Passive Latex Agglutination Test Research Articles

Staphylococcus aureus Genotypes of Subclinical Bovine Mastitis Milk in the Middle Western Anatolia

Background: Staphylococcus aureus is the most common etiological pathogen of bovine mastitis. Subclinical mastitis is characterised by a non-alteration of the milk but can cause food poisoning by production of enterotoxins in milk. Knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments to prevent subclinical mastitis and provide useful data for epidemiological studies. The aim of this study was to characterize the genetic nature of the S. aureus cultured from subclinical bovine mastitis occurring in 16 farms in the middle western Anatolia. Methods: Two hundred sixty eight milk samples positive with California Mastitis Test (CMT) suggesting the subclinical mastitis of lactating cows in 16 different farms in the Middle Western Anatolia were collected and S. aureus were isolated. Identification was carried out by traditional tests and ribotyping confirmed the identification. Staphylococcal Enterotoxins (SE) were detected and typed by Staphylococcal Enterotoxin Test Reversed Passive Latex Agglutination (SET-RPLA) test kit. Genetic characterisation of the isolates was carried out by both ribotyping and pulsed field gel electrophoresis (PFGE). Results: A total of 77 isolates of S. aureus were purified and analysed by both biochemical identification and genotyping. Only 4 isolates (5.19 %) of S. aureus were recorded as enterotoxin positive. Genetic characterisation of the isolates was carried out by ribotyping revealed eight ribotypes while pulsed field gel electrophoresis (PFGE) was more discriminative representing 19 pulsotypes. Conclusion: This study shows no significant association between enterotoxin production, ribogroup and pulsotype profile of the S. aureus isolates collected from the Middle Western Anatolia.

Department of Microbiology, Bangladesh Agricultural University, Mymensingh

The study was done to detect different exotoxins among the strains of Staphylococcus aureus isolated in the department of Microbiology, Mymensingh Medical College in collaboration with the Department of Medicine under the Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh between the periods from July, 2006 to June, 2007. A total of 40 S. aureus isolates investigated in this study were identified by standard microbiological techniques. Antimicrobial susceptibility of the isolates to Oxacillin was carried out by disk diffusion method as per recommendation of the National Committee for Clinical Laboratory Standards. Any isolate showing resistance to Oxacillin was tested again by agar dilution method to determine minimum inhibitory concentration (MIC) of Methicillin. All strains were also tested for mecA gene by Polymerase Chain Reaction (PCR) for confirmation of Methicillin resistance. Enterotoxin (A-D) and Toxic Shock Syndrome Toxin-1 (TSST-1) were detected by Reverse Passive Latex Agglutination (RPLA) test. Out of 40 S. aureus isolates, 7 (17.5%) Methicillin Resistant S. aureus (MRSA), 1 (2.5%) Methicillin Sensitive S. aureus (MSSA) produced Staphylococcal Enterotoxin A (SE-A) and 1 MRSA isolate was positive for TSST-1. In case of combined toxin production among the S. aureus isolates, 2 (5%) MSSA were found to produce SE-A and SE-B, 2 (5%) MSSA produced SE-C and SE-D, and 1 (2.5%) MRSA, 1 (2.5%) MSSA produced SE-C and TSST-1.Bangladesh J Med Microbiol 2008; 02 (02): 3-6

Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in Escherichia coli O157:H7.

Shiga-like toxin (Stx) is an important factor in the pathogenesis of Escherichia coli O157:H7 infection and is responsible for some severe complications. Stx2 is usually associated with hemolytic uremic syndrome in humans. Its expression is regulated by elements located upstream of the stx2 gene, including stx2-promoter sequence, ribosome binding site, and the antiterminator q gene. The present study aimed to find the correlation between regulatory elements and the expression level of Stx2 in two local isolates of E. coli O157:H7. Two local E. coli O157:H7 strains SM-25(1) and KL-48(2), originating from human and cattle feces, respectively, and an E. coli reference strain, ATCC 43894, were investigated. The complete stx2 gene covering the sequences of promoter, ribosome binding site, and open reading frame and q gene of each strain was analyzed. The magnitude of Stx2 production was detected with a reverse passive latex agglutination method and Stx mediated cellular damage was determined with the Vero cell assay. A comparison of the complete stx2 gene contained stx2-promoter, ribosome binding site, and q genes of two local strains KL-48(2) and SM25(1), and the E. coli ATCC 43894 showed that the amino acid sequences were identical. Both local isolates were Stx negative in the reverse passive latex agglutination test and nontoxic in the Vero cell assay. The expression level of Shiga-like toxin of the two local isolates of E. coli O157:H7 did not only depend on the regulatory elements of the stx2 gene.

