Reverse Passive Latex Agglutination Research Articles

The level of fecal carriage and the toxic potential of Clostridium perfringens in the feces of a Taiwan subpopulation

Clostridium perfringens ranks among the most common agents of food poisoning in industrialized nations. Little is known about the level of fecal carriage and the toxic potential of this microorganism in Taiwan. One of the criteria to confirm that C. perfringens was the causative agent of food poisoning is to isolate 105 organisms/g from stools of food poisoning patients. The aim of the present study was to determine the carriage of C. perfringens in the feces of a Taiwan subpopulation. The percentage of strains that have toxic potential was also determined by using polymerase chain reaction (PCR) assay and reverse passive latex agglutination (RPLA). As a result, 30 (60%) of the 50 fecal samples carried C. perfringens. The spore counts were between 50 and 2.5×108 CFU/g. The average spore count of all the test samples was 4.3×102 CFU/g. Eleven (22%) of the samples carried more than 105 spores per gram. Only three (1.8%) isolates from fecal samples were cpe gene positive. The RPLA results were in accordance with the genotypic results. Our findings emphasize that the culture of C. perfringens from fecal samples needs to be supplemented with presence of cpe gene or enterotoxin production in the outbreak situation.

Contrasting Results from Two Commercial Kits Testing for the Presence of Clostridium perfringens Enterotoxin in Feces from Norovirus-Infected Human Patients.

Detection of Clostridium perfringens enterotoxin (CPE) is critical for disease surveillance; however, commercial testing kits produce contrasting results. We examined the cause of the differing results from a reversed passive latex agglutination (RPLA) assay (PET-RPLA Toxin Detection Kit) and an enzyme-linked immunosorbent assay (C. perfringens Enterotoxin ELISA Kit) using 73 human norovirus-positive fecal samples from gastroenteritis patients across 22 episodes in Japan. CPE was detected in 39/73 samples using the RPLA method; however, ELISA-based examination of 10 RPLA-positive samples produced negative results. Moreover, cpe was not detected in any of the RPLA-positive (n = 32) or -negative (n = 5) samples, and C. perfringens was only isolated from one RPLA-positive sample. An ELISA-based testing approach may be more reliable than RPLA assays for CPE detection from human fecal samples. These findings may also be applicable to the detection of other foodborne diseases.

Analysis of enterotoxin production by Staphylococcus aureus in Minas Frescal Cheese under different intrinsic and extrinsic factors

Staphylococcus aureus is a foodborne associated pathogen, mainly in dairy products due to the enterotoxin production. This study analyzed the production of classical toxins in cheese by S. aureus , in different temperatures, atmospheres, and levels of contamination. Minas Frescal Cheese were classified according to their microbiota. The cheese samples were classified as low or high level of contamination according to the concentration of that bacteria. An aliquot of 10 5 CFU of each strain (producing enterotoxin A, B, C, or D) in BHI was inoculated into two types of commercial cheeses presenting low and high contamination. Therefore, the samples were incubated at 8 °C, 15 °C, and 35 °C for 24, 48 and 72 hours in aerobic and anaerobic condition. The detection and quantification of enterotoxins were tested against toxin A, B, C and D using reverse passive latex agglutination. There was no enterotoxin production at 8 °C in all treatment, consequently, no statistically significant difference was observed. At 15 °C, the SEA production was significantly higher in BHI broth than in cheese with low contamination in all three times evaluated. Regarding SEB, in aerobic condition at 15 °C after 72 hours, the BHI broth showed significantly higher production than the low contaminated cheese. S. aureus produced a different range of enterotoxin according to the substrate. In BHI, the production was higher than in cheese, suggesting that food substrates more appropriate to analyze the real enterotoxin production. Besides, the microbiota presents can interfere in its production.

