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 bla_CTX-M genes. Methods: Antimicrobial 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 bla_CTX-M genes: bla_CTX-M-1 (4 strains), bla_CTX-M-2 (2 strains), bla_CTX-M-3 (4 strains), bla_CTX-M-9 (1 strain), bla_CTX-M-14 (5 strains), bla_CTX-M-15 (11 strains), bla_CTX-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 ISEcp1 was located upstream of the 5′ end of the bla_CTX-M gene. Among these strains, in five isolates, ISEcp1 was disrupted by insertion sequences such as IS26 (in three of them) or IS1 or IS10. Insertion sequence IS903 was found downstream of bla_CTX-M-14 or bla_CTX-M-24. Examination of the other three bla_CTX-M genes (two bla_CTX-M-2 and one bla_CTX-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 bla_CTX-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.