CTX-M Enzymes: Origin and Diffusion

Abstract

CTX-M β-lactamases are considered a paradigm in the evolution of a resistance mechanism. Incorporation of different chromosomal bla(CTX-M) related genes from different species of Kluyvera has derived in different CTX-M clusters. In silico analyses have shown that this event has occurred at least nine times; in CTX-M-1 cluster (3), CTX-M-2 and CTX-M-9 clusters (2 each), and CTX-M-8 and CTX-M-25 clusters (1 each). This has been mainly produced by the participation of genetic mobilization units such as insertion sequences (ISEcp1 or ISCR1) and the later incorporation in hierarchical structures associated with multifaceted genetic structures including complex class 1 integrons and transposons. The capture of these bla(CTX-M) genes from the environment by highly mobilizable structures could have been a random event. Moreover, after incorporation within these structures, β-lactam selective force such as that exerted by cefotaxime and ceftazidime has fueled mutational events underscoring diversification of different clusters. Nevertheless, more variants of CTX-M enzymes, including those not inhibited by β-lactamase inhibitors such as clavulanic acid (IR-CTX-M variants), only obtained under in in vitro experiments, are still waiting to emerge in the clinical setting. Penetration and the later global spread of CTX-M producing organisms have been produced with the participation of the so-called "epidemic resistance plasmids" often carried in multi-drug resistant and virulent high-risk clones. All these facts but also the incorporation and co-selection of emerging resistance determinants within CTX-M producing bacteria, such as those encoding carbapenemases, depict the currently complex pandemic scenario of multi-drug resistant isolates.

Keywords: ISCR1; ISEcp1; Kluyvera spp.; antibiotic selective force; bacterial clones; blaCTX-M genes; gene mobilization; plasmid.

Figure 1

Factors fueling the emergence, maintenance, and spread of the CTX-M extended-spectrum β-lactamases (ESBLs).

Figure 2

Hierarchical complexity of bla_CTX-M genes within genetic structures and bacterial clones participating in the mobilization, spread, and maintenance of these genes (see the text for further explanation).

Figure 3

Maximum likelihood trees of bla_CTX-M genes and 16S rDNA of Kluyvera spp. in order to compare their respective phylogenetic topologies. (A) Phylogenetic tree of bla_CTX-M genes (n = 109 and 17 chromosomal genes from Kluyvera) was obtained using PhyML_3.0 program (846 nt). The Tamura Nei nucleotide substitution model, used as evolutionary model was selected with the jModeltest program. The robustness of the relevant nodes was estimated with 1000 bootstrap pseudorandom replicates. We considered nodes valid when bootstrap value was >95%. (B) Phylogenetic tree of 16s rDNA of Kluyvera spp. (n = 55) and related species download of www.ncbi.nlm.nih.gov, using PhyML_3.0 (1310 nt). The Hasegawa–Kishino–Yano was the evolutionary model inferred by jModeltest program and invariant site rate of 77.6% (HKY + I + G). We considered nodes valid when bootstrap value was >85%. The phylogenetic trees were represented using MEGA 5.0 program.

Figure 4

Schematic representations of genetic environments surrounding bla_CTX-M genes corresponding to suspected cases of mobilization events within different CTX-M groups (CTX-M-1, CTX-M-2, CTX-M-9, and CTX-M-25).

Figure 5

Alignment of upstream sequences in different bla_CTX-M gene clusters. Different bla_CTX-M genes belonging to the same cluster share an identical DNA sequence in upstream region, suggesting the same origin (A). Nevertheless, the loss of alignment among different upstream sequences from different bla_CTX-M genes belonging to different clusters (included in the box squared) but theoretically derived from the same ancestral source in Kluyvera georgiana suggesting that these clusters had different sources (B).