New CTX-M Group Conferring β-Lactam Resistance: A Compendium of Phylogenetic Insights from Biochemical, Molecular, and Structural Biology

Simple Summary CTX-M β-lactamases are a growing group among extended-spectrum β-lactamases (ESBLs), not only in number, diversity, and functional kinetics. Until now, it has been well-accepted five main clusters within CTX-M β-lactamases. These canonical clusters are CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. In the present study, we propose a new sixth cluster, CTX-M-151. Abstract The production of extended-spectrum β-lactamases (ESBLs) is the main defense mechanism found in Gram negative bacteria. Among all the ESBLs, the CTX-M enzymes appear as the most efficient in terms of dissemination in different epidemiological contexts. CTX-M enzymes exhibit a striking plasticity, with a large number of allelic variants distributed in several sublineages, which can be associated with functional heterogeneity of clinical relevance. This observational analytical study provides an update of this family, currently with more than 200 variants described, from a phylogenetic, molecular, and structural point of view through homology in amino acid sequences. Our data, combined with described literature, provide phylogenetic and structural evidence of a new group. Thus, herein, we propose six groups among CTX-M enzymes: the already stablished CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 clusters, as well as CTX-M-151 as the new cluster.


Introduction
Penicillin, discovered by Alexander Fleming in 1928, represented a turning point for humanity, revolutionizing Medicine by significantly reducing the number of deaths from bacterial infections worldwide [1][2][3]. However, in 1940, a bacterial penicillinase was identified, foreshadowing the clinical findings that have been occurring since then until today [3,4]. The introduction of antimicrobial agents in clinical practice was followed by the gradual appearance of resistant bacterial strains, whose threat of dissemination constitutes a worrying reality in Public Health [4,5]. In this matter, it is estimated that approximately 25,000 patients die each year in the European Union because of infections caused by multidrug-resistant bacteria, and the associated costs are estimated circa 1.5 billion euros/per year) [6]. more specifically: (i) Clustal Omega for sequences alignment; (ii) Jalview for visualization, analysis and comparison of previously aligned sequences; and (iii) NGPhylogeny.fr for the construction of the phylogenetic tree (dendrogram), following the UPGMA (Unweighted Pair Group Method with Arithmetic mean) method, which assumes evolutionary rates for all taxonomic units, meaning it groups the shortest distances and recalculates new distances using arithmetic means for a new grouping [39][40][41]. This bioinformatics analysis allowed all CTX-M variants to be clustered, by comparing their amino acid sequences' homology and by their phylogenetic relationship. For this purpose, we defined the cut-off value for the phylogenetic identity as ≥95% for the several taxonomic units belonging to the same cluster. Furthermore, the comparative study of protein sequences allowed the measurement of possible mutational hot spots. To this end, it is important to mention that the sequences were compared individually by analogy with the lowest numbered variant within the cluster.

Results
As previously described, the analysis of the CTX-M enzymes' phylogeny showed conserved regions of amino acids, further supporting the idea of a common ancestral protein identity. In addition, these amino acids are also the structural requirements of CTX-M β-lactamases, which are necessary for the presence of their typical profiles (phenotypic as well as biochemical).

Phylogenetic Analysis
The phylogenetic trees obtained are presented ( Figure 1). Clusters may be observed by colors (in black, the CTX-M-1 cluster; in red, the CTX-M-2; in grey, the CTX-M-8; in orange, the CTX-M-9; in green, the CTX-M-25. In addition, this analysis returned a new and apparently more distant group, the CTX-M-151 enzyme (blue line). Considering these results, a multiple sequence alignment approach was performed to assess the amino acid sequence.

Molecular and Structural Study
The amino acid sequence was explored by means of multiple sequence analysis using Clustal Omega (The EMBL-EBI search and sequence analysis tools APIs, 2019). With this approach, alignments between classic Bonnet representative CTX-M enzyme members included CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. Moreover, CTX-M-151 amino acid sequence was included for comparison purposes (Figure 2).

