Interspecies spread of CTX-M-32 extended-spectrum β-lactamase and the role of the insertion sequence IS1 in down-regulating blaCTX-M gene expression

Objectives: To characterize the extended-spectrum beta-lactamases (ESBLs) as well as their genetic environment in different isolates of Enterobacteriaceae from a patient with repeated urinary tract infections. Methods: Two isolates of Escherichia coli and one Proteus mirabilis, all with ESBL phenotypes, were studied. Conjugation experiments and restriction fragment length polymorphisms (RFLPs) were performed. Cloning of the bla genes was by plasmid restriction and fragments ligation. Antibiotic susceptibility testing was by Etest. The genetic environment was analysed by direct sequencing of the DNA surrounding the bla gene. RT-PCR was performed to study the differences in the bla(CTX-M) gene expression. Results: The bla gene was transferred by conjugation from the three clinical isolates, which by RFLP showed the same plasmid. The bla gene and surrounding sequences were cloned, an similar to 9 kbp Accf fragment was sequenced and the bla(CTX-M-32) gene was identified. The MiCs of ceftazidime for transconjugants and transformants bearing the bla(CTX-M-32) gene were lower than those previously reported. Analysis of the DNA surrounding the ESBL gene revealed a new genetic structure with two insertion sequences, IS5 and IS1, located immediately upstream of the bla(CTX-M-32) gene; IS1 was located between the bla gene and IS5, and within the -10 and -35 promoter boxes of the b/aCTX-M-32 gene. Microbiological and biochemical studies revealed lower bla(CTX-M-32) gene expression in bacterial isolates with IS 1 between the promoter boxes. Conclusions: Data suggest putative in vivo horizontal bla(CTX-M-32) gene transfer between two different genera of Enterobacteriaceae. A new complex structure, IS5-IS1, was detected upstream of the bla gene and IS1 negatively modulated expression of the bla(CTX-M-32) gene because its location modified the bla promoter region.