Toxigenic properties and stx phage characterization of Escherichia coli O157 isolated from animal sources in a developing country setting

BackgroundIn many Asian countries including Bangladesh E. coli O157 are prevalent in animal reservoirs and in the food chain, but the incidence of human infection due to E. coli O157 is rare. One of the reasons could be inability of the organism from animal origin to produce sufficient amount of Shiga toxin (Stx), which is the main virulence factor associated with the severe sequelae of infection. This study aimed to fill out this knowledge gap by investigating the toxigenic properties and characteristics of stx phage of E. coli O157 isolated from animal sources in Bangladesh.ResultsWe analysed 47 stx2 positive E. coli O157 of food/animal origin for stx2 gene variants, Shiga toxin production, presence of other virulence genes, stx phage insertion sites, presence of genes associated with functionality of stx phages (Q933 and Q21) and stx2 upstream region. Of the 47 isolates, 46 were positive for both stx2a and stx2d while the remaining isolate was positive for stx2d only. Reverse Passive Latex Agglutination assay (RPLA) showed that 42/47 isolates produced little or no toxin, while 5 isolates produced a high titre of toxin (64 to 128). 39/47 isolates were positive for the Toxin Non-Producing (TNP) specific regions in the stx2 promoter. Additionally, all isolates were negative for antiterminator Q933while a majority of isolates were positive for Q21 gene suggesting the presence of defective stx phage. Of the yehV and wrbA phage insertion sites, yehV was found occupied in 11 isolates while wrbA site was intact in all the isolates. None of the isolates was positive for the virulence gene, cdt but all were positive for hlyA, katP, etpD and eae genes. Isolates that produced high titre Stx (n = 5) produced complete phage particles capable of infecting multiple bacterial hosts. One of these phages was shown to produce stable lysogens in host strains rendering the Stx2 producing ability.ConclusionDespite low frequency in the tested isolates, E. coli O157 isolates in Bangladesh carry inducible stx phages and have the capacity to produce Stx2, indicating a potential risk of E. coli O157 infection in humans.

Detection of Staphylococcus aureus enterotoxigenic strains in bovine raw milk by reversed passive latex agglutination and multiplex polymerase chain reaction.

Aim:This review gives an outline of the assessment of enterotoxigenic Staphylococcus aureus tainting levels in raw milk from different sources in Egypt and characterization of enterotoxigenic strains utilizing a technique in light of PCR to identify genes coding for the production of staphylococcal enterotoxin (SE). The obtained data were compared with results from the application of the reversed passive latex.Materials and Methods:Multiplex PCR and reversed passive latex agglutination (RPLA) were used. A total of 141 samples of raw milk (cow’s milk=33, buffalo’s milk=58, and bulk tank milk=50) were investigated for S. aureus contamination and tested for enterotoxin genes presence and toxin production.Results:S. aureus was detected in 23 (16.3%) samples phenotypically and genotypically by amplification of nuc gene. The S. aureus isolates were investigated for SEs genes (sea to see) by multiplex PCR and the toxin production by these isolates was screened by RPLA. SEs genes were detected in six isolates (26.1%) molecularly; see was the most observed gene where detected in all isolates, two isolates harbored seb, and two isolates harbored sec. According to RPLA, three isolates produced SEB and SEC.Conclusion:The study revealed the widespread of S. aureus strains caring genes coding for toxins. The real significance of the presence of these strains or its toxins in raw milk and their possible impact a potential hazard for staphylococcal food poisoning by raw milk consumption. Therefore, detection of enterotoxigenic S. aureus strains in raw milk is necessary for consumer safety.

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.

Housefly (Musca domestica) as Carrier of Enterotoxigenic Staphylococcus aureus in Broiler Farms in Iran: Is it Important for Public Health?

Background: During the last decade, the prevalence of insect borne diseases due to contaminated food as well as the outbreaks of diseases due to enterotoxin-producing Staphylococcus aureus (ETSA) strains has increased. Objectives: This study was conducted to determine the prevalence rate, enterotoxigenecity, and antimicrobial resistance of S. aureus isolated from M. domestica collected from the residential areas of nine districts broiler farms in Zabol, Iran. Materials and Methods: The flies were captured with a sterile nylon net and washed twice with distilled water. The contents were streaked onto selective media and S. aureus was identified using the conventional biochemical tests. Antimicrobial susceptibility testing was performed using CLSI guideline. Staphylococcal enterotoxins (SEs), SEA, SEB, SEC and SED, was detected by the reverse passive latex agglutination (PRLA) method. Coagulase typing was performed with coagulase typing kit. Results: A total of 87 (17%) S. aureus was isolated from 450 samples. The ability to synthesize staphylococcal enterotoxins (SEs) was determined in 62 of 87 (71%) isolates. SE type B was the most common enterotoxin found in the isolated S. aureus (45%), followed by SE type A (26%), SE type C (5%), SE type D (8%), SEA + SEC (7%) and SEA + SEB (7%). Among the antibiotic tested, Penicillin was the most resistant antibiotic tested. Sixty seven percent of isolates belonged to coagulase type III, VI, VII, VIII, IV, and V. Conclusions: Our results suggest that S. aureus, including ETSA, is being carried by house flies such as M. domestica and may contribute to the spread of pathogenic isolates, with an impact on public health.

