Production of enterotoxins and toxic shock syndrome toxin by Staphylococcus aureus isolated from bovine mastitis in Brazil

The production of toxic shock syndrome toxin 1 (TSST-1) and enterotoxins (SE) A, B, C and D by bovine mastitis isolates of Staphylococcus aureus was evaluated by immunodiffusion using the Optimum-Sensitivity Plate method. S. aureus strains were isolated from bovine mastitis in 23 dairy herds in the state of Minas Gerais, Brazil, during 1994-9. Of 127 isolates, 83 (65.04%) produced one or several toxins, and among them production of SE was found in 54 (43.0%) isolates, of which 1138 (29.09%) secreted enterotoxin identified as type D. TSST-1 was found in 5829 (45.723.0%) isolates.     

Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci

The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.     

Screening of staphylococci for the toxic shock syndrome toxin-1 (TSST-1) gene

Dot blot hybridization was used to screen 820 staphylococci for the presence of the gene coding for TSST-1. The DNA of 33 strains among 70 Staph. aureus strains isolated from suspected toxic shock syndrome (TSS) cases hybridized with the probe. These results agreed perfectly with those obtained with a phenotypic method (immunodiffusion). Among 608 Staph. aureus strains isolated over a period of one month from hospitalized patients, 66 (11%) hybridized with the probe; of these strains, 64 (97%) were found to produce TSST-1 in vitro. None of 145 coagulase-negative staphylococcal strains harboured DNA hybridizing with the probe. The data indicate that this genotypic assay is suitable for epidemiological studies.     

Detection of enterotoxins and TSST-1 secreted by Staphylococcus aureus isolated from ruminant mastitis. Comparison of ELISA and immunoblot.

The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 (TSST-1) was studied in 81 strains of Staphylococcus aureus isolated from cases of mastitis in cattle, goats and sheep. SE and TSST-1 were detected by two techniques: ELISA double antibody sandwich, and an immunoblot technique combined with a semiautomated electrophoresis system. More Staph. aureus strains isolated from sheep produced enterotoxins than those from goats and cattle. SEC was the predominant type in all isolates from these animal species. The highest proportion of strains producing TSST-1 were obtained from sheep, twice as many as those from goats or cows. The two techniques gave similar results, as all the strains positive by immunoblot were also positive by ELISA, and only three were positive by ELISA but negative by immunoblot.     

Toxic shock syndrome toxin 1 (TSST-1) production by staphylococci isolated from goats and presence of specific antibodies to TSST-1 in serum and milk.

The ability of staphylococcal strains isolated from different anatomical sites in 133 healthy goats to produce toxic shock syndrome toxin 1 (TSST-1) and the presence of antibodies to this toxin in serum and milk were studied. The enzyme-linked immunosorbent assay method was used to detect both the toxin and the presence of antibodies. Of a total of 342 staphylococcal strains studied, 86 (25.2%) were found to produce TSST-1. Specific antibodies to TSST-1 were found in the serum of 57 (42.9%) of the animals studied and the milk of 63 (47.4%) of the animals. These results suggest that goats are frequently in contact with staphylococci that produce TSST-1, a toxin usually associated with Staphylococcus aureus strains isolated from cases of toxic shock syndrome in humans.     

Detection of specific antibodies in cord blood, infant and maternal saliva and breast milk to staphylococcal toxins implicated in sudden infant death syndrome (SIDS)