Staphylococcus aureus y resistencia antimicrobiana en dos hospitales de Tegucigalpa, 2011-2012

El objetivo de esta investigación es determinar la resistencia antimicrobiana del Staphylococcus aureus en dos hospitales de Tegucigalpa. Para lograrlo se siguió una metodología de estudio descriptivo, transversal. Se recolectaron todas las muestras de Staphylococcus aureus aisladas de pacientes, de los laboratorios de dos hospitales, durante un periodo de 5 meses, en cada institución; las que se trasladaron al Laboratorio de Bacteriología de la Escuela de Microbiología de la UNAH; para el estudio de sensibilidad antibiótica y la identificación de los SARM, mediante el método de Kirby-Bauer y según normas establecidas por el Clinical and Laboratory Standards Institute (CLSI); para los que se usaron diferentes antibióticos. También se realizó la determinación de la concentración inhibitoria mínima (CIM) a OX usando E- test y la determinación de la producción de PBP2a, por el método de aglutinación en látex. Los datos fueron analizados con estadística descriptiva con el paquete estadístico EPI-INFO, versión 3.5.1. para Windows. Se obtuvieron 90 aislamientos de S. aureus del laboratorio del Hospital Escuela y 106 de S. aureus en el laboratorio del IHSS. La mayoría de las bacterias aisladas provenían de exudados, los cuales presentaron resistencia a diferentes antibióticos y ninguna bacteria aislada presentó resistencia a Vancomicina. La resistencia a Oxacilina o SARM fue de un 14.3 % (IC95 % = 9.7 a 20). Se confirmó la resistencia antimicrobiana de S. aureus a Oxacilina o SARM por el método de Kirby-Bauer, Etest, PBP2.En conclusión, la prevalencia de SARM no es tan alta, basada en los parámetros para los países en vías de desarrollo y no se encontró resistencia a Vancomicina. DOI: http://dx.doi.org/10.5377/rct.v0i13.1712 Revista Ciencia y Tecnología, No. 13, diciembre 2013: 30-40

Molecular identification and safety of Bacillus species involved in the fermentation of African oil beans (Pentaclethra macrophylla Benth) for production of Ugba

Molecular identification of Bacillus spp. involved in the fermentation of African oil bean seeds for production of Ugba, as well as ability of the Bacillus spp. isolated to produce toxins, were investigated. Forty-nine bacteria were isolated from Ugba produced in different areas of South Eastern Nigeria and identified by phenotyping and sequencing of 16S rRNA, gyrB and rpoB genes. Genotypic diversities at interspecies and intraspecies level of the isolates were screened by PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR) and repetitive sequence-based PCR (rep-PCR). The ability of the bacteria to produce toxins was also investigated by detection of genes encoding production of haemolysin BL (HblA, HblC, HblD), non-haemolytic enterotoxin (NheA, NheB, NheC), cytotoxin K (CytK) and emetic toxin (EM1) using PCR with specific primers. Moreover, a Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) was used to screen ability of the isolates to produce haemolysin in broth and during fermentation of African oil bean seeds.The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. They were identified as Bacillus cereus sensu lato (42), Lysinibacillus xylanilyticus (3), Bacillus clausii (1), Bacillus licheniformis (1), Bacillus subtilis (1), and Bacillus safensis (1). B. cereus was the predominant Bacillus species and was present in all samples studied. Using ITS-PCR, interspecies diversity was observed among isolates, with six clusters representing each of the pre-cited species. Rep-PCR was more discriminatory (eight clusters) and allowed further differentiation at intraspecies level for the B. cereus and L. xylanilyticus isolates with two genotypes for each species. Genes encoding production of non-haemolytic enterotoxin (NheA, NheB, NheC) and cytotoxin K (CytK) genes were detected in all B. cereus isolates, while Hbl genes (HblA, HblC, HblD) were detected in only one isolate. The emetic-specific gene fragment was not detected in any of the isolates studied. None of the toxin genes screened was detected in isolates belonging to other Bacillus species. Using RPLA, haemolysin production was detected in one isolate of B. cereus, which showed positive amplicons for Hbl genes, both during cultivation in broth and during fermentation of oil bean seeds.

