Heat resistance and presence of genes encoding staphylococcal enterotoxins evaluated by multiplex-PCR of Staphylococcus aureus isolated from pasteurized camel milk.
ABSTRACT: Milk pasteurization eliminates vegetative pathogenic microorganisms and reduces microorganisms associated with spoilage. Camel milk is a well-accepted, traditionally consumed food in Arab countries. The present study aimed to investigate the microflora of pasteurized camel milk sold in Riyadh City, Saudi Arabia. The heat resistance of the microflora was tested in culture medium and lab-sterilized milk, and its composition was verified by multiplex polymerase chain reaction (PCR) using specific primers. Further verification was performed by using separate specific primers. The identified strain survived heat treatment at 65, 72, 80, 85, and 90°C for 30, 15, 10, 5, and 2 min, respectively. An unanticipated result was obtained when an enterotoxin producing strain of Staphylococcus aureus showed abnormal resistance to heat treatment. The enterotoxin gene within the PCR fragment was identified as enterotoxin C by DNA sequencing. During Basic Local Alignment Search Tool (BLAST) analysis, the isolated enterotoxin C genes showed >99% similarity to published database sequences of the Staphylococcus aureus strain SAI48 staphylococcal enterotoxin C variant v4 (sec) gene. The decimal reduction value (D-value) at 90°C (D90) was determined after 10 s. This is the first time to report this abnormally heat resistant and enterotoxin-producing strain of Staphylococcus aureus. The use of ultra-high temperatures (UHTs) is preferable for reducing or killing bacteria in camel milk, especially if this problem is encountered in many camel milk factories.
Project description:Antibiotic- and heat-resistant bacteria in camel milk is a potential public health problem. Staphylococcus aureus (S. aureus) is an opportunistic pathogen in humans, dairy cattle and camels. We characterized the phenotype and genotype of methicillin-resistant staphylococcal strains recovered from pasteurized and raw camel milk (as control) distributed in the retail markets of Saudi Arabia. Of the 100 samples assessed between March and May 2016, 20 S. aureus isolates were recovered from pasteurized milk, 10 of which were resistant to cefoxitin, and as such, were methicillin-resistant. However, raw camel milk did not contain methicillin-resistant S. aureus (MRSA). Antimicrobial susceptibility tests showed that the resistance ratio for other antibiotics was 60%. We performed a polymerase chain reaction (PCR) assay using primers for the methicillin-resistant gene mecA and nucleotide sequencing to detect and verify the methicillin-resistant strains. Basic local alignment search tool (BLAST) analysis of the gene sequences showed a 96-100% similarity between the resistant isolates and the S. aureus CS100 strain's mecA gene. Ten of the methicillin-resistant isolates were heat-resistant and were stable at temperatures up to 85°C for 60 s, and three of these were resistant at 90°C for 60 or 90 s. The mean decimal reduction time (D85-value) was 111 s for the ten isolates. Sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis (PAGE) showed that there was no difference in the total protein profiles for the ten methicillin heat-resistant S. aureus (MHRSA) isolates and for S. aureus ATCC 29737. In conclusion, a relatively high percentage of the tested pasteurized camel milk samples contained S. aureus (20%) and MHRSA (10%).
Project description:Staphylococcal enterotoxins (SEs) are a family of 17 major serological types of heat-stable enterotoxins that are one of the leading causes of gastroenteritis resulting from consumption of contaminated food. SEs are considered potential bioweapons. Many Staphylococcus aureus isolates contain multiple SEs. Because of the large number of SEs, serological typing and PCR typing are laborious and time-consuming. Furthermore, serological typing may not always be practical because of antigenic similarities among enterotoxins. We report on a microarray-based one-tube assay for the simultaneous detection and identification (genetic typing) of multiple enterotoxin (ent) genes. The proposed typing method is based on PCR amplification of the target region of the ent genes with degenerate primers, followed by characterization of the PCR products by microchip hybridization with oligonucleotide probes specific for each ent gene. We verified the performance of this method by using several other techniques, including PCR amplification with gene-specific primers, followed by gel electrophoresis or microarray hybridization, and sequencing of the enterotoxin genes. The assay was evaluated by analysis of previously characterized staphylococcal isolates containing 16 ent genes. The microarray assay revealed that some of these isolates contained additional previously undetected ent genes. The use of degenerate primers allows the simultaneous amplification and identification of as many as nine different ent genes in one S. aureus strain. The results of this study demonstrate the usefulness of the oligonucleotide microarray assay for the analysis of multitoxigenic strains, which are common among S. aureus strains, and for the analysis of microbial pathogens in general.