The common bacterial toxins hypothesis of sudden infant death syndrome (SIDS) is that nasopharyngeal bacterial toxins can trigger events leading to death in infants with absent/low levels of antibody that can neutralise the toxins. The aim of this study was to investigate nasopharyngeal carriage of Staphylococcus aureus and determine levels of immunity in the first year of life to toxic shock syndrome toxin (TSST-1) and staphylococcal enterotoxin C (SEC). Both toxins have been implicated in SIDS cases. Seventy-three mothers and their infants (39 males and 34 females) were enrolled onto the study. The infants had birth dates spread evenly throughout the year. In infants, S. aureus carriage decreased significantly with age (P<0.001). Between 40% and 50% of infants were colonised with S. aureus in the first three months of life and 49% of the isolates produced one or both of the staphylococcal toxins. There was a significant correlation between nasopharyngeal carriage of S. aureus in mothers and infants in the three months following the birth (P<0.001). Carriage of S. aureus in infants and their mothers was not significantly associated with levels of antibody to TSST-1 or SEC in cord blood, adult saliva or breast milk. Infants colonised by S. aureus had higher levels of salivary IgA to TSST-1 than infants who were culture negative. Analysis of cord blood samples by a quantitative ELISA detected IgG bound to TSST-1 and SEC in 95.5% and 91.8% of cases respectively. There was a marked variation in levels of maternal IgG to both TSST-1 and SEC among cord blood samples. Maternal age, birth weight, and seasonality significantly affected the levels of IgG binding to TSST-1 or SEC. Analysis of infant saliva samples detected IgA to TSST-1 and SEC in the first month after birth; 11% of samples tested positive for salivary IgA to TSST-1 and 5% for salivary IgA to SEC. By the age of two months these proportions had increased to 36% and 33% respectively. More infants who used a dummy tested positive for salivary IgA to TSST-1 compared to infants who did not use a dummy. Levels of IgA to TSST-1 and SEC detected in the breast-milk samples varied greatly among mothers. There was a trend for infants receiving breast milk with low levels of antibody to TSST-1 or SEC to have higher levels of salivary antibody to the toxins. In conclusion, passive immunity to toxins implicated in SIDS cases varies greatly among infants. Infants are able to mount an active mucosal immune response to TSST-1 and SEC in the first month of life.     

Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin

Staphylococcus aureus colonizes and infects humans as well as animals. In the present study, 17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well-established multilocus sequence typing (MLST) technique and a diagnostic microarray covering 185 S. aureus virulence and resistance genes were used for genetic and epidemiological analyses. Virulence gene expression studies were performed by analyzing the extracellular protein pattern of each isolate on 2-D gels. By this way, a pronounced heterogeneity of the extracellular proteome between the bovine isolates has been observed which was attributed to genome plasticity and variation of gene expression. Merely 12 proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc, PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen extracellular proteins were highly variably expressed and only present in less than 20% of the isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC-bov, SEL and TSST-1. Compared to human isolates we identified at least six proteins with significantly different expression frequencies. While SAB0846 was expressed more frequently in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these isolates.     

Enterocolitis caused by methicillin-resistant Staphylococcus aureus: molecular characterization of respiratory and digestive tract isolates.

We investigated the mechanism of outbreak of enterocolitis caused by methicillin-resistant Staphylococcus aureus (MRSA). Five epidemiological markers [coagulase type, enterotoxin type, toxic shock syndrome toxin-1 (TSST-1) production, beta-lactamase production and pulsed-field gel electrophoresis (PFGE)] of 45 strains of MRSA isolated simultaneously from the respiratory tract (nasal cavity and/or pharynx and/or sputum) and stool (plus one sample of gastric juice) in 13 patients (8 males and 5 females, mean age, 77.1 years) were compared retrospectively. Forty-four of the 45 isolates of MRSA were positive for enterotoxin C and TSST-1 production, and the remaining isolate was positive for enterotoxin A and negative for TSST-1 production. All isolates were coagulase type II, and 27 showed beta-lactamase production. The patterns of coagulase type, enterotoxin type, TSST-1 and beta-lactamase production of MRSA isolated from the respiratory tract were similar to those of MRSA isolated from the intestine in 12 of 13 patients. Molecular typing by PFGE demonstrated that the pattern of respiratory tract isolates was identical to those of stool isolates in 9 (69.2%), similar in 3 (23.1 %), and different in 1 (7.7%). The data suggested that enterocolitis might be caused by the MRSA colonized in the respiratory tract and incorporated into the digestive tracts. Therefore, we propose that early eradication of MRSA in the respiratory tract is important for protection of patients against the development of enterocolitis, particularly in susceptible patients, e.g., immunocompromised or pre-operated patients with digestive diseases, especially malignant disease.     