Enterotoxigenicity of Staphylococcus aureus Isolated from Food Handlers during Hajj Season in Saudi Arabia

Food poisoning during Hajj season is one of the main hazardous issues where most of the health services in Saudi Arabia are targeting to minimize every year during Hajj seasons. Ordinarily, food handlers are subjected to medical examination before assignment to work. However, they are mostly lacking proper training in food handling operations, mass feeding, and sanitary practices. This situation may encourage causing food poisoning especially with staphylococcus enterotoxins. 1516 clinical specimens from food handlers of different nationalities in Makkah were microbiologically investigated for bacterial pathogens during the hajj seasons of 2001-2002 in Makkah, Saudi Arabia. 129 Staphylococcus aureus isolates were isolated. Of which, 35% produced enterotoxins A, B, C and D singly or in pairs, when such enterotoxins were evaluated by Reversed Passive Latex Agglutination test (RPLA). Enterotoxins C and A, elaborated by 15.5% and 12.4%, isolates respectively, which showed the highest percentage. They were mostly isolated from nasal swabs than throat swabs. All isolates were resistant to Penicillin G. On the other hand, they were sensitive to Clindamycin, Oxacillin and Gentamicin when tested by Kirby-Bauer method. The (RPLA) method yielded satisfactory results.

The Liposome PCR Assay Is More Sensitive than the Vibrio cholerae Enterotoxin and Escherichia coli Heat-Labile Enterotoxin Reversed Passive Latex Agglutination Test at Detecting Cholera Toxin in Feces and Water

Practical detection of cholera toxin (CT) by a liposome PCR (LPCR) immunoassay was compared to that of an established V. cholerae enterotoxin and Escherichia coli heat-labile enterotoxin reversed passive latex agglutination (VET-RPLA) assay. LPCR detected CT in the range of 10 pg/ml to 100 ng/ml in simulated feces and environmental water. Detection by VET-RPLA required at least 4 to 19 ng/ml CT.

Genetic Variations in Shiga Toxin‐Producing Abilities of Bovine and Human Escherichia coli O157:H7

Cattle are a primary reservoir of Escherichia coli O157:H7, a major foodborne pathogen. The organism causes haemorrhagic colitis which can lead to serious complications, including haemolytic-uraemic syndrome. Although E. coli O157:H7 is widely prevalent in cattle and cattle environments, the number of human cases remain relatively low, suggesting possible strain diversity and differences in virulence between human and bovine strains. Shiga toxins, Stx1 and Stx2, are the major virulence factors. Differences in Stx2 production between human and bovine strains have been demonstrated previously, and isolates possessing the stx2 gene, but not producing Stx2 [toxin non-producing (TNP) strains] have been identified. In this study, 150 isolates (56 human, 94 bovine) were tested by PCR for stx2 upstream regions associated with TNP and the Q933 gene, which has been previously associated with toxin production. A reverse passive latex agglutination test was used to evaluate 107 isolates (50 human, 57 bovine) for Stx1 and Stx2 production. The percentages of human and bovine isolates positive for presence of the TNP regions were similar (57.1% and 53.1% respectively), while a higher percentage of human isolates was positive for Q933 gene (89.3% versus 54.3%). Stx2 production of ≥ 1:8 was found in 86.0% of human isolates compared with 26.3% of bovine isolates. Bovine isolates with the presence of the TNP regions were associated with significantly lower Stx2 production (P < 0.05), while the Q933 gene was associated with higher Stx2 production (P < 0.05). However, the presence of the TNP region was not associated (P > 0.05) with low Stx2 production in human isolates. Therefore, Q933 was a better indicator of high Stx2 production by human and bovine isolates and may be a useful screening method to assess their potential to cause human disease.

