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

Abstract

To estimate the diversity of extended-spectrum beta-lactamases in Brazil, 18 strains from different species of the family Enterobacteriaceae exhibiting a positive double-disk synergy test were collected by a clinical laboratory 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, and C. amalonaticus isolates harbored a bla gene which was identified by cloning and sequencing as a bla(CTX-M) gene. E. coli HB101 transconjugants and the E. coli DH5alpha transformant harboring a recombinant plasmid produced a CTX-M beta-lactamase with an isoelectric point of 7.6 conferring a resistance phenotype characterized by a higher level of resistance to cefotaxime than to ceftazidime, as observed with the other CTX-M enzymes. The deduced protein sequence showed a novel Ambler class A CTX-M enzyme, named CTX-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 enzymes. The evolutionary distances between the four types of CTX-M were large, suggesting that the four clusters branched off early from a distant unknown enzyme and that intermediate enzymes probably existed.

FIG. 1

Map of the EcoRI CTX-M-8-encoding insert of pC1Rio-2. Arrows indicate the strategy used to establish the nucleotide sequence.

FIG. 2

Nucleotide sequence of the 1,184-bp fragment of pC1Rio-2 containing bla_CTX-M-8. The deduced amino acid sequence is designated in single-letter code below the nucleotide sequence. The potential promoter sequences (−10 and −35 regions) and ribosome-binding site (RBS) are shown. Solid arrows, inverted repeat sequences possibly acting as terminators. Conserved residues in serine β-lactamases (SXXK, SDN, E, KTG) are boxed.

FIG. 3

Alignments of the CTX-M-8 amino acid sequence with those of CTX-M-1 (4, 6), CTX-M-2 (5), CTX-M-3 (19), CTX-M-4 (18), CTX-M-5 (11), CTX-M-6 (15), CTX-M-7 (15) (previously designated CTX-M-5), Toho-1 (19), and Toho-2 (27). Dots, amino acids identical to those of CTX-M-8; boldface letters, specific amino acid residues of CTX-M-8 in the CTX-M family; italic letters, peptide signal of CTX-M-8 as determined by a hydopathy plot; boxes, peptide signals determined by amino acid sequencing; roman numerals, boxes described by Joris et al. (23). Amino acids are numbered according to the standard numbering scheme for the class A β-lactamases of Ambler et al. (1).

FIG. 4

Dendrogram of CTX-M family. Branch lengths are to scale and are proportional to the numbers of amino acid changes. The percentages at the branch points refer to the numbers of times particular nodes were found in 100 bootstrap replications (underlined numbers). The distance along the vertical axis has no significance. ∗, previously designated CTX-M-5 (15).