Asma Haque, Abdul Haque, Yasra Sarwar, Aamir Ali, Saira Bashir, Ayesha Tariq, Mushkoor Mohsin.
Identification of drug resistance genes in clinical isolates of Salmonella typhi for development of diagnostic multiplex PCR.
Pak J Med Sci Jan ;21(4):402-7.

Background: Typhoid is one of the most common infectious diseases in the developing countries including Pakistan. In recent years, rapid emergence of multiple drug resistant (MDR) strains of Salmonella typhi has been a major problem. Objectives: To develop a multiplex PCR method that can be subsequently improved in a second step to diagnose Salmonella typhi and genes responsible for causing resistance against commonly used anti typhoid drugs, i.e. chloramphenicol, ampicillin, sulphamethoxazole and ciprofloxacin. Methods: Drug resistance patterns against the above mentioned drugs were determined by standard drug diffusion methods. Primers were synthesized to target various genes that could be responsible for the drug resistance. Amplicons indicating presence of gyrA gene which is responsible for ciprofloxacin resistance were obtained from resistant Escherichia coli strains and cloned into Salmonella typhi. The transformation was confirmed by sequencing. Results: In 23 MDR isolates of Salmonella typhi tested by disc diffusion method against ciprofloxacin, chloramphenicol, co-trimoxazole, and ampicillin, resistance was observed in 0 (0%), 17 (73.9%), 17 (73.9%), and 22 (95.7%) isolates respectively. The genes responsible for drug resistance were identified to be catP (chloramphenicol), tem (ampicillin), and sul 2 (co-trimoxazole). An amplification product representing gyrA gene (ciprofloxacin resistance) was cloned and successfully transformed into a Salmonella typhi isolate (confirmed by amplification of fliC gene) showing resistance to other three drugs. Confirmation was obtained by sequencing. This transformed strain was used to develop a multiplex PCR that gave amplification products of 707(sul2) 623 (cat P) 363 (fliC) 313 (gyrA) and 311 (tem) bps respectively. Conclusion: A multiplex PCR for simultaneous detection of Salmonella typhi and resistance against anti-typhoid drug has been successfully developed that can be easily improved for direct application on clinical samples.

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