Canine serum C‐reactive protein detected by means of a near‐patient test for human C‐reactive protein

The aim of the study was to evaluate the reliability of a rapid human C-reactive protein near-patient slide reversed passive latex agglutination test (Randox) for the semi-quantitative determination of canine serum C-reactive protein. The concentration of C-reactive protein was determined in 244 canine serum samples by an established automated immunoturbidimetric method and in various predilutions by a commercially available reversed passive latex agglutination test for human C-reactive protein. The results were compared to assess if the reversed passive latex agglutination test reflected the results of the established method with special emphasis on the reversed passive latex agglutination test's ability to identify samples characterised as positive or negative by the established method. The reversed passive latex agglutination test reflected the C-reactive protein concentration in canine serum samples at all the tested predilutions (undiluted, 1:4, 1:8 and 1:16). When applying a predilution of 1:8, the positive and negative analytical predictive values for discriminating between positive and negative samples (according to the established quantitative method) were high (0.94 [0.82 to 0.99] and 0.97 [0.93 to 0.99], respectively). In conclusion, this near-patient test was able to reflect the serum C-reactive protein concentration in canine samples in a reliable and clinically useful manner and could be applicable for general practice for evaluating C-reactive protein levels in canine serum.

Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments

The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean (Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth and during the fermentation of African locust bean using the Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDEVIA). Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific of emetic toxin producers was also investigated using PCR with single pair and multiplex primers. Of 41 isolates, 29 Bacillus belonging to the species of B. cereus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus showed haemolysis on blood agar. Using RPLA, enterotoxin production was detected for three isolates of B. cereus in broth and all B. cereus (9) in fermented seeds. Using BDEVIA, enterotoxin production was detected in broth as well as in fermented seeds for all B. cereus isolates. None of the isolates belonging to the other Bacillus species was able to produce enterotoxins either by RPLA or BDEVIA. Nhe genes were detected in all B. cereus while Hbl and CytK genes were detected respectively in five and six B. cereus strains. A weak presence of Hbl (A, D) and CytK genes was detected in two isolates of B. subtilis and one of B. licheniformis but results were inconsistent, especially for Hbl genes. The emetic specific gene fragment EM1 was not detected in any of the isolates studied.

Ocorrência de Bacillus cereus em leite integral e capacidade enterotoxigênica das cepas isoladas

Pesquisaram-se a presença de Bacillus cereus e a produção de enterotoxinas produzidas por esses microrganismos em 120 amostras de diversos tipos de leite. Bacillus cereus foi isolado e identificado em 22 (73,3%), 15 (50,0%), 29 (96,7%) e quatro (13,3%) amostras de leite em pó, cru, pasteurizado e UAT (longa vida), respectivamente. Para a detecção de enterotoxinas pela técnica da alça ligada de coelho, foram positivos, respectivamente, três (13,6%), um (7,1%) e 10 (35,7%) microrganismos isolados das amostras de leite em pó, leite cru e leite pasteurizado. Pelo teste de aumento de permeabilidade vascular, dois (9,1%), um (7,1%), um (3,6%) e um (4,0%) microrganismos isolados de leite em pó, cru, pasteurizado e UAT apresentaram-se enterotoxigênicos, respectivamente. O uso da técnica de aglutinação passiva em látex demonstrou a produção da toxina diarréica por três (33,3%), sete (63,6%), quatro (30,8%) e oito (80,0%) microrganismos isolados, respectivamente, de leite em pó, cru, pasteurizado e UAT. Os resultados indicam um risco potencial, podendo colocar em risco a saúde dos consumidores desses produtos.