Toxic shock syndrome toxin 1 (TSST-1) production by staphylococci isolated from goats and presence of specific antibodies to TSST-1 in serum and milk

The ability of staphylococcal strains isolated from different anatomical sites in 133 healthy goats to produce toxic shock syndrome toxin 1 (TSST-1) and the presence of antibodies to this toxin in serum and milk were studied. The enzyme-linked immunosorbent assay method was used to detect both the toxin and the presence of antibodies. Of a total of 342 staphylococcal strains studied, 86 (25.2%) were found to produce TSST-1. Specific antibodies to TSST-1 were found in the serum of 57 (42.9%) of the animals studied and the milk of 63 (47.4%) of the animals. These results suggest that goats are frequently in contact with staphylococci that produce TSST-1, a toxin usually associated with Staphylococcus aureus strains isolated from cases of toxic shock syndrome in humans.     

Detection of enterotoxins and TSST-1 secreted by Staphylococcus aureus isolated from ruminant mastitis. Comparison of ELISA and immunoblot

J.A. ORDEN, J. GOYACHE, J. HERNÁNDEZ, A. DOMÉNECH, G. SUÁREZ AND E. GÓMEZ-LUCÍA. 1992. The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 (TSST-1) was studied in 81 strains of Staphylococcus aureus isolated from cases of mastitis in cattle, goats and sheep. SE and TSST-1 were detected by two techniques: ELISA double antibody sandwich, and an immunoblot technique combined with a semiautomated electrophoresis system. More Staph. aureus strains isolated from sheep produced enterotoxins than those from goats and cattle. SEC was the predominant type in all isolates from these animal species. The highest proportion of strains producing TSST-1 were obtained from sheep, twice as many as those from goats or cows. The two techniques gave similar results. as all the strains positive by immunoblot were also positive by ELISA, and only three were positive by ELISA but negative by immunoblot.     

The effect of Staphylococcus aureus carriage in late pregnancy on antibody levels to staphylococcal toxins in cord blood and breast milk

We investigated the effect of carriage of Staphylococcus aureus in the later stages of pregnancy on levels of antibody specific to the S. aureus toxins, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC) and toxic shock syndrome toxin-1 (TSST-1), in cord blood and breast milk and also explored the relationship between levels of antibody in antenatal serum and cord blood. Nasopharyngeal swabs and stool samples were collected on two occasions, from 96 women, during the last 6 weeks of pregnancy. Samples were cultured and S. aureus isolates were identified. Antenatal and cord blood samples from the same women and their infants were analysed for IgG antibody to SEB, SEC and TSST-1 by enzyme-linked immunosorbent assay. Breast milk samples were analysed for IgA antibody to the same toxins. We found that S. aureus carriage in pregnancy is common and exposure to a toxin-producing isolate boosts immunity. Over 89% of women and infants have some protective antibody to the toxins, and antitoxin IgG levels are higher in cord blood samples compared with antenatal samples. Levels of cord blood IgG and breast milk IgA specific for the staphylococcal toxins vary. Some infants lack protection and could be at risk of toxin-induced disease.     

Occurrence of selected enterotoxins and toxic shock syndrome toxin genes among sensitive and resistant to methicillin Staphylococcus aureus isolated from clinical samples

The aim of study was to estimate frequency of occurrence of enterotoxins (sea-sed) and TSST-1 (tst) genes. One hundred seven methicillin-sensitive and one hundred three methicillin-resistant strains of S. aureus isolated from hospital patients in 21 medical centers, in majority from the region of Gdansk were examined. The presence of selected toxins genes was detected by multiplex PCR. The results showed that almost 80% of MRSA strains were positive for sea gene, in contrast to MSSA (17,8%). Both MSSA and MRSA strains were rarely positive for the presence of other enterotoxins genes seb, sec, sed (less than 10%) and a tst gene was detected in about 15% of them. No correlation between presence of the particular genes and clinical samples was observed.     

