Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia).

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C.jejuni for hippuricase, GenBank: Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.

Improving the Quality and Safety of Fresh Camel Meat Contaminated with Campylobacter jejuni Using Citrox, Chitosan, and Vacuum Packaging to Extend Shelf Life.

Camel meat is one of the most consumed meats in Arab countries. The use of natural antimicrobial agents to extend the shelf life of fresh camel meat, control Campylobacter jejuni contamination, and preserve meat quality is preferred. In this study, we determined the antimicrobial effects of using 1% or 2% Citrox alone or in combination with 1% chitosan on the survival of C. jejuni in vitro and on camel meat samples during storage at 4 or 10 °C for 30 days in vacuum packaging. We determined the total viable count (TVC (cfu/g)), total volatile base nitrogen (TVB-N) content, and pH of the treated camel meat samples every three days during storage. The shelf lives of camel meat samples treated with 2% Citrox alone or in combination with 1% chitosan were longer than those of camel meat samples treated with 1% Citrox alone or in combination with 1% chitosan at both the 4 and 10 °C storage temperatures, with TVCs of <100 cfu/g after the first ten days and six days of storage at 4 and 10 °C, respectively. The addition of Citrox (1% and 2%) and 1% chitosan to camel meat samples and the application of vacuum storage were more effective than using Citrox (1% and 2%) alone and led to a reduction in C. jejuni in approximately 4.0 and 3.5 log cycles at 4 and 10 °C, respectively. The experimental results demonstrated that using a Citrox-chitosan combination improved the quality of camel meat and enhanced the long-term preservation of fresh meat for up to or more than 30 days at 4 °C.

Prevalence of methicillin-resistant (mecA gene) and heat-resistant Staphylococcus aureus strains in pasteurized camel milk.

Staphylococcus aureus is a significant opportunistic pathogen in humans, dairy cattle, and camels. The presence of antibiotic-resistant and heat-resistant bacteria in camel milk has become a potential public health issue. The phenotypic and molecular characterization of methicillin-resistant staphylococcal strains recovered from pasteurized camel milk distributed in retail markets of Saudi Arabia was assessed. A total of 100 samples were collected between March and May 2017. Out of the 20 S. aureus isolates that were recovered from the pasteurized camel milk, 10 were found to be resistant to cefoxitin (30 µg) and, thus, were designated as methicillin-resistant strains. The resistance ratio of methicillin-resistant S. aureus isolates for a different class of antibiotics was determined by performing the antimicrobial susceptibility test and was estimated to be approximately 60%. Polymerase chain reaction assay was performed to amplify the methicillin-resistant gene mecA, and furthermore, nucleotide sequencing was performed to detect and verify the presence of methicillin-resistant strains. Upon sequencing the putative S. aureus methicillin-resistant strains, we obtained 96 to 100% similarity to the penicillin-binding protein 2a gene (mecA) of the S. aureus strain CS100. Moreover, the 10 methicillin-resistant S. aureus isolates were also identified to be heat resistant and were stable at temperatures up to 85°C for 60 s, with 3 isolates being heat resistant even at 90°C for 60 or 90 s. The mean decimal reduction time (D85 value) was 111 s for all the 10 isolates. No difference was observed in the profile of total protein between the 10 methicillin- and heat-resistant S. aureus isolates and the S. aureus strain ATCC 29737, which was determined by sodium dodecyl sulfate-PAGE analyses. Therefore, we could conclude that a relatively high percentage of the tested pasteurized camel milk samples were contaminated with S. aureus (20%) and methicillin- and heat-resistant S. aureus (10%).

Molecular detection of methicillin heat-resistant Staphylococcus aureus strains in pasteurized camel milk in Saudi Arabia.

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%).

Heat resistance and presence of genes encoding staphylococcal enterotoxins evaluated by multiplex-PCR of Staphylococcus aureus isolated from pasteurized camel milk.

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.