Multiplex PCR for bla(CTX-M) & bla(SHV) in the extended spectrum beta lactamase (ESBL) producing gram-negative isolates.

Background & Objective: Phenotyping is commonly used for detection of extended spectrum beta lactamase (ESBL) production in gram-negative isolates. ESBLs are mainly coded for by three important genes, namely bla(TEM), bla(SHV) and bla(CTX-M). In this study we used a multiplex PCR as a rapid method to identify two common genes (bla(CTX-M) & bla(SHV)) responsible for extended spectrum beta lactamase production in members of Enterobacteriaceae family isolated from different clinical samples from a specialty hospital at Chennai.

Methods: A total of 260 non repetitive clinical isolates from 240 patients (some patients had more than one organism also), was selected for the study. Of these 33 were from sputum, 64 from urine, 46 from blood, 28 from pus aspirates, 58 from endotracheal secretions and 31 from other miscellaneous specimens. Phenotypic identification for ESBL production was confirmed by double disk synergy test (DDST) and phenotypic confirmatory double disk test (PCDDT) according to CLSI guidelines. Multiplex PCR for bla(CTX-M) and bla(SHV) was performed for the ESBL positive isolates.

Results: bla(SHV) like genes were found in 6 of 42 E.coli (14%), 7 of 46 Enterobacter species (15%), 28 of 62 Klebsiella species (45%) and bla(SHV) was not detected in any of the 50 isolates of non-fermenting gram-negative isolates. (Pseudomonas and Acinetobacter species) bla(CTX-M) like genes were found in 21 of 42 E. coli (50%), 13 of 46 Enterobacter species (28%), 25 of 62 (40%) Klebsiella species and 1 of 50 nonfermenting gram-negative bacilli (2%).

Interpretation & Conclusion: Our study demonstrated rapid detection of bla(SHV) and bla(CTX-M) in isolates belonging to Enterobacteriaceae and other non-fermenting clinical isolates using multiplex PCR. This genotypic method provided a rapid and efficient differentiation of ESBLs in the laboratory.