Occurrence of Enteric Pathogens on Fresh Produce Grown on Irrigated Soils

Aims: To assess the potential health risks of fresh produce grown on irrigated soils treated with manure in Kano State, a large produce region in Nigeria. Methodology: Fresh produce irrigated with wastewater on manure treated soils were assessed for the prevalence, serotype distribution and toxigenicity of Escherichia coli O157, Salmonella spp and Vibrio cholerae in a large produce region in Nigeria. A total of 230 samples obtained from five designated produce locations were examined using selective isolation method with prior enrichment. Fresh produce comprised carrots, cabbage, cucumber, lettuce, tomatoes, spinach, green peas and spring onions. Suspect isolates were identified and characterized by conventional biochemical methods and Microbact 24E (Oxoid, UK) kit. Confirmed isolates were serotyped and E. coli O157 and Vibrio cholerae O1 were assayed for their toxigenic potentials using the Reverse Passive Latex Agglutination kit. The enterotoxigenicity of Salmonella spp was determined by detection of stn gene using polymerase chain reaction (PCR) techniques. Original Research Article Abakpa et al.; BMRJ, 6(1): 13-23, 2015; Article no.BMRJ.2015.054 14 Results: Results obtained showed that overall, Salmonella spp and Escherichia coli O157 had the highest prevalence of 17.0% and 10.9%, respectively. Both were most commonly detected from fresh produce. The serotypes of Salmonella detected include Salmonella typhi (51.3%), Salmonella paratyhpi (20.5%) and Salmonella typhimurium (28.2%); strains of Vibrio cholerae O1 detected include Vibrio cholerae O1 of the Ogawa, Inaba and the Hikojima serotypes. Conclusion: The use of untreated irrigation water in vegetable production represents a significant route of transmission of diarrheal pathogen to humans and hence represents a public health risk. We recommend proper and adequate wastewater treatment before use.

Effect of rifampicin and gentamicin on Shiga toxin 2 expression level and the SOS response in Escherichia coli O104:H4.

A novel pathotype, Shiga toxin-producing Escherichia coli O104:H4, was the cause of a severe outbreak that affected European countries, mainly Germany, in 2011. The effect of different regimens of rifampicin and gentamicin were evaluated to determine possible treatment modes for the novel strain, and to evaluate the SOS response and its effect on toxin release. Pulsed-field gel electrophoresis (PFGE) was performed on the novel E. coli O104:H4 pathotype and two pre-outbreak E. coli O104:H4 CDC strains. Transcript levels of the stx2 and recA gene (SOS response inducer) were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) in the novel E. coli O104:H4 samples subjected to different regimens of rifampicin and gentamicin. Consequently, reverse passive latex agglutination (RPLA) was used to determine the Stx2 titers in these samples. Western blot was performed to determine the LexA levels (SOS response repressor) in E. coli O104:H4. The efficacy of treatment with antimicrobial agents was assessed in BALB/c mice. The outbreak and pre-outbreak strains are closely related as shown by PFGE, which demonstrated slight genomic differences between the three strains. The transcription level of the stx2 gene in the new pathotype was 1.41- and 1.75-fold that of the 2009 EL-2050 and 2009 EL-2071 pre-outbreak strains, respectively. Moreover, the transcription level of the stx2 gene in the new pathotype was substantially decreased as a result of treatment with the different concentrations of the antimicrobial agents, but was enhanced when the antibiotics were administered at two subinhibitory levels. RPLA data were in accordance with the qRT-PCR results. E. coli O104:H4 exposed to gentamicin at both sub-minimum inhibitory concentration (MIC) levels led to high transcription levels of the recA gene and lack of expression of the LexA protein, implying that the SOS response was activated. Rifampicin at both sub-MIC levels resulted in low transcript levels of the recA gene, indicating that the SOS response was not induced. In vivo, the highest survival rate in BALB/c mice was observed in the group that was treated with the minimum bactericidal concentration (MBC) of gentamicin. The use of antimicrobial agents in E. coli O104:H4 infection seems to be effective at the MIC and MBC levels. This provides a promising ground for treatment of E. coli O104:H4.

