One hundred and ninety eight samples of human and animal sources from different areas in El-Sharkia Governorate were used in this study to isolate Staphylococcus species and to determine the presence of coagulase ( coa) gene ,a virulence gene detected in S. aureus (CoPS), by PCR. Primary isolation on mannitol salt agar, βhaemolysis, tube coagulase test (TCT) and other biochemical characterization revealed 32.8% (65/198) of the total samples were infected with staphylococci, only 35 isolates were biotyped as coagulase-positive Staphylococci (Cops) and represented as 8 (8.8%), 10 (22.2%) and 17 (26.9%) in milk, meat and human samples, respectively.Meanwhile, Coagulase-negative Staphylococci (CoNS) were obtained only from cows milk. Fifteen CoPS (5 milk samples, 4 meat products and 6 human samples) were subjected to analysis by PCR for detection of coa gene. The resulted amplicons were 648, 723, 812 and 913 bp. According to coa gene polymorphism, milk and meat isolates were grouped into 5 groups, while human isolates were grouped into 4 groups. It was noted that two of the strains, classified as coagulase negative by tube coagulase test, were found to be positive with PCR amplification of the gene which clearly emphasizes the use of molecular methods in detecting S. aureus. In conclusion, the coa amplification has been considered as a simple and accurate method for typing of S. aureus.
Aims This study was applied to evaluate the usefulness of a high‐throughput sample preparation protocol prior to the application of quantitative real‐time PCR (qPCR) for the early diagnosis of bloodstream and pyogenic infections in humans and animals compared to matrix‐assisted laser desorption ionization–time of flight mass spectrometry (MALDI‐TOF MS) and classical culture. Methods and Results Saponin‐mediated selective host cell lysis combined with DNase‐1 was applied for processing of whole blood and pus clinical samples collected from suspected cases of septicaemia and pyogenic infections in humans and animals. The pre‐PCR processing strategy enabled the recovery of microbial cells with no changes in their colony forming units immediately after the addition of saponin. DNase‐1 was efficient for removing the DNAs from the host cells as well as dead cells with damaged cell membranes. The metagenomic qPCR and MALDI‐TOF MS could identify the bacterial community of sepsis at species level with a concordance of 97·37% unlike the conventional culture. According to qPCR results, Staphylococcus aureus (24·24%) was predominated in animal pyogenic infections, whereas Klebsiella pneumonia (31·81%) was commonly detected in neonatal sepsis. Conclusions Saponin combined with DNase‐1 allowed the efficient recovery of microbial DNA from blood and pus samples in sepsis using qPCR assay. Significance and Impact of the Study Metagenomic qPCR could identify a broad range of bacteria directly from blood and pus with more sensitivity, higher discriminatory power and shorter turnaround time than those using MALDI‐TOF MS and conventional culture. This might allow a timely administration of a prompt treatment.
Foodborne infections due to bacterial pathogens are increasing worldwide. Given the surreptitious nature of viable but nonculturable (VBNC) bacteria, they largely remain a threat to public health and food safety due to their non-detectability through conventional plate count techniques. Hence, species-specific quantitative real-time polymerase chain reaction (PCR) (qPCR) alone and combined with the use of propidium monoazide (PMA) was used along with the plate count method to quantify VBNC Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and Enterobacteriaceae in fresh and processed meat samples. The major bacterial pathogen isolated was S. aureus (93%) followed by Enterobacteriaceae (80.33%), C. perfringens (26.33%), and B. cereus (21.33%); their total viable counts were mostly recorded in raw meat than examined meat products. PMA quantitative real-time PCR (PMA qRT-PCR) could detect and quantify VBNC bacteria in 90.48% of culture-negative samples. It affirmed the presence of VBNC S. aureus (n = 10), B. cereus (n = 8), C. perfringens (n = 6), and Enterobacteriaceae (n = 12) in either single or mixed bacterial contamination. The log mean values of VBNC bacterial counts were highly reported for C. perfringens and S. aureus (9.60 ± 0.449 and 8.27 ± 0.453 CFU/g, respectively) followed by Enterobacteriaceae (6.95 ± 0.564 CFU/g) and B. cereus (6.69 ± 0.749 CFU/g). Sequencing of rpoB gene of Enterobacteriaceae enabled the identification of Klebsiella pneumoniae complex, Enterobacter cloacae complex, and Salmonella Typhi, which have been reported to be capable of entry into the VBNC state. To our knowledge, this is the first report at least in Egypt that records the presence of VBNC cells in meat samples representing a strong threat to public health and food safety. Moreover, PMA qRT-PCR allowed a quick and unequivocal way of enumeration of VBNC foodborne bacteria.
Introduction: Avian mycoplasmas impose a significant economic burden to the poultry industry. In recent years, macrolide-resistant Mycoplasma gallisepticum have occasionally been encountered in Egypt. Methodology: This study was designed to document the involvement of macrolide-resistant M. gallisepticum in respiratory organs of chickens suffering respiratory problems. Concurrently, an exhaustive molecular characterization of the intrinsic resistance of recovered isolates to macrolides was done. Results: Of 120 chickens showing respiratory problems, 14 (11.67%) M. gallisepticum were isolated and genetically identified; 8 of them were recovered from air sacs, 4 from lungs, and 2 from tracheas. Broth microdilution of all M. gallisepticum isolates showed various degrees of minimum inhibitory concentrations (MICs) against macrolides: erythromycin (0.25-32 µg/mL), tylosin (0.0625-4 µg/mL), and tiamulin (0.031-2 µg/mL). Nucleotide sequencing of domain V (peptidyl transferase region) of the 23S rRNA gene of macrolide-resistant M. gallisepticum isolates revealed transition mutations at positions 2068 and 2069 (corresponding to 2058 and 2059 in Escherichia coli numbering) in an isolate and at position 2067 (corresponding to 2057 in E. coli numbering) in three isolates as hot spots for macrolide resistance. Surprisingly, a transversion mutation at position 2621 (corresponding to 2611 in E. coli numbering) was reported in one of the recovered isolates as a first report. Conclusion: Generation of new mutations is evidence for persistence of M. gallisepticum despite macrolide treatment. Periodic surveys to monitor for the possible appearance of resistant strains are recommended.
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