Project description:Food poisoning caused by Staphylococcus aureus (S. aureus) toxins is considered one of the foremost public health threat that usually occurs through the ingestion of raw milk contaminated with staphylococcal enterotoxins. The current study spotlights on the prevalence, antibiogram and genetic diversity of S. aureus enterotoxin genes. One hundred and fifty of raw milk (90) and ice cream (60) samples were randomly collected from local markets from Sadat city, Egypt. S. aureus was recovered from 44% of raw milk and 20% of ice cream samples. The identification for the obtained S. aureus isolates was confirmed through targeting the nuc gene. Antibiogram pattern of 32 S. aureus isolates showed high resistance to Cefoxitin, Sulpha/Trimethoprim, Tetracycline, Norfloxacin, Penicillin and Cephradine. However, high susceptibility to Gentamycin and Vancomycin were observed. Multiplex PCR was a competent practise for the recognition of Staphylococcus enterotoxin (SE) genes (SEA, SEB and SED). The phylogenetic analysis of the SED gene of enterotoxigenic S. aureus strains showed identical similarity with 100% to each other and high similarity with other international isolates in GenBank from different localities and sources. The frequency of enterotoxigenic S. aureus strains in milk products could have serious hazardous effects on humans. These results suggested possible strains transmission between different geographical areas through the food and milk product trades.
Project description:Antibacterial peptides were isolated and purified from whey proteins of camel milk (CaW) and cow milk (CoW) and their antimicrobial activities were studied. The whey proteins were hydrolyzed using trypsin, and the degree of hydrolysis was identified by gel electrophoresis. The whey hydrolysate (WH) was purified using ultrafiltration and Dextran gel chromatography to obtain small peptides with antibacterial activity. The effect of the antimicrobial peptides on the morphology of bacterial strains was investigated using transmission electron microscopy. Their amino acid composition and antimicrobial activities were then determined. Polypeptides CaWH-III (<3 kDa) and CoWH-III (<3 kDa) had the strongest antibacterial activity. Both Fr.A2 (CaWH-?'s fraction 2) and Fr.B1 (CoWH-?'s fraction 1) had antibacterial effects toward Escherichia coli and Staphylococcus aureus, with minimum antimicrobial mass concentrations of 65 mg/mL and 130 mg/mL for Fr.A2, and 130 mg/mL and 130 mg/mL for Fr.B1, respectively. The highly active antimicrobial peptides had high amounts of alkaline amino acids (28.13% in camel milk Fr.A2 and 25.07% in the cow milk Fr.B1) and hydrophobic amino acids. (51.29% in camel milk Fr.A2 and 57.69% in the cow milk Fr.B1). This results showed that hydrolysis of CaW and CoW using trypsin produced a variety of effective antimicrobial peptides against selected pathogens, and the antibacterial activity of camel milk whey was slightly higher than that of cow milk whey.
Project description:BACKGROUND:In the previous studies using the commercial ELISA kit, the existence of staphylococcal superantigens has been reported in synovial fluid of patients with rheumatoid arthritis (RA). OBJECTIVES:This study aimed to design molecular methods to detect staphylococcal enterotoxin C in synovial fluid of patients with rheumatoid arthritis. MATERIALS AND METHODS:In this experimental study, Staphylococcus aureus strain producing enterotoxin C was used as the reference strain. The polymerase chain reaction (PCR) was set up by design a specific pair of primers. Besides bacterial culture, 50 synovial fluid samples of patients with rheumatoid arthritis were subjected to DNA extraction, and then PCR amplification was carried out according to the protocol. All samples were examined by ELISA method for enterotoxin C. The data were descriptively analyzed. RESULTS:The results of bacterial culture were negative for all samples. The results showed that 66% (33 cases) of samples contained entC gene and only 46% (23 cases) have also enterotoxin C. The interesting finding was that the results of ELISA and PCR were the same and have shown only 22 positive cases (44%samples) for staphylococcal enterotoxin C. CONCLUSIONS:Based on the findings of this study, S. aureus enterotoxin C (SEC) has been detected in synovial fluid of patients with rheumatoid arthritis by PCR and ELISA methods. These valuable findings may describe the exact etiology of the RA and as well as change the methods of its diagnosis and treatment. This is the first research, which has shown the staphylococcal entC gene in synovial fluid of RA patients. However, S. aureus strains can produce more than 20 types of enterotoxins. Therefore, its involvement on rheumatoid arthritis pathogenesis makes an important challenge in the future. In this regard, further investigation on the other enterotoxins is necessary.
