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

Abstract

Background: Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous bacterium which can be found in most moisture places such as soil, water, food, plants, and animals including humans. Due to genetic flexibility among strains, there is no standard molecular identification for P. aeruginosa from different sources, particularly for human colonizing P. aeruginosa or commensal isolates. Materials and Method: Monoplex PCR using published primers of oprL, algD, nfxB gene were compared for their specificity and sensitivity in identification of commensal P. aeruginosa. Twenty- nine human colonizing Pseudomonas isolates including 16 P. aeruginosa isolates and 13 P. aeruginosa-like isolates were used in the study. To improve the detection, two new primer pairs targeting oprL gene was designed (oprL-pp1 and oprL-pp2), and oprL-algD duplex PCR was carried out with newly designed oprL primer pairs. Result and conclusion: PCR targeting algD or nfxB genes has the same sensitivity of 93.75% and specificity of 100%, while oprL-specific PCR using published primers is more sensitive (100%) but less specific (0%). Duplex PCR yielded high sensitivity and specificity in detecting P. aeruginosa. Both oprL-pp1/algD and oprL-pp2/algD duplex-PCR had 93.75% sensitivity (15/16 P. aeruginosa isolates) and 100% specificity (0/13 P. aeruginosa-like isolates). Besides, oprL-pp2 primers were more specific than oprL-pp1 primers, with only 2 over 13 P. aeruginosa-like isolates detected, while oprL-pp1 primers detected all P. aeruginosa-like isolates. Compared to the monoplex PCR that only targeted oprL gene, the duplex-PCR utilizing oprL-pp2 and algD primer can identify 15/16 P. aeruginosa isolates (93.75% specificity) and reduce the number of oprL-positive isolates required further exact identification. Additionally, the duplex-PCR used in this study was negative for non-Pseudomonas species including E. coli, V. cholera, V. parahaemolyticus, S. aureus. In conclusion, our duplex PCR targeting oprL and algD could be a valuable tool for P. aeruginosa screening.