Detection of Pseudomonas aeruginosa by a triplex polymerase chain reaction assay based on lasl/R and gyrB genes

Pseudomonas aeruginosa is a nosocomial pathogen, which, due to its inherent and acquired resistance to a wide range of antibiotics, causes high mortality rates. Therefore, rapid detection of the bacterium with high specificity and sensitivity plays a critical role in the control of the pathogenic bacterium. The aim of this study was to evaluate the accuracy and specificity of a prompt detection of the bacterium based on a triplex polymerase chain reaction that amplifies the last, lasR and gyrB genes. For this purpose, 30 clinical isolates of P. aeruginosa and 30 wound biopsy samples were retrieved from clinical diagnostic laboratories. After the extraction of the chromosomal DNA, the desired genes were amplified using uniplex and triplex PCR with appropriate primers. The specificity of the primers was evaluated by a comparison of the PCR results for P. aeruginosa clinical samples and non-Pseudomonas species control samples. The sensitivity of the primers was determined using a serial dilution of the genomic DNA template [100ng to 100fg] and by a comparison of the PCR and bacterial culture results. The results showed that the triplex PCR assay was positive for all of the samples [100%], while the PCR identifications were negative for non-Pseudomonas species. Additionally, at 10[-4] and 10[-5] diluted genomic DMA from P. aeruginosa [10 pg and 1 pg], the triplex PCR test was positive for the Las and gyrB genes in all of the samples, respectively. Based on these results, the designed primers can be used for the rapid, specific and sensitive diagnosis of P. aeruginosa in a triplex PCR assay

Antibacterial activity of ethyl acetate and aqueous extracts of Mentha longifolia L. and hydroalcoholic extract of Zataria multiflora Boiss. plants against important human pathogens

OBJECTIVE@#To determine the potential antibacterial activity of ethyl acetate and aqueous extracts from Mentha longifolia L. (M. longifolia) and hydroalcoholic extract of Zataria multiflora Boiss. (Z. multiflora) against important human pathogens.@*METHODS@#Pseudomonas aeruginosa, Shigella dysenteriae, Klebsiella pneumoniae (K. pneumonia), Enterobacter cloacae, Salmonella typhi, Proteus mirabilis, Serratia marcescens, Bacillus cereus, Staphylococcus saprophyticus and Staphylococcus aureus were kinds of pathogenic bacteria to determine the antibacterial effect of aqueous and ethyl acetate extracts of M. longifolia and hydroalcoholic extract of Z. multiflora using broth microdiluation method.@*RESULTS@#The lowest minimum inhibitory concentration and minimum bactericidal concentration values for K. pneumonia and Pseudomonas aeruginosa (1.25 and 2.5 mg/mL) were observed by the hydroalcoholic extract of Z. multiflora and the lowest minimum inhibitory concentration and minimum bactericidal concentration values for K. pneumonia and Serratia marcescens (2.5 and 5 mg/mL) were observed by the aqueous extracts of M. longifolia.@*CONCLUSIONS@#In conclusion, it seems that Z. multiflora and M. longifolia extracts could inhibit the growth of all of the mentioned bacteria.

Antibacterial activity of ethyl acetate and aqueous extracts of Mentha longifolia L. and hydroalcoholic extract of Zataria multiflora Boiss. plants against important human pathogens

Objective: To determine the potential antibacterial activity of ethyl acetate and aqueous extracts from Mentha longifolia L. (M. longifolia) and hydroalcoholic extract of Zataria multiflora Boiss. (Z. multiflora) against important human pathogens. Methods: Pseudomonas aeruginosa, Shigella dysenteriae, Klebsiella pneumoniae (K. pneumonia). Enterobacter cloacae, Salmonella typhi, Proteus mirabilis, Serratia marcescens, Bacillus cereus, Staphylococcus saprophyticus and Staphylococcus aureus were kinds of pathogenic bacteria to determine the antibacterial effect of aqueous and ethyl acetate extracts of M. longifolia and hydroalcoholic extract of Z. multiflora using broth microdiluation method. Results: The lowest minimum inhibitory concentration and minimum bactericidal concentration values for K. pneumonia and Pseudomonas aeruginosa (1.25 and 2.5 mg/mL) were observed by the hydroalcoholic extract of Z. multiflora and the lowest minimum inhibitory concentration and minimum bactericidal concentration values for K. pneumonia and Serratia marcescens (2.5 and 5 mg/mL) were observed by the aqueous extracts of M. longifolia. Conclusions: In conclusion, it seems that Z. multiflora and M. longifolia extracts could inhibit the growth of all of the mentioned bacteria.

Cloning and Expression of N-terminal Region of IpaD from Shigella dysenteriae in E. coli

Genus Shigella is one of the important members of the family Enterobacteriaceae. There are numerous antigens in Shigella carrying by a 220 kb plasmid. Among them, IpaD is the key virulence factor of S. flexneri. Apart from having effectors function that is essential for host cell invasion and intracellular survival, this protein also controls the secretion and translocation of other effector proteins into eukaryotic host cells. In the present study, we have cloned and expressed the ipaD in E. coli. The ipaD gene was amplified by PCR. Prokaryote expression vector pET-28a [+] - ipaD was constructed, and used to transform E. coli BL21DE3 plySs. The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of IpaD-His by a rabbit monoclonal antibodies against his-tag. SDS-PAGE demonstrated that the constructed prokaryotic expression efficiently produced IpaD at the 1 mmol/L of IPTG. IpaD protein was able to react with the rabbit monoclonal antibody against His-tag. IpaD is essential for Shigella spp invasion. N-terminal region is most significant functional fragment of IpaD. Purification of IpaD from the wild type of Shigella is difficult furthermore profound study on a specific domain on the N-terminal of IpaD by using the wild type of purified IpaD is not feasible