BIOCHEMICAL, SEROLOGICAL AND MOLECULAR STUDIES FOR. COLI ISOLATED from STUDIES FOR SHEEP AND GOAT SHEEP AND GOAT

Staphylococcus aureus is one of the most significant pathogens causing bovine mastitis. Among the secreted staphylococcal virulence factors, enterotoxins which can induce food poisoning, toxic shock syndrome (TSS) and bovine mastitis. A total of 140 gram-positive, catalase-positive cocci were considered as an etiological agent of clinical and subclinical bovine mastitis, being tested for sugar and mannitol fermentation, urease production, citrate utilization, egg yolk factor production, haemolytic properties and coagulase production. All 140 strains were identified as Staphylococcus aureus. Staphylococcal Enterotoxin Test - Reverse Passive Latex Agglutination (SET-RPLA) is the most commonly used method for the determination of enterotoxins of S. aureus. It has several advantages at present, including high specificity, simplicity and economy. It was found that six strains produced enterotoxin D, whereas, other strains didn’t produce enterotoxins as detected by the SET-RPLA. The relationship between enterotoxigenicity and coagulase production demonstrated that all enterotoxigenic isolates showed positive reaction for coagulase production, but not all coagulase positive staphylococcal isolates were enterotoxigenic

Prevalence of staphylococcal enterotoxins inStaphylococcus pseudintermediusisolates from dogs with pyoderma and healthy dogs

To investigate the role of staphylococcal enterotoxins (SEs) produced by Staphylococcus pseudintermedius in the pathogenesis of pyoderma, isolates from dogs with pyoderma and healthy dogs were analyzed. According to reverse passive latex agglutination, 14/184 isolates (7.6%) from dogs with pyoderma and 9/87 (10.3%) from healthy dogs produced SEs (SEA, SEC or SED). According to multiplex PCR, 99 isolates (53.7%) from dogs with pyoderma and 97 (90.8%) from healthy dogs possessed one or more se genes. There was no significant difference regarding ses between dogs with pyoderma and healthy dogs. Therefore, SEs may not be a direct virulence factor in pyoderma.

Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products

Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2ng/ml within 15min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1ng/ml in milk; the SEs were detected within 12min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products.

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.

Classical enterotoxins of coagulase-positive Staphylococcus aureus isolates from raw milk and products for raw milk cheese production in Ireland

Toxin-producing Staphylococcus aureus can be present in raw milk and therefore in cheese made from raw milk. To determine the number and type of toxin producers in raw milk used for raw milk cheese production in Ireland, 117 samples of raw milk and related products from five raw milk suppliers, to four raw milk cheesemakers in the South of Ireland, were analysed for coagulase positive S. aureus. Enumeration, using ISO 688-2 and plating on Baird Parker Rabbit Plasma Fibrinogen selective agar showed samples were within limits set by EC regulations. Isolates (151 from 81 positive samples) were characterised for production of staphylococcal enterotoxins (SEs) SEA, SEB, SEC and SED by reverse passive latex agglutination (SET-RPLA) and by multiplex polymerase chain reaction for the sea, seb, sec, sed and see genes. The results showed 83.2% of the isolates did not contain the se genes or the toxin producing capability tested for. From only one supplier, 26 isolates contained the sec gene and produced SEC. Within these 26 isolates, there were only two PFGE types. One SEC-producing isolate showed no toxin production when grown in sterile 10% reconstituted skim milk at 10 °C and 12 °C for 96 and 74 h, respectively. Low concentrations of SEC were produced at 14 °C and 16 °C after 74 and 55 h, respectively. The results of this survey indicate that milk used for raw milk cheese production in Ireland poses a limited risk to public health, although further studies on occurrence of toxin producing S. aureus should be undertaken.

