Differentiation of common gram negative pathogens by PCR- ribotyping

ABSTRACT

Gram negative bacteria especially members of family Enterobacteriaceae are among the most frequently isolated organisms from the clinical specimens. Rapid diagnosis of the pathogen in a clinical sample is always very important. Conventional methods are time-consuming. Among molecular techniques, PCR is very useful but unless very specific primers are used, non-specific amplifications are a problem. PCR-ribotying is a technique that gives very specific multiple bands by use of a single primer set. This study was designed to establish patterns for five common pathogens of Enterobacteriaceae, namely Escherichia coli, Salmonella enterica serovar Typhi [Salmonella Typhi], Proteus vulgaris, Klebsiella aerogenes, and Cirtobacter freundii along with another very common and problematic gram negative pathogen Pseudomonas aeruginosa. Each species gave a specific ribotyping pattern. Escherichia coli gave four amplification products of 1200, 850, 800, and 700 bps. Four amplification products of different sizes were also observed in Citrobacter freundii [3000, 850, 700, and 580 bps], Proteus vulgaris [900, 800, 750 and 700 bps], and Klebisella aerogenes [3000, 870, 700 and 520 bps]. More discrimination with five amplification products was seen in Salmonella Typhi [3000, 1200, 900, 850, and 700 bps]. On the other side of spectrum was Pseudomonas aeruginosa only a single amplification product of 750 bps was observed. PCR-ribotyping can very efficiently and specifically differentiate between opportunistic gram negative human pathogens