were investigated by ELISA. Enterotoxigenic S. aureus isolates produced one to three enterotoxins, with the most frequently produced types being enterotoxin A and C. There was a correlation of 72.1% between production of a specific toxin and the presence of the respective genes. PFGE analysis was used to identify genetic-relatedness of enterotoxigenic S. aureus isolates and the results revealed that 13 groups of isolates from different or the same origin that contained the same genes showed 100% homology with indistinguishable band patterns. The other enterotoxigenic isolates showed related band patterns with 72-86% homology in sea-, 61-90% homology in sec-, 80-96% homology in seh-, and 69-96% homology in sep-positive isolates. To our knowledge, this is the first study to examine enterotoxins and related gene contents of S. aureus food isolates in the Marmara Region of Turkey.

Sensitive and specific detection of staphylococcal epidermolysins A and B in broth cultures by flow cytometry-assisted multiplex immunoassay.

Two of the most common bacterial skin infections of young infants and children are bullous impetigo due to Staphylococcus aureus and its more acute form, staphylococcal scalded skin syndrome. Epidermolysin A (ETA), ETB and, possibly, ETD are responsible for these diseases, which may appear as epidemics in pediatric patients. We tested the reliability of a flow cytometry-assisted multiplex immunoassay (Bio-Plex system) for the detection of ETA and ETB. The Bio-Plex system was found to be highly specific and highly sensitive for toxin concentrations of between 2 and 80,000 pg/ml. The results of this assay were 100% identical to the results of a PCR-based method. We demonstrated that this test did not generate any cross-reactions with ETD-producing isolates. The level of detection of ETB by this test differed according to culture conditions and from isolate to isolate; these results must be taken into account for diagnostic purposes.

A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus.

AIMS: To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). METHODS AND RESULTS: A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. CONCLUSIONS: This study has provided a new tool for detection of toxigenic Staph. hyicus and a more comprehensive picture of the prevalence of the Staph. hyicus exfoliative toxins in Danish pig herds. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex PCR can be used in studies on the prevalence of toxigenic Staph. hyicus elucidating the epidemiology of EE in pigs. The multiplex PCR is currently being used for selection of Staph. hyicus isolates for production of autogenous vaccine.

Recent developments in staphylococcal scalded skin syndrome.

Staphylococcal scalded skin syndrome describes a spectrum of superficial blistering skin disorders caused by the exfoliative toxins of Staphylococcus aureus. In its severe form, the exfoliation can spread to cover the entire body surface area. Two S. aureus exfoliative toxin serotypes affecting humans have been identified, but their purpose and mechanism of action have remained elusive. Based on their interaction with human and mouse epidermis, their three-dimensional structure and site-directed mutagenesis studies, it is speculated that they act as atypical serine proteases, and desmoglein-1 has now been identified as the specific epidermal substrate. Recent studies also suggest that the toxins may have a unique superantigenic activity. Clinically, new rapid diagnostic tests have been developed, including one that is able to detect the toxins directly from serum. With early diagnosis and appropriate management, mortality in children remains low and long-term complications are rare because the lesions are superficial and heal rapidly without scarring. In adults, however, the condition carries a mortality of almost 60% despite aggressive treatment, usually because of serious underlying illness. The recent developments in our understanding of the exfoliative toxins should lead to new and improved diagnostic and therapeutic strategies, including the use of specific antixoxins to prevent exfoliation.

Development and evaluation of detection systems for staphylococcal exfoliative toxin A responsible for scalded-skin syndrome.

Staphylococcal scalded-skin syndrome is usually diagnosed clinically by its characteristic exfoliating rash. Isolation of Staphylococcus aureus from the patient further supports the diagnosis. Several detection systems have been developed to determine whether the isolated strain produces exfoliative toxin, but none are routinely available in hospital laboratories. In a novel approach, we used computer models to predict the structure of the exfoliative toxins based on other serine proteases and to identify surface epitopes for the production of antibodies that specifically bound the exfoliative toxin A (ETA) serotype. Several rapid immunologically based diagnostic tests for ETA were developed with these antibodies and compared with existing systems. Our results showed that Western blot analysis using these antibodies was in complete correlation with PCR, which has been validated against the "gold standard" mouse model. On the other hand, the double-antibody enzyme-linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion assay gave unacceptably high false-positive results due to interference by staphylococcal protein A. This problem was successfully overcome by the development of a F(ab')(2) fragment ELISA, which was rapid and reproducible and was as sensitive and specific as PCR and Western blot analysis. The F(ab')(2) fragment ELISA is superior to existing diagnostic systems because it is quantitative, which may be related to the severity of the condition, and can detect amounts of exfoliative toxin in the picogram range directly from serum. This is the first detection system with the potential to confirm the diagnosis of staphylococcal scalded-skin syndrome from a routine blood test within 3 h of presentation.