Project description:Bacillus cereus sensu lato species, as well as Staphylococcus aureus, are important pathogenic bacteria which can cause foodborne illness through the production of enterotoxins. This study characterised enterotoxin genes of these species and examined growth and enterotoxin production dynamics of isolates when grown in milk or meat-based broth. All B. cereus s. l. isolates harboured nheA, hblA and entFM toxin genes, with lower prevalence of bceT and hlyII. When grown at 16 °C, toxin production by individual B. cereus s. l. isolates varied depending on the food matrix; toxin was detected at cell densities below 5 log10(CFU/mL). At 16 °C no staphylococcal enterotoxin C (SEC) production was detected by S. aureus isolates, although low levels of SED production was noted. At 30 °C all S. aureus isolates produced detectable enterotoxin in the simulated meat matrix, whereas SEC production was significantly reduced in milk. Relative to B. cereus s. l. toxin production, S. aureus typically required reaching higher cell numbers to produce detectable levels of enterotoxin. Phylogenetic analysis of the sec and sel genes suggested population evolution which correlated with animal host adaptation, with subgroups of bovine isolates or caprine/ovine isolates noted, which were distinct from human isolates. Taken together, this study highlights the marked differences in the production of enterotoxins both associated with different growth matrices themselves, but also in the behaviour of individual strains when exposed to different food matrices.
Project description:We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureus that utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.
Project description:To investigate the distribution of staphylococcal enterotoxin (SE) A to I (SEA to SEI) genes (sea to sei) in Staphylococcus aureus, 146 isolates obtained in Japan from humans involved in and samples from food poisoning outbreaks, healthy humans, cows with mastitis, and bovine raw milk were analyzed by multiplex PCR. One hundred thirteen (77.4%) S. aureus isolates were found to be positive for one or more se genes. The se genotype was classified into 14 genotypes. seg and sei coexisted in the same S. aureus strain. The newly developed sandwich enzyme-linked immunosorbent assay showed that most seh-harboring S. aureus isolates were able to produce a significant amount of SEH. However, most of the S. aureus isolates harboring seg and about 60% of the isolates harboring sei did not produce a detectable level of SEG or SEI, while reverse transcription-PCR analysis proved that the mRNAs of SEG and SEI were transcribed in S. aureus strains harboring seg and sei genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning.
Project description:We present the first complete genome sequence of a Staphylococcus aureus strain assigned to clonal complex 12. The strain was isolated in a food poisoning outbreak due to contaminated potato salad in Switzerland in 2009, and it produces staphylococcal enterotoxin B.
Project description:BACKGROUND: Widespread in the environment, Staphylococcus spp. infect animals and humans as normal flora or pathogens. By extending our recent report of multi-drug resistant (MDR) S. aureus in dairy goats, this study investigated the staphylococcal infection and characterized the MDR-S. aureus and methicillin-resistant S. aureus (MRSA) isolates collected from goats in 2008 to elucidate the appearance of MRSA in goats and the mastitis associated staphylococcus enterotoxin (SE) types. A total of 555 samples were collected from six goat parts and three environmental sources among four dairy goat farms in southern Taiwan. Coagulase-positive and negative Staphylococcus spp. (CPS and CNS, respectively) were also identified. Furthermore, predominant SE genes of nine enterotoxin genes sea through sej along with antimicrobial resistance and genetic variations were determined. RESULTS: In total, 137 staphylococcal strains were identified and found predominantly in milk, and in the vagina, anus, and nasal cavity. The most prevalent species was S. lentus, followed by S. aureus, S. epidermidis, and S. xylosus. Enterotoxin genes were not identified in any CNS isolates, however sec and see were identified only in S. aureus associated with mastitis in goat. In compared to the isolates from 2006 to 2007, 27 S. aureus isolates from 2008 were found to be more resistant to ampicillin, cephalothin, oxacillin, oxytetracycline, penicillin G, and tetracycline. Eleven MRSA isolates were identified and belonged to SCCmec type III (nine isolates) as the major type and SCCmec type II (two isolates). These MRSA isolates revealed pulse-field gel electrophoresis (PFGE) pattern A (five isolates), C (one isolate), and D (one isolate) of human isolates. The other two isolates without pulsotypes belonged to ST59. CONCLUSION: The prevalence and infection sites of CNS differed from those of CPS. Genetic analyses indicated that genetic divergence, possible zoonotic transfer of MRSA, and the involvement of sec as important virulence factors for of S. aureus that lead to mastitis in goats.