Variations in amount of TSST-1 produced by clinical methicillin resistant Staphylococcus aureus (MRSA) isolates and allelic variation in accessory gene regulator (agr) locus

Background  

Staphylococcus aureus (S. aureus) is an important pathogen associated with both nosocomial and community-acquired infections and its pathogenicity is attributed to its potential to produce virulence factors. Since the amount of toxin produced is related to virulence, evaluating toxin production should be useful for controlling S. aureus infection. We previously found that some strains produce relatively large amounts of TSST-1; however, no reports have described the amount of TSST-1 produced by clinical isolates.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts

We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2', showing that methicillin-resistant S. aureus was not present in the samples.     

Detection and toxin production of Staphylococcus aureus in sudden infant death cases in Hungary

The potential role of microbial agents was investigated in 13 cases of Sudden Infant Death Syndrome and in 9 non-SIDS cases in Budapest between September 1996 and May 1998. Autopsy, histological examination and microbiological tests were performed on samples of blood, cerebrospinal fluid, pharyngeal samples and lung tissue from infants under one year died suddenly, without previous diseases. The multifactorial pathomechanism of SIDS was suggested by the isolation of toxin producing Staphylococcus aureus-, Enterobacteriaceae and Candida albicans strains in large number and by the detection of Parainfluenza Type 2 virus antigen. S. aureus proved the predominant bacteria in the SIDS cases. Nasopharyngeal microbial flora and S. aureus carrier of 100 age matched healthy infants were tested during the same period. S. aureus was isolated from 54% of SIDS cases and 37% from healthy infants /OR = 1.986 (95% Confidence interval = 0.55-7.33), p = 0243/. The enterotoxin and TSST-1 toxin producing activity of S. aureus showed the characteristic difference. The toxigenic S. aureus was detected in 46% of SIDS cases and 16% of healthy infants /OR = 4.5 (95% CI = 1.15-17.72), p = 0.010/. The distribution of toxigenic and nontoxigenic isolates was 86% in SIDS cases and 43% in healthy infants /OR = 7.875 (CI = 0.78-191.89), p = 0.041/.     

Serotyping of Dutch Staphylococcus aureus strains from carriage and infection

International epidemiological studies have shown that clinical isolates of Staphylococcus aureus are usually capsulated with either type 5 or 8 capsular polysaccharides (CPs). Because all noncapsulated strains were found to be cross-reactive with polysaccharide 336 (336PS) antibodies, the noncapsulated strains were denoted as type 336PS. The capsular types of 162 Dutch methicillin-susceptible S. aureus strains derived from individuals living in the Rotterdam area were determined. The serotype distribution was 28.4% serotype 5, 53.7% type 8, and 17.9% type 336PS. Serotyping was in agreement with genotyping by amplified fragment length polymorphism (AFLP) and multi locus sequence typing (MLST). Among 49 nasal carriage isolates from healthy children 24.5% belonged to serotype 5, 67.3% were type 8 and 8.2% were type 336PS. For 28 adult patients on chronic ambulatory peritoneal dialysis (CAPD) the serotype incidences among carriage isolates obtained from the nose, catheter exit-site, and abdominal skin were 45.1%, 41.2% and 13.7%, respectively. Among S. aureus strains deriving from blood cultures, the serotype incidences were 17.7% serotype 5, 53.2% type 8, and 29.0% type 336PS. Apparently, type 336PS strains are more prevalent (P=0.017) among bacteraemia isolates as compared with the nasal carriage isolates obtained from healthy children and CAPD patients. In conclusion, all Dutch S. aureus isolates belonged to types 5, 8, or 336PS, which is in agreement with data from other countries. Thus, addition of the 336PS conjugate to a type 5- and type 8-CP protein conjugate vaccine would significantly extend the vaccine coverage.     