Prevalence of toxin producing strains of Staphylococcus aureus in a pediatric burns unit

The aims of the study were to determine the sites in a pediatric burns unit that were contaminated with Staphylococcus aureus. Samples from the environment in bedrooms and the common room were taken monthly for 6 months using blood agar for total counts and Baird-Parker agar for S. aureus. The air was sampled using an air-sampling device and settle plates. Hard and soft surfaces including bed, blanket, sofa, chair, taps, bathtub, soft toys, locker and cupboard in the same rooms were sampled using contact plates. Swabs were taken from staff monthly for 3 months. S. aureus isolates were tested for production of enterotoxins A–D and toxic shock syndrome toxin-1 using a reverse passive latex agglutination test. The results showed that S. aureus was recovered more frequently using settle plates than using the air sampler. All surfaces sampled were contaminated with S. aureus and contamination was greatest in frequently occupied rooms. A variety of toxin producing isolates were found with enterotoxin C isolates, either alone or in combination with TSST-1 (toxic shock syndrome toxin-1) dominant. The staff were transiently colonised with S. aureus strains with a different toxin production pattern. The results show that airborne transmission may be a route for infection by S. aureus and is responsible for contaminating the environment.

Determination of Toxigenic Potentials of Bacillus Strains Isolated from Okpehe, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.

INCIDENCE AND CHARACTERIZATION OF BACILLUS CEREUS IN MEAT AND MEAT PRODUCTS CONSUMED IN TURKEY

A total of 100 retail samples of meat and meat products were examined for Bacillus cereus using mannitol egg yolk polymyxin (MYP) agar as a selective isolation medium. Only 22.4% of the samples contained detectable levels of B. cereus, with counts ranging from log10 0.69 to 4.80 cfu/g, but a large number of other organisms up to log10 9.06 cfu/g were sometimes observed on the plates and may have masked the presence of B. cereus or inhibited growth. Two samples of soudjouck contained significant levels of B. cereus, sufficient enough to create a public health hazard. Selected isolates were tested for diarrheal enterotoxin production by a reversed passive latex agglutination (RPLA) test kit. Results showed no difference in the toxin production of B. cereus between beef, ground meat, soudjouck and pastrami samples. Plasmid-profile analysis and susceptibility to the six commonly used antimicrobial agents were done on selected B. cereus isolates. About 96.4% of the isolates contained plasmids. Plasmid sizes ranged between 2.5 and 3.5 kb. The isolates showed a high rate of resistance to oxacillin (OXA) and amoxicillin (AMC) and a low frequency of resistance to the other antimicrobial agents, with all of them being susceptible to vancomycin (VAN). Approximately 54% of the isolates showed multiple resistance. There was no apparent relationship between drug resistance and carriage of plasmids.

Conservation of cholera toxin gene in a strain of cholera toxin non-producing Vibrio cholerae O1.

BT23, a Vibrio cholerae O1 E1 Tor isolate, possesses the cholera toxin (CT) gene as determined by PCR. However, CT was not detected in the culture medium by the reversed passive latex agglutination test, nor in the whole cell lysate as examined by Western blotting. The toxin-coregulated pilus (TCP) was not detected by Western blotting. This suggests the presence of defects in the regulatory cascade. toxR, toxS and toxT, members of the regulatory cascade, were examined by PCR. toxR and toxS were conserved but toxT was not. CT and TCP production was complemented by transformation of toxT. The lack of toxT was suspected to be the cause of the undetectable production of CT in strain BT23.

Bacillus cereus may produce two or more diarrheal enterotoxins.

Bacillus cereus strains were tested for production of diarrheal enterotoxin by the reverse passive latex agglutination test and for presence of B. cereus enterotoxin gene (bceT) by polymerase chain reaction. About 50% of 56 B. cereus strains reacted positive in broth culture in the reverse passive latex agglutination test, while the bceT gene was detected in 41.1%. Sixteen percent of the strains were positive for both diarrheal enterotoxin and bceT gene. A 741 bp probe prepared from the polymerase chain reaction product detected bceT gene in all strains that were positive with the polymerase chain reaction. This study indicates a likelihood of two or more enterotoxins being produced by B. cereus which may be involved in causing diarrheal type food poisoning.

