Identification of Pseudomonas aeruginosa strains isolated from Dorper sheep milk with subclinical-mastitis infection by uniplex PCR using 16S rRNA, lasI/R, gyrB and ecfX genes

Abstract

Pseudomonas aeruginosa is an opportunistic and versatile pathogenic bacterium that can adapt in various environmental condition, which play a role in multiple virulence factor and resistance to antibiotics. Moreover, molecular identification techniques using single target gene is more susceptible to error and false positive. Thus, the detection of this strain with high specificity and sensitivity is crucial in order to control this pathogenic bacterium. The aim of this study was to evaluate six bacteria strains (13-1, 66-1, 00-1, 46-1, 10-R and 67-1) isolated from Dorper sheep milk and two P. aeruginosa ATCC strains (ATCC BAA-2108 and ATCC27853) for prompt identification of the strains based on uniplex polymerase chain reaction which targeting PA-SS, PA-GS, lasI/R, gyrB and ecfX genes. In the present study, the Dorper sheep milk’s samples (n = 32) were collected and tested with California mastitis test (CMT). Out of 32 milk’s samples, six of the samples were detected with strong mastitis, and thus were continued with inoculation process on selective media Pseudomonas Agar P (for pyocyanin) or F (fluorescein) and MacConkey agar for differentiation. After extraction of the bacteria chromosomal DNA, uniplex PCR amplification were carried out by using 16S rRNA (PA-SS and PA-GS) primers and specific P. aeruginosa genes (lasI/R, gyrB and ecfX) primers. The specificity of the primers was also examined by non-Pseudomonas species as a control for data comparison. Sequence analysis has revealed that six of the isolated samples were confirmed as P. aeruginosa strains with the respective genes sequence confirmed by BLAST and Clustal Omega. From this study, lasI/R, gyrB and ecfX were highly reliable primers with the percentage of identification of more than 95.0% as compared to PA-SS and PA-GS which were less than 90.0%. This study concludes that highly specific and sensitive assay has been developed using lasI/R, gyrB and ecfX targeted genes for the detection of P. aeruginosa strains isolated from fresh sheep milk samples.

Identification of commensal Pseudomonas aeruginosa isolates using duplex PCR targeting the oprL and algD genes

“…However, the specificity of this multiplex PCR against P. aeruginosa-like isolates was not fully considered. Another PCR assay using the same genes was effective in identifying 95% of P. aeruginosa isolates from Dorper sheep milk 21 . Multiplex PCR can also be developed to allow for more in-depth diagnostics, as multiple bands and single bands can be interpreted differently, minimizing false positives and false negatives 22 .…”