Evolutionary trajectories of beta-lactamase CTX-M-1 cluster enzymes: predicting antibiotic resistance

Abstract

Extended-spectrum beta-lactamases (ESBL) constitute a key antibiotic-resistance mechanism affecting Gram-negative bacteria, and also an excellent model for studying evolution in real time. A shift in the epidemiology of ESBLs is being observed, which is characterized by the explosive diversification and increase in frequency of the CTX-M-type beta-lactamases in different settings. This provides a unique opportunity for studying a protein evolutionary radiation by the sequential acquisition of specific mutations enhancing protein efficiency and fitness concomitantly. The existence of driver antibiotic molecules favoring protein divergence has been investigated by combining evolutionary analyses and experimental site-specific mutagenesis. Phylogenetic reconstruction with all the CTX-M variants described so far provided a hypothetical evolutionary scenario showing at least three diversification events. CTX-M-3 was likely the enzyme at the origin of the diversification in the CTX-M-1 cluster, which was coincident with positive selection acting on several amino acid positions. Sixty-three CTX-M-3 derivatives containing all combinations of mutations under positively selected positions were constructed, and their phenotypic efficiency was evaluated. The CTX-M-3 diversification process can only be explained in a complex selective landscape with at least two antibiotics (cefotaxime and ceftazidime), indicating the need to invoke mixtures of selective drivers in order to understand the final evolutionary outcome. Under this hypothesis, we found congruent results between the in silico and in vitro analyses of evolutionary trajectories. Three pathways driving the diversification of CTX-M-3 towards the most complex and efficient variants were identified. Whereas the P167S pathway has limited possibilities of further diversification, the D240G route shows a robust diversification network. In the third route, drift may have played a role in the early stages of CTX-M-3 evolution. Antimicrobial agents should not be considered only as selectors for efficient mechanisms of resistance but also as diversifying agents of the evolutionary trajectories. Different trajectories were identified using a combination of phylogenetic reconstructions and directed mutagenesis analyses, indicating that such an approach might be useful to fulfill the desirable goal of predicting evolutionary trajectories in antimicrobial resistance.

Figure 1. Phylogenetic tree of bla _CTX-M sequences (n = 73) inferred by Bayesian analysis.

Support for relevant nodes in the tree (indicated by a red dot) was estimated by approximate likelihood ratio, bootstrapping (1000 replicates) with maximum-likelihood reconstruction, bootstrapping (1000 replicates) with neighbor-joining reconstruction, and Bayesian posterior probability, color-coded with increasing density of green with the following equivalences: white <0.80, lightest green ≥0.80, light green ≥0.90, medium green ≥0.95 and dark-green ≥0.99. Kluyvera spp. bla sequences (n = 12) from putative ancestors were included in the analyses and the tree was arbitrarily rooted with Kluyvera cryocrescens. The discrepancy detected between this topology and the one generated by Barlow et al., consisting of the position of the bla _CTX-M-11 sequence, might be due to the existence of two different entries in the PubMed corresponding to this bla gene: GenBank accession number AY005110 (this study) and AJ310929 , differing in six nucleotides and four amino acid changes, probably sufficient to ascribe these variants to different branches. ^a bla _CTX-M-14 and bla _CTX-M-18 genes show identical nucleotide sequences; ^b bla _CTX-M-64 gene corresponds to a hybrid sequence, probably resulting from a homologous recombination event between bla _CTX-M-15 and bla _CTX-M-14 genes .

Figure 2. Phylogenetic reconstruction of CTX-M-1 cluster enzymes.

Using the MESQUITE 2.0 program, the amino acid sequence of the putative CTX-M-1 ancestor was identified, and subsequently all CTX-M-1 cluster variants were positioned in the generated phylogeny. The amino acid changes acquired in each consecutive step are indicated in each branch. Numbers (n = 1–6) up in the figure represent the cumulative number of amino acid changes present in CTX-M variants with respect to the ancestor sequence. Underlined CTX-Ms represent the variants with activity on both cefotaxime and ceftazidime.

Figure 3. Schematic representation of most CTX-M-3 derivative mutants containing P167S or D240G amino acid changes.

Light grey dots represent P167S-carrying mutants, while dark grey dots represent D240G-containing mutants. The size of the dots is directly proportional to the number of mutants represented in each position (1–2). Their corresponding cefotaxime and ceftazidime MIC values (µg/ml) are shown in the “x” and “y” axis respectively.

Figure 4. Mutational pathways showing the diversification of CTX-M-3 towards the most evolved variants, determined by step-by-step site-specific mutagenesis: the P167 pathway.

Different CTX-M-3 evolutionary scenarios are shown depending on the acquisition of P167S (Figure 4) or D240G (Figure 5) mutations in the first step or in the absence of these changes (Figure 7). Each rectangle represents a particular CTX-M-3 derivative obtained by introduction of mutations predicted to be under positive selection (A77V, N114D, P167S, D240G and D288N) and A140S in cumulative steps towards the most complex mutant (containing all mutations analyzed in each pathway). The binary code indicates the content of mutations in each CTX-M variant in this order: A77V, N114D, A140S, P167S, D240G and D288N. Variants identical or closely related with enzymes already detected in nature appear with the corresponding CTX-M designation. Numbers inside the rectangles indicate MIC values (µg/ml) of ceftazidime (A) or both cefotaxime and ceftazidime (B) observed for MI1443 E. coli cells carrying a recombinant plasmid containing each corresponding mutant. Possible trajectories identified are shown with arrows. Filled arrows indicate evolutionary steps predicted to be favored by selection, whereas dotted arrows represent steps associated with neutral events and thus fixed by drift. Deleterious steps (associated with negative changes in fitness) are not shown. Asterisks indicate significant (using Mann-Whitney tests) increases in MIC between the corresponding mutants.

Figure 5. Mutational pathways showing the diversification of CTX-M-3 towards the most evolved variants, determined by step-by-step site-specific mutagenesis: the D240 pathway.

See legend for Figure 4.

Figure 6. Median-joining network inferred for CTX-M-1 group amino acid sequences.

Numbers next to the segments connecting nodes denote the amino acid position inferred to have changed between the two connected alleles. Large dots represent CTX-M variants detected in natural contexts, which differ in the presence of mutations in positions evolving under positive selection (A77V, N114D, P167S, D240G, D288N) as follows: blue dots represent variants containing the P167S mutation alone (light blue) or associated with others (dark blue); green dots represent alleles containing the D240G change alone (light green) or associated with others (dark green); yellow dots represent variants containing mutations A77V, N114D and/or D288N; white dots represent variants containing mutations in other positions presumptively not subjected to positive selection. Alleles whose existence is postulated by the analysis but which have not yet been identified are represented by small red dots in the corresponding nodes.

Figure 7. Diversification of CTX-M-3 towards CTX-M-1 in the absence of P167S and D240G.

See legend for Figure 4.

Figure 8. Interconnectedness of three CTX-M-3 diversification routes.

Circles represent CTX-M-3 mutant derivatives associated with the different evolutionary routes towards the most evolved variants: i) P167 pathway (blue), ii) D240 pathway (green) and iii) evolution towards CTX-M-1 (yellow). The color gradient represents the direction of evolution.

Figure 9. Median-joining network inferred for all CTX-M amino acid sequences studied.

Numbers next to the segments connecting nodes denote the number of amino acid positions inferred to have changed between the two connected alleles, when these are larger than one. The major groups identified with this analysis correspond to the groups also defined in the phylogenetic tree shown in Figure 1.