Relationship between stx Genotype and Stx2 Expression Level in Shiga Toxin-Producing Escherichia coli O157 Strains

To determine the expression level of Shiga toxin (Stx) 2-related toxins (Stx2 and Stx2c) produced by each of 33 Stx-producing Escherichia coli (STEC) O157 strains, stx2 and stx2c mRNAs (stx2-related mRNA) were measured using real-time PCR with primers that recognize sequences common to stx2 and stx2c. The amount of Stx2 and Stx2c protein was measured using a reversed passive latex agglutination (RPLA) kit. Expression of stx2-related mRNA was significantly higher in STEC O157 strains carrying the stx2 gene (i.e., stx2, stx1/stx2, or stx2/stx2c) than in most strains that carried the stx2c gene but not the stx2 gene (i.e., stx2c or stx1/stx2c). RPLA might not measure the precise amount of each toxin variant; nevertheless, stx2-inclusive strains had 40-fold higher mean toxin titers than did strains that carried the stx2c gene but not the stx2 gene, with the exception of 1 stx2c strain. Interestingly, 1 stx2c strain that was isolated from a patient with severe hemorrhagic diarrhea had the highest stx2-related mRNA expression and the highest toxin titer of all 33 STEC O157 strains. Taken together, these findings indicated that measurement of stx2-related mRNA expression could reflect differences in production levels of toxins among STEC strains.

Verotoxigenic Escherichia coli isolated from human samples in Slovenia.

BACKGROUND Shiga toxin-producing E. coli or Vero cytotoxin-producing E. coli (VTEC) are characterised by the ability to produce either one or both cytotoxins referred to as Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). VTEC infection may result in life-threatening conditions such as haemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Due to different methods of monitoring and identification of these bacteria in recent years, the existing data on reported cases of VTEC infections probably do not reflect reality. Our study of VTEC strains isolated in different regions of Slovenia, showed serogroups, major virulence factors and specific epidemiological data that can serve as a basis for further laboratory and epidemiological surveillance of VTEC infections. METHODS A total of 66 VTEC strains, isolated from stool samples of patients with diarrhoea from the year 1993 to 2009, were collected at NIPH (National Institute of Public Health). The data of patient’s age and gender, onset of illness and clinical manifestation of disease were gathered. The serogroups of isolated strains were determined with antisera following manufacturer’s instructions. The ability to produce verocytotoxins was tested using the reversed passive latex agglutination method. The presence of genes for intimin (eae), enterohaemolysin (ehxA) and verotoxins (vtx1 and vtx2) were determined by polymerase chain reaction (PCR). RESULTS Infection with VTEC was encountered throughout the year, but most people were ill in the summer and autumn months. More than half of patients (57.6 %) were younger than five years. Collected VTEC strains belonged to serogroups O17, O26, O91, O103, O111, O113, O126, O128, O145, O148 and O157 (the most frequent were O157 and O26). A high percentage of VTEC strains showed the presence of intimin (86.4 %) and enterohaemolysin (86.4 %) genes. The gene for vtx1 was found in 22.7 % of strains, the vtx2 in 57.6 % of strains, while the presence of both genes was determined in 19.7 % of strains. The presence of the vtx2 gene was determined in all strains associated with HUS and TTP, most of them possessed the eae and ehxA genes too. These patients were mostly older people and young children. CONCLUSIONS Most infections with VTEC occurred in the warmer months of the year, most patient were small children. The severity of VTEC infection is determined by several factors such as the E. coli serogroup, the type of Shiga toxin produced and presence of other virulence genes. The most common serogroups among the study strains were O157 and O26. VTEC O26 has been the most commonly isolated serogroup in recent years, nevertheless more and more different serological groups began to emerge. In all strains associated with HUS and TTP, the vtx2 gene was determined. Further typing of verocytotoxin encoding genes will contribute to assess the risk for complications with VTEC infection. The study provides insights about the age of the patients, seasonal distribution of disease, serogroups and genotypes of the agent.

Phenotype characterization of Staphylococcus species strains isolated from buffalo (Bubalus bubalis) milk

The objective of the current study was to isolate and identify phenotypes of Staphylococcus spp. strains derived from buffalo (Bubalus bubalis) milk. A total of 548 milk samples from 137 buffalo were cultured in Columbia agar enriched with 5% defibrinated sheep blood. Determination of the capacity of Staphylococcus aureus to produce enterotoxins A-D and toxic shock syndrome toxin-1 (TSST-1) was achieved by reverse passive latex agglutination (RPLA). Antimicrobial sensitivity of S. aureus strains was evaluated using the disk diffusion technique, and β-lactamase detection was achieved using the chromogenic test with paper discs impregnated with nitrocefin. From all the mammary quarters examined, 36 (10.8%) were positive for Staphylococcus spp., 83.3% were coagulase-negative staphylococcus (CNS), 11.1% were coagulase-positive staphylococcus (CPS), and 5.6% were of CPS+CNS positive. All isolates of S. aureus produced at least 1 toxin and 5 out of 6 isolates (83.0%) produced β-lactamase. One hundred percent of S. aureus isolates were sensitive to methicillin and amoxicillin + clavulanic acid, and resistant to ampicillin, penicillin, and oxacillin. Analysis of the results obtained in the current study highlight the epidemiologic importance of buffalo milk regarding the production of enterotoxins and TSST-1 and the potential risk to public health.

