DNA sequence analysis of the genetic environment of various blaCTX-M genes

Abstract

Objectives: Over a 3 year period (2000-2003) 21 Escherichia coli, 5 Klebsiella pneumoniae, 1 Serratia marcescens and 1 Proteus mirabilis producing CTX-M-type β-lactamase were collected from five different hospitals in Paris, France. This study was conducted to analyse the genetic environment of these 28 blaCTX-M genes. Methods: An timicrobial susceptibility testing was performed by the disc diffusion method and MICs of various β-lactams were determined by an agar dilution method. PCR was used to detect and sequence alleles encoding CTX-M, TEM, SHV and CMY enzymes. The genetic environment was analysed by amplification and direct sequencing using various set of PCR primers or cloning in pBK-CMV. Results: Sequence analysis revealed that these isolates contained seven different blaCTX-M genes: blaCTX-M-1 (4 strains), blaCTX-M-2 (2 strains), blaCTX-M-3 (4 strains), blaCTX-M-9 (1 strain), blaCTX-M-14 (5 strains), blaCTX-M-15 (11 strains), blaCTX-M-24 (1 strain). TEM-1 was associated with CTX-M-type enzymes in 15 isolates. Two strains produced both CTX-M-15 and SHV-2 or CTX-M-14 and CMY-2. In 25 strains the insertion sequence IS Ecp1 was located upstream of the 5' end of the blaCTX-M gene. Among these strains, in five isolates, IS Ecp1 was disrupted by insertion sequences such as IS 26 (in three of them) or IS 1 or IS 10. Insertion sequence IS 903 was found downstream of blaCTX-M-14 or blaCTX-M-24. Examination of the other three blaCTX-M genes (two blaCTX-M-2 and one blaCTX-M-9) by cloning, sequencing and PCR analysis revealed the presence of complex Class 1 integrons, In35, an integron similar to In60 and a novel integron. Conclusions: This work further confirmed the predominant role of ISEcp1 in the mobilization of blaCTX-M genes of the CTX-M-1 cluster and the presence of In35, of an integron similar to In60 and a novel complex Class 1 integron.