[Neonatal staphylococcal epidermolysis due to maternal-fetal transmission]. / Epidermolyse staphylococcique néonatale à transmission materno-foetale.

INTRODUCTION: The staphylococcal scalded skin syndrome (SSSS) is due to exfoliative toxins A or B excreted by some strains of Staphylococcus aureus. This syndrome is exceptional in the first hours of life. We report a case of SSSS due to materno-fetal infection. CASE REPORT: At 31 weeks of pregnancy a 40-year-old mother was febrile (39 degrees C) and a premature rupture of the amniotic sac occurred the following day. SSSS was diagnosed at 6 hours of life in the newborn, a 1760 g female. Staphylococcus aureus grew on the blood and vaginal bacterial cultures of the mother, as well as, from cultures of skin, nose, throat, and umbilical catheter in the newborn. The strains of Staphylococcus aureus isolated in the mother and the child had identical characteristic antibiotype and genotype by Random-PCR. The genes for both exfoliations A and B were present. Epidermization was rapidly obtained and no septicemia or septic complication was noted. DISCUSSION: Staphylococcus aureus is usually responsible for nosocomial infections which occur in the early newborn period. In most cases, the infection is transmitted by a carrier who manipulates the child (family, visitors, nurse or medical staff). In our case, onset of SSSS early after birth suggested a perinatal transmission, due to lower genital tract infection in the mother. The presence of SSSS in the child and not in the mother may be explained by a massive perinatal infection and low elimination of the toxin in the newborn resulting in higher concentrations of exfoliative toxins in the blood.

Evaluation of the staphylococcal exotoxins and their specific IgE in childhood atopic dermatitis.

BACKGROUND: Superantigenic exotoxins produced by Staphylococcus aureus and their specific IgE antibodies are thought to be important precipitating factors of atopic dermatitis (AD), but there are few reports evaluating these 2 factors at the same time. OBJECTIVE: We examined whether the presence of the exotoxins sampled from the skin of patients with AD and the levels of anti-exotoxin IgE antibodies in their sera correlated with their severity of AD. METHODS: Patients with mild-to-severe AD, 1 to 22 years of age, were evaluated by using Leicester's scoring system. Specific IgE antibodies against these exotoxins were determined by using ELISA. S aureus was isolated from 3 different areas of the skin. We examined whether the exotoxin (staphylococcal enterotoxin [SE]A, SEB, SEC, SED, and toxic shock syndrome toxin-1) could be detected. RESULTS: The levels of SEB-specific IgE were correlated with the severity of AD. Five of 6 patients having very high SEB-specific IgE antibody titers were under 6 years of age, and SEB was most frequently isolated (41%). There was no difference in severity between patients with or without exotoxin-producing S aureus. The severity of 9 patients who had both exotoxin-producing S aureus on the skin and specific IgE antibody against the same exotoxin in sera was significantly higher than that of the other patients. CONCLUSIONS: Anti-SEB IgE titers correlate well with the severity of AD. The presence of exotoxin-producing S aureus may precipitate AD through its specific IgE antibody.

Molecular epidemiology of staphylococcal scalded skin syndrome in premature infants.

BACKGROUND: Outbreaks of nosocomial staphylococcal scalded skin syndrome (SSSS) in infants have been well-described associated with the well baby nursery or delivery room. We describe two cases of SSSS in very low birth weight infants in a neonatal intensive care unit (NICU) and the success of infection control strategies used to prevent an outbreak. METHODS: Staphylococcal scalded skin syndrome was diagnosed in two infants in the NICU: Case I (a 47-day-old, formerly 530-g female); and Case II diagnosed 48 h later (a 41-day old, formerly 706-g female). Multiple infection control measures were implemented: (1) isolation and intravenous antibiotic treatment of cases; (2) placement of exposed infants into a cohort; (3) prophylactic mupirocin treatment of the anterior nares of all infants in the NICU and staff colonized with Staphylococcus aureus; and (4) personnel hand washing with hexachlorophene. Detection of exfoliative toxin A and studies to determine the genetic relatedness of S. aureus strains isolated from patients and staff were performed. RESULTS: In addition to the two SSSS cases, S. aureus was isolated from 2 of 12 (17%) exposed asymptomatic infants, 2 of 20 (10%) ancillary staff, 8 of 30 (27%) nurses and 6 of 24 (25%) physicians. Exfoliative toxin A-producing strains were isolated from both cases and one asymptomatic infant. No toxin was expressed by strains isolated from staff. Pulse field gel electrophoresis demonstrated genetically identical strains of S. aureus from the two SSSS cases and the asymptomatic infant, whereas three staff members harbored strains genetically related to the case strain. Unexpectedly two additional unique clusters of genetically related S. aureus strains were identified from the surveillance cultures. CONCLUSIONS: This report documents the rare occurrence of nosocomial SSSS attributed to transmission in the NICU among extremely low birth weight infants. Multiple infection control strategies were effective in limiting the outbreak. Molecular epidemiology investigation supported a unique S. aureus strain responsible for this event and the presence of bidirectional spread between staff and patients of non-toxin-producing strains.