Binding of type-I and type-II collagens to Staphylococcus aureus strains isolated from patients with toxic shock syndrome compared to other staphylococcal infections

Toxic shock syndrome toxin-1 (TSST-1) producing strains of Staphylococcus aureus isolated from 18 patients with toxic shock syndrome (TSS) and from 56 patients with other diagnoses were compared for capacity to interact with various serum and connective tissue proteins. TSS associated isolates showed significantly stronger binding of Type-I collagen (Cn-I) and Cn-II than non-TSS strains, in a particle agglutination assay (PAA) as well as in 125I labelled Cn uptake experiments. 125I Cn-IV binding, was similar between the two groups, whereas in PAA, a stronger interaction was observed for non-TSS than TSS associated strains. The median binding of 125I Cn to TSS-associated strains were 52.2 (Cn-I), 30.6 (Cn-II) and 20.0 (Cn-IV) compared to 20.0 (Cn-I), 14.4 (Cn-II) and 24.4 (Cn-IV) values of non-TSS strains. A saturation with 125I Cn-I and Cn-II binding was established for TSS (30 min) and non-TSS (15 min) strains. 125I Cn-IV binding reached a saturation in 10 min and 90 min with TSS and non-TSS strains respectively. Finally, the binding profiles of TSS associated and non-TSS strains to fibronectin, fibrinogen, laminin and IgG did not differ in both PAA and radioisotope assays. In scanning electron microscopy, cells of TSS associated strains bound to the reprecipitated native Cn-I fibrils. In contrast, most cells of non-TSS strains were localized to the distal end or were trapped between the Cn fibrils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence and clonal relatedness of sec/tst-gene positive Staphylococcus aureus isolates of quartermilk samples of cows suffering from mastitis

AIMS: To investigate the prevalence of sec/tst-gene positive Staphylococcus aureus in bovine mastitis and to get information about the clonal relatedness of these clinical isolates. METHODS AND RESULTS: A total of 533 Staph. aureus strains isolated from bovine mastitic quartermilk samples at 493 randomized dairy farms in Hessia, Germany, from January 1997 until June 1998 were examined for enterotoxin C (sec) gene and toxic shock syndrome toxin (tst) gene by multiplex polymerase chain reaction. Fifty-three (9.3%) of the strains were sec/tst-gene positive. Phenotypic TSST-1 production was found in all positive strains by reversed passive latex agglutination test. With DNA macrorestriction analysis, sec/tst-gene positive strains were divided into five different macrorestriction types. Type I (10 isolates) and III (40 isolates) were found to be the predominant types in terms of frequency of isolation in the investigated area. These DNA macrorestriction types differed in only two bands in the 500 and 270 bp region. CONCLUSIONS: Closely related Staph. aureus strains seem to be responsible for an unusual large proportion of bovine mastitis cases in geographically widely distinct locations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first reports about the relatedness of sec/tst-gene positive Staph. aureus clinical isolates from bovine mastitis.     

Synthetic DNA probes for detection of genes for enterotoxins A, B, C, D, E and for TSST-1 in staphylococcal strains.

A dot blot hybridization technique with oligonucleotide probes was developed for the specific detection of the TSST-1 gene and the staphylococcal enterotoxin (SE) genes A, B, C, D and E. For each toxin gene a probe sequence was chosen from the previously determined sequence. A total of 145 staphylococcal strains (133 Staphylococcus aureus and 12 coagulase-negative staphylococci (CNS) were studied by this genotypic method and by two phenotypic assays (gel immunodiffusion and ELISA). An excellent correlation (96%) was observed between the genotypic and phenotypic assays. DNA from two CNS strains hybridized with a probe without detection of the corresponding toxin (SEB for one strain and SEC for the other strain). One Staph. aureus strain was shown to be an SEC producer, but was not detected by the corresponding probe. Gene probe and immunological assays seem to be complementary methods for studies of staphylococcal strains producing (or potentially producing) TSST-1 or enterotoxins.