MOLECULAR DETECTION OF Salmonella typhi STRAINS & THEIR DRUG RESISTANCE PATTERN

Abstract

Typhoid fever has become a major problem due to the emergence of MDR strains of Salmonella enterica serovar Typhi, the causative agent, at an alarming rate during recent years. The situation is worsened due to lack of quick, sensitive and reliable diagnostic tools for determining the drug resistance pattern. Conventional methods are time consuming and lack sensitivity. It was envisaged that a multiplex PCR diagnosing typhoid and detecting resistance against standard typhoid drugs, ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole and ciprofloxacin would be very useful. After determining drug resistance patterns by standard disc diffusion method on a pool of 23 MDR S. Typhi isolates, a PCR amplification technique was used for various drug resistance related genes found universally in S. Typhi. These were tem, catP, and sul2 genes. None of these isolates was resistant to ciprofloxacin, so a fragment of gyrA gene (related to ciprofloxacin resistance) was amplified from an MDR E.coli isolate, cloned, and transformed into an MDR S. Typhi isolate that was naturally resistant to other drugs. A regular multiplex PCR was subsequently developed by using this cloned bacterium which was followed by the development of a nested multiplex PCR for increasing specificity and sensitivity. This diagnostic multiplex PCR has been successfully optimized to be directly applicable to clinical samples.