Transcription levels of Pseudomonas aeruginosa exotoxin a gene and severity of symptoms in patients with otitis externa.

Polymerase chain reaction (PCR)-based detection and transcription of the gene encoding a potent virulence factor, the exotoxin A, were done on 32 isolates of Pseudomonas aeruginosa belonging to 23 genotypes. These isolates were obtained from 22 patients who were admitted to the emergency room in a medical center during a 5-month period with the diagnosis of either unilateral or bilateral otitis externa. Patients showed symptoms that ranged from mild to severe. PCR amplification of a 396-bp fragment of the gene encoding the exotoxin A was done on extracted DNA. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was performed on extracted RNA to detect exotoxin A gene mRNA transcripts. Quantitation of RT-PCR amplicons from P. aeruginosa isolates associated with mild and severe symptoms was determined by end-point titration of c-DNA and scanning of amplicons with the Storm Gel and Blot Imaging System. Data have shown that all of the 32 isolates of P. aeruginosa carry the exotoxin A gene, and all isolates with the exception of two had the exotoxin A transcription demonstrated by the production of a 396-bp amplicon from RT-PCR-amplified RNA. The remaining two isolates amplified fragments that were slightly smaller than the expected size. Additional studies are needed to characterize these two mRNA transcripts. Transcription levels of exotoxin A gene associated with severe symptoms were significantly more elevated than those associated with mild to moderate symptoms. Studies are under way to determine expression of P. aeruginosa exotoxin A by detecting quantitatively levels of the translated exotoxin A protein produced by isolates associated with severe and mild to moderate symptoms.