A novel CTX-M beta-lactamase (CTX-M-8) in cefotaxime-resistant Enterobacteriaceae isolated in Brazil

To estimate the diversity of extended-spectrum beta-lactamases in Brazil, 1 8 strains from different species of the family Enterobacteriaceae exhibitin g a positive double-disk synergy test were collected by a clinical laborato ry from several hospitals in Rio de Janeiro, Brazil, in 1996 and 1997. Four strains (Proteus mirabilis, Enterobacter cloacae, Enterobacter aerogenes, and Citrobacter amalonaticus) hybridized with a 550-bp CTX-M probe. The P. mirabilis strain produced a CTX-M-2 enzyme. The E. cloacae, E. aerogenes, a nd C. amalonaticus isolates harbored a bla gene which was identified by clo ning and sequencing as a bla(CTX-M) gene. E. coli HB101 transconjugants and the E. coli DH5 alpha transformant harboring a recombinant plasmid produce d a CTX-M beta-lactamase with an isoelectric point of 7.6 conferring a resi stance phenotype characterized by a higher level of resistance to cefotaxim e than to ceftazidime, as observed with the other CTX-M enzymes. The deduce d protein sequence showed a novel Ambler class A CTX-M enzyme, named CTS-M- 8, which had 83 to 88% identity with the previously described CTX-M enzymes . The phylogenic study of the CTX-M family including CTX-M-8 revealed four CTX-M types, CTX-M-8 being the first member of a new phylum of CTX-M enzyme s. The evolutionary distances between the four types of CTX-M were large, s uggesting that the four clusters branched off early from a distant unknown enzyme and that intermediate enzymes probably existed.