Molecular and Structural Study
The amino acid sequence was explored by means of multiple sequence analysis Clustal Omega (The EMBL-EBI search and sequence analysis tools APIs, 2019)  304 amino acids. "*" represents fully conserved residues, "." indicates c vation between groups of strongly similar properties and ":" indicates conservation between g of weakly similar properties.
As observed in Figure 2, sequence variability among CTX-M enzymes is extra nary. There are several nonconserved amino acid substitutions and it is reasonable pect great impact in the three-dimensional structure of these enzyme, since in most tions amino acid substitutions change its class, e.g., changing from polar amino aci 304 amino acids. "*" represents fully conserved residues, "." indicates conservation between groups of strongly similar properties and ":" indicates conservation between groups of weakly similar properties.
As observed in Figure 2, sequence variability among CTX-M enzymes is extraordinary. There are several nonconserved amino acid substitutions and it is reasonable to expect great impact in the three-dimensional structure of these enzyme, since in most positions amino acid substitutions change its class, e.g., changing from polar amino acids for negatively charged amino acids, the substitution for an aromatic polar amino acid for a positively charged one, and so on. Another important aspect observed in Figure 2 is that CTX-M-151 presents 304 amino acids, unlike the remaining CTX-M enzymes aligned, which present 291 amino acids. In Figure 3, we compare the tridimensional (3D) structure of CTX-M-1, here used as an example of the CTX-Ms with 291 amino acids, with CTX-M-151 s 3D structure, highlighting in red the extra 13 residues. Interestingly, when we look at these enzymes' structures, their sequences' variability seems unnoticeable, as they display notorious similarities when overlapped ( Figure 3A). negatively charged amino acids, the substitution for an aromatic polar amino acid for a positively charged one, and so on. Another important aspect observed in Figure 2 is that CTX-M-151 presents 304 amino acids, unlike the remaining CTX-M enzymes aligned, which present 291 amino acids. In Figure 3, we compare the tridimensional (3D) structure of CTX-M-1, here used as an example of the CTX-Ms with 291 amino acids, with CTX-M-151′s 3D structure, highlighting in red the extra 13 residues. Interestingly, when we look at these enzymes' structures, their sequences' variability seems unnoticeable, as they display notorious similarities when overlapped ( Figure 3A). As we can see from Figure 3, when we overlap the respective 3D structures of CTX-M-1 and CTX-M-151, the similarities stand out, despite the extension of amino acid substitutions between both enzymes that account for more than 50% ( Figure 3A). The residues corresponding to the Omega loop show two substitutions: Thr168 and Thr171 in CTX-M-1 that correspond to Asn176 and Ser179 in CTX-M-151, respectively ( Figure 3B). However, these substitutions do not seem to induce any structural alteration, conserving the Omega loop that is transversal to all CTX enzymes. Five major hot spots are found in the cluster (M1 to M5). These hot spots include five key substitutions: Val80Ala, Asp117Asn, Ser143Ala, Asp242Gly, and Asn289Asp. In Table As we can see from Figure 3, when we overlap the respective 3D structures of CTX-M-1 and CTX-M-151, the similarities stand out, despite the extension of amino acid substitutions between both enzymes that account for more than 50% ( Figure 3A). The residues corresponding to the Omega loop show two substitutions: Thr168 and Thr171 in CTX-M-1 that correspond to Asn176 and Ser179 in CTX-M-151, respectively ( Figure 3B). However, these substitutions do not seem to induce any structural alteration, conserving the Omega loop that is transversal to all CTX enzymes.  Subsequently, although having more than one amino acid mutation, some enzymes differ by the occurrence of one or more variant-specific mutations. These are listed below in Table 2. Five major hot spots are found in the cluster (M1 to M5). These hot spots include five key substitutions: Val80Ala, Asp117Asn, Ser143Ala, Asp242Gly, and Asn289Asp. In Table 1 is listed the CTX-M-1-like enzymes by their enzyme index. For example, Val80Ala may be seen in indexes 3, 10, 12, 15 . . . 232. This means that mutation Val80Ala is also present in CTX-M-3, CTX-M-10, CTX-M-12, CTX-M-15, and CTX-M-232 among all others listed in Table 1.

CTX-M Clusters
All 106 enzymes belonging to this group present a protein sequence with 291 amino acids, except CTX-M-211 (with 294 amino acids) where the insertion of three amino acids at the beginning of the sequence (methionine, valine, lysine) is observed.
Through the observation of Figure 4, it is possible to verify that the enzymes in this group suffer more than one mutation, with the exception of CTX Subsequently, although having more than one amino acid mutation, some enzymes differ by the occurrence of one or more variant-specific mutations. These are listed below in Table 2. Table 1. Distribution of CTX-M-1-like enzymes by major hotspot mutation sites. M1 to M5 represent the five hotspots in CTX-M-1 cluster; For each hotspot, the substitution of the respective amino acid is represented; The numbers for each hotspot represent the indexes of all the enzymes from the CTX-M-1 cluster that present the corresponding hotspot. Additionally, as it can be observed, five mutations occur in many variants (hotspots). As such, they have been identified from M1 to M5.       that the number of amino acid substitutions is quite small (only four substitutions). It is also noted that, except for Ser274Asn that occurs in the CTX-M-63 enzyme, all other substitutions occur simultaneously in both variants.