Superantigen production by Staphylococcus aureus in psoriasis.

BACKGROUND: Activation of T cells is believed to play a critical role in the pathogenesis of psoriasis. Recently, it has been proposed that psoriasis is a T-cell-mediated autoimmune reaction triggered by bacterial superantigen. OBJECTIVE: We investigated whether patients with chronic plaque psoriasis bear superantigen-producing Staphylococcus aureus on the skin or the throat. METHODS: S. aureus producing exfoliative toxin, staphylococcal enterotoxin B or toxic shock syndrome toxin 1 was isolated from the skin and throat of 100 psoriasis patients using Western blot analysis and polymerase chain reaction. RESULTS: Only 5, 4 and 9 patients had super-antigen producing S. aureus identified on lesional skin, nonlesional skin and throat, respectively. The vast majority of patients did not bear superantigen-producing S. aureus. CONCLUSION: We believe that superantigens are not essential in sustaining disease activity but may, instead, be exacerbating or triggering factors for some psoriasis patients.

Exfoliative toxin detection using reversed passive latex agglutination: clinical and epidemiologic applications.

A rapid and simple method for detecting exfoliative toxin serotypes A and B from clinical isolates has been developed as a test kit (EXT-RPLA; Denka Seiken Co. Ltd., Niigata, Japan). This method is based on reversed passive latex agglutination. The detection limit of the EXT-RPLA observed for purified exfoliative toxin serotypes A and B was 1 ng/ml. We evaluated the clinical and epidemiologic uses of the EXT-RPLA. A total of 381 isolates of Staphylococcus aureus, 292 from various clinical specimens and 89 from the skin of dermatologic patients, were studied. The EXT-RPLA detected 19 exfoliative toxin producers, including 16 serotype A producers and 3 serotype B producers, but no double producers. The sensitivity and specificity of the EXT-RPLA were confirmed by the newborn mouse bioassay and a PCR assay for the structural genes for exfoliative toxin serotypes A and B (eta and etb, respectively). The overall positivity rate of exfoliative toxin producers was 5.0% (19 of 381), including 16 serotype A isolates and 3 serotype B isolates. Of the 89 isolates from the skin of dermatologic patients, 12 (13.5%) were positive for exfoliative toxin production. Only 2 (1.3%) of the 153 methicillin-resistant S. aureus isolates produced exfoliative toxin, while 17 (7.5%) of the 228 methicillin-sensitive isolates produced exfoliative toxin. The EXT-RPLA assay is a simple and reliable method for detecting exfoliative toxin, and we recommend its use for the rapid diagnosis of staphylococcal scalded skin syndrome. We also recommend its use for detection of this syndrome so that effective control measures can be taken against the spread of this syndrome.

Staphylococcal scalded skin syndrome in an immunocompromised adult.

Staphylococcal scalded skin syndrome, a generalized exfoliative dermatitis complicating infections by exfoliative toxin-producing strains of Staphylococcus aureus, is rarely observed in adults. In contrast to mortality in infants, mortality in adults is usually high. A case of generalized staphylococcal scalded skin syndrome in an immunocompromised woman is reported. Culture of skin biopsy and pleural fluid yielded identical strains of staphylococcus aureus belonging to phage group II. Exfoliative toxins A and B were detected in both isolates. As far as can be determined, this is the first reported case of generalized staphylococcal scalded skin syndrome in an adult with detection of exfoliate toxins A and B in which the patient was treated successfully.

Staphylococcal scalded skin syndrome in an adult. Influence of immune and renal factors.