Staphylococcal toxins in patients with psoriasis, atopic dermatitis, and erythroderma, and in healthy control subjects

The aggravating role of Staphylococcus aureus superantigens is well known in atopic dermatitis (AD) but has not yet been proven in psoriasis (PS). We investigated the distribution of S aureus in the skin and nares of patients with AD, PS vulgaris, erythroderma, skin infections, and sepsis, and in healthy control subjects. A Staphylococcal enterotoxin test-reversed passive latex agglutination (SET-RPLAR) test was performed to determine Staphylococcal enterotoxins A, B, C, and D. S aureus was cultivated from lesional skin of 22 of 25 patients with AD and 15 of 25 patients with PS. Isolated strains were toxigenic in 44% for patients with AD and in 36% for patients with PS. The activity of disease in AD and PS according to the Severity Scoring of Atopic Dermatitis (SCORAD) or Psoriasis Area and Severity Index score, respectively, correlated significantly (P = .001) with an isolated toxigenic strain in both diseases. S aureus from skin infections was toxigenic in half of the patients. All patients with erythroderma harbored S aureus, mostly on their skin. In AD, sepsis and skin infections, toxin C and in PS toxin B was most often detected. S aureus was cultured in 12% of healthy persons. These strains were toxin negative. The limitations of these investigations are that other potentially acting enterotoxins, such as toxic shock syndrome toxin-1, which may play a role in aggravating disease, were not investigated with our latex agglutination test. In this study, S aureus was present in more than 50% of patients with AD and PS. We found that the severity of AD and PS significantly correlated to enterotoxin production of the isolated S aureus strains.

Incidence and Characterization of Bacillus cereus Isolated from Traditional Fermented Meals in Nigeria

The aim of this study was to examine the presence of Bacillus cereus in fermented meals used in food seasoning in Nigeria. The microbial profiles of iru and ogiri, two Nigerian fermented vegetable proteins, were examined for presence of B. cereus. In the 50 samples tested, B. cereus was detected in all the samples, with the level of detection ranging from log 6.3 to log 8.3 g−1 sample. Phenotypic characteristics of the B. cereus isolates showed that all of them could not ferment many sugars, most especially mannitol, but they utilized propionate citrate as a source of carbon and grew anaerobically. The isolates do not produce gas from glucose but hydrolyzed starch, casein, and gelatin. API-50CHB combined with API-20E identified the isolates as B. cereus. The diarrheal enterotoxin was detected by a reversed passive latex agglutination test kit. Results showed no significant difference in toxin production between ogiri and iruB. cereus isolated from different sources; all the isolates also demonstrated positive hemolytic activity. The API-ZYM enzyme profile showed that the strains have poor hydrolytic enzyme potential; hence, their possible contributions to the fermentation of vegetable protein is doubtful. This study established the proliferation of B. cereus in fermented protein meal and determined the diarrheal toxin production potential of the organism.

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

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. 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. Closely related Staph. aureus strains seem to be responsible for an unusual large proportion of bovine mastitis cases in geographically widely distinct locations. This is one of the first reports about the relatedness of sec/tst-gene positive Staph. aureus clinical isolates from bovine mastitis.

Detection and genotyping by real-time PCR of the staphylococcal enterotoxin genes sea to sej

This paper describes real-time fluorescence PCR assays for detecting and toxinotyping nine enterotoxin genes from Staphylococcus aureus. A universal set of primers allowed sea, seb, sec, sed, see, seg, seh, sei, sej enterotoxin genes from S. aureus to be detected in a single real-time PCR assay with the LightCycler (LC) instrument. Using the universal forward primer and a type-specific reverse primer, real-time PCR assays allowed the S. aureus enterotoxin genes to be specifically genotyped. A collection of S. aureus isolates ( n=83) was detected and further characterised for sea, seb, sec, sed, see, seg, seh, sei, sej, using real-time PCR assays, and data were compared with those obtained by conventional block cycler PCR. Isolates were also tested for their ability to produce staphylococcal enterotoxins A, B, C and D by a commercial reversed passive latex agglutination (RPLA) test. Real-time PCR assays developed on the LightCycler system (LC-PCR) are a powerful tool for rapid detection and toxinotyping of the enterotoxin genes sea to sej from S. aureus. The work offers a very quick, reliable and specific alternative to conventional block cycler PCR assays to identify the enterotoxin profile of toxigenic S. aureus.