Decrease in Shiga toxin expression using a minimal inhibitory concentration of rifampicin followed by bactericidal gentamicin treatment enhances survival of Escherichia coli O157:H7-infected BALB/c mice

BackgroundTreatment of Escherichia coli O157:H7 infections with antimicrobial agents is controversial due to an association with potentially fatal sequelae. The production of Shiga toxins is believed to be central to the pathogenesis of this organism. Therefore, decreasing the expression of these toxins prior to bacterial eradication may provide a safer course of therapy.MethodsThe utility of decreasing Shiga toxin gene expression in E. coli O157:H7 with rifampicin prior to bacterial eradication with gentamicin was evaluated in vitro using real-time reverse-transcription polymerase chain reaction. Toxin release from treated bacterial cells was assayed for with reverse passive latex agglutination. The effect of this treatment on the survival of E. coli O157:H7-infected BALB/c mice was also monitored.ResultsTranscription of Shiga toxin-encoding genes was considerably decreased as an effect of treating E. coli O157:H7 in vitro with the minimum inhibitory concentration (MIC) of rifampicin followed by the minimum bactericidal concentration (MBC) of gentamicin (> 99% decrease) compared to treatment with gentamicin alone (50-75% decrease). The release of Shiga toxins from E. coli O157:H7 incubated with the MIC of rifampicin followed by addition of the MBC of gentamicin was decreased as well. On the other hand, the highest survival rate in BALB/c mice infected with E. coli O157:H7 was observed in those treated with the in vivo MIC equivalent dose of rifampicin followed by the in vivo MBC equivalent dose of gentamicin compared to mice treated with gentamicin or rifampicin alone.ConclusionsThe use of non-lethal expression-inhibitory doses of antimicrobial agents prior to bactericidal ones in treating E. coli O157:H7 infection is effective and may be potentially useful in human infections with this agent in addition to other Shiga toxin producing E. coli strains.

Detection of enterotoxins produced by B. cereus through PCR analysis of ground and roasted coffee samples in Rio de Janeiro, Brazil

Coffee is one of the most appreciated drinks in the world. Coffee ground is obtained from the fruit of a small plant that belongs to the genus Coffea. Coffea arabica and Coffea canephora robusta are the two most commercially important species. They are more commonly known as arabica and robusta, respectively. Two-thirds of Coffea arabica plants are grown in South and Central America, and Eastern Africa - the place of origin for this coffee species. Contamination by microorganisms has been a major matter affecting coffee quality in Brazil, mainly due to the harvesting method adopted. Brazilian harvests are based on fruits collected from the ground mixed with those that fall on collection cloths. As the Bacillus cereus bacterium frequently uses the soil as its environmental reservoir, it is easily capable of becoming a contaminant. This study aimed to evaluate the contamination and potential of B. cereus enterotoxin genes encoding the HBL and NHE complexes, which were observed in strains of ground and roasted coffee samples sold in Rio de Janeiro. The PCR (Polymerase Chain Reaction) results revealed high potential of enterotoxin production in the samples. The method described by Speck (1984) was used for the isolation of contaminants. The investigation of the potential production of enterotoxins through isolates of the microorganism was performed using the B. cereus enterotoxin Reverse Passive Latex Agglutination test-kit (BCET-RPLA, Oxoid), according to the manufacturer's instructions. The potential of enterotoxin production was investigated using polymerase chain reaction (PCR) methods for hblA, hblD and hblC genes (encoding hemolysin HBL) and for nheA, nheB and nheC genes (encoding non-hemolytic enterotoxin - NHE). Of all the 17 strains, 100% were positive for at least 1 enterotoxin gene; 52.9% (9/17) were positive for the 3 genes encoding the HBL complex; 35.3% (6/17) were positive for the three NHE encoding genes; and 29.4% (5/17) were positive for all enterotoxic genes.