We report a case of staphylococcal scalded skin syndrome in a 77-year-old man with an infected surgical wound. The patient was immunocompetent and had only mildly impaired renal function. The pathogenic and aetiological factors of the condition are discussed.

ELISA estimation of the binding of epidermolytic toxin to neonatal mouse skin.

A sandwich ELISA, with antisera from rat and rabbit, was used to determine epidermolytic toxin (ET) to a limit of about 0.01 ng at 0.1 ng/ml. The binding of ET to the epidermis of skin discs was measured in vitro. The ability of the assay to discriminate between the two forms of the toxin was used to demonstrate that there was a saturable component of toxin binding to the epidermis. The rate of uptake, the effect of the inhibitor EGTA and comparative experiments with the inactive nitrated toxin confirmed that the observed binding is associated with toxigenesis. From measurements at toxin concentrations from 0.25 microgram/ml to 100 micrograms/ml, it was calculated that the saturable binding component has a Kd of about 2 micrograms/ml (approximately 60 nM) and a capacity of 0.5 ng per skin disc (1 ng per cm2 of epidermis).

Incidence and characterization of Staphylococcus aureus from the tongues of children.

Three hundred and seven children who had no diseases other than dental disease were examined for their oral carriage of Staphylococcus aureus, the most common persistent human pathogen. Eighty-four percent of them were positive for staphylococci, and 33% were positive for S. aureus. Among the 100 strains of S. aureus isolated, 40 strains produced enterotoxin, and 19 strains produced exfoliative toxin. Their susceptibility to antibiotics was also investigated: Six strains demonstrated resistance to methicillin (MIC greater than or equal to 12.5 microgram/mL), and 50% of the isolates were borderline resistant (MIC of 3.13 to 6.25 micrograms/mL) to the drug. These data suggest that the mouths of children could be reservoirs of pathogenic S. aureus.

Purification of staphylococcal exfoliative toxin by high pressure liquid chromatography.

Exfoliative toxin (ET) isolated from a clinical strain of Staphylococcus aureus was purified to homogeneity, using a 3-step HPLC system. NH2-terminal 20 amino residues of purified ET was found to be identical with ETA of S. aureus TA (7), S. aureus TC16 (9) and S. aureus ZM (10), but stability of purified ET was completely different from that of ETA. This purification system gave a high yield of pure ET, which exhibited higher purity than specimens purified by more complicated and time-consuming procedures. It is useful for small-scale purification for the comparative study of ET and easy to scale up for preparative purification.

Synthetic exfoliative toxin A and B DNA probes for detection of toxigenic Staphylococcus aureus strains.

Two methods for the detection of exfoliative toxin (ET) from Staphylococcus aureus were compared: (i) a phenotypic assay, electrosyneresis, and (ii) a genotypic assay, staphylococcal DNA hybridization with oligodeoxynucleotide probes. The probes were chosen from the previously determined sequences of serotype A and B of ET, one probe for serotype A and another for serotype B. Strains exhibiting ET production in electrosyneresis always possessed the ET gene(s). Conversely, some strains not exhibiting ET production in electrosyneresis harbored the ET gene(s). The latter strains produced levels of ET. ET-negative phage group 2 strains of S. aureus as well as tested coagulase-negative staphylococci did not possess the ET gene(s). The sensitivity of the DNA hybridization technique was 10(6) bacteria or 100 ng of genomic DNA.

Detection of staphylococcal exfoliative toxin by slide latex agglutination.

A simple and rapid method in which slide latex agglutination was used was developed to detect the exfoliative toxin (ET) elaborated by clinical isolates. ET types A and B (ET-A and ET-B) were purified by plate gel isoelectrofocusing, and anti-ET sera were obtained by immunizing rabbits. A specific immunoglobulin G antitoxin was then prepared from the immunized rabbit sera by fast protein liquid chromatography, and latex particles were coated with the antitoxin. Of 74 staphylococcal strains isolated from patients with staphylococcal scalded skin syndrome, 61 strains were found to produce ET by the newborn mouse bioassay. All 61 strains were shown to be positive for ET-A and ET-B production by the slide latex agglutination method. The lowest concentration of ETs detected by the latex agglutination method was 0.5 microgram/ml, which was much lower than that detected by the double immunodiffusion method, with a sensitivity of 50 micrograms/ml. It is crucial to prove ET production by clinical isolates for the diagnosis and surveillance of staphylococcal scalded skin syndrome. The latex agglutination method is a sensitive, simple, and rapid test which can be used as an alternative to the newborn mouse bioassay.