Hotspot
Additionally, a methionine deletion is observed at position 1 in the CTX-M-40 sequence, as well as in the CTX-M-63 sequence. For this reason, in this group, only the CTX-M-8 enzyme has 291 amino acids, considering that the other variants have minus one amino acid.   Therefore, all other enzymes in this group have the Ala234Val substitution. Of these, it is possible to verify that some have one or more mutations exclusive to themselves, as presented in Table 3: Table 3. Enzymes from CTX-M-9 cluster enzymes and their respective exclusive mutations. It should also be noted that CTX-M 110, in addition to the two substitutions mentioned above, is the only enzyme that presents the insertion of an amino acid at the end of the sequence (insertion of a leucine in position 292).

Sequence Mutations in
The remaining enzymes also have more substitutions common to other enzymes, and some even have variant-exclusive mutations. Thus, it appears that, of these, there are 15 enzymes with the Asp242Gly substitution, seven enzymes with Ala80Val, five with Pro170Ser, and seven with Ser275Arg.
Thus, among those with the Asp242Gly substitution, it appears that CTX-M-27 is the only one with only that substitution. Still analyzing Figure 7, it is possible to verify that the enzymes that present more mutations in relation to CTX-M-9 are CTX-M-137 and CTX-M-221, accumulating 19 and 21 different substitutions, respectively. Between them, they share 18 amino acid substitutions (Gln195Asn, His200Lys, Glu204Asp, Thr205Ser, Leu214Met, Arg225Gln, Thr230Ala, Thr233Val, Ala234Val, Gln255Lys, Gly256Asp, Val261Ile, Gln271Pro, Asn272Lys, Arg285Lys, Ile287Val, Ala288Thr, and Glu289Asp). Thus, they mainly differ because the CTX-M-137 also has the Asp242Gly replacement, as mentioned above, and another replacement, Val6Met (simultaneous with the CTX-M-13). In turn, CTX-M-221 has a replacement of a proline for a serine at position 170 and a replacement of a threonine with an isoleucine at position 264.
The CTX-M-13 has, as previously described, the replacement Val6Met (common with the CTX-M-137), the Ala157Glu substitution (common with the CTX-M-122), and two other exclusive substitutions (Ala56Lys and Ala57Glu). In parallel, in the set of enzymes with the Analyzing CTX-M-49 and CTX-M-50, it appears that both have the Ala51Pro substitution, but the first additionally features the Gly46Arg (in common with the CTX-M-44). Finally, regarding the substitutions already described as influencers of the resistance pro-file, the following are verified in this group: Ala80Val, Pro170Ser, and Asp242Gly. Analyzing CTX-M-49 and CTX-M-50, it appears that both have the Ala51Pro substitution, but the first additionally features the Gly46Arg (in common with the CTX-M-44). Finally, regarding the substitutions already described as influencers of the resistance profile, the following are verified in this group: Ala80Val, Pro170Ser, and Asp242Gly.

Sequence Mutations in Cluster CTX-M-25
The diagram in Figure 8 represents all the amino acid mutations that occur within CTX-M-25-like members when compared with the CTX-M-25 sequence. However, Val80ala and Gly242Ser substitutions are not exclusive to the abovementioned variants. They occur together in a substantial number of other variants, they appear in seven and eight different enzymes, respectively. However, Val80ala and Gly242Ser substitutions are not exclusive to the abovementioned variants. They occur together in a substantial number of other variants, they appear in seven and eight different enzymes, respectively. Therefore, the remaining variants occur by the presence of more than one amino acid mutation, which can be seen in more than one enzyme of this group. For example, CTX-M-91 has two amino acid substitutions, one of which has already been evidenced previously

Phylogenetic Study
Regarding the phylogenetic studies, Figures 1 and 2 show the CTX-M enzymes clustered in groups, with each group being named based on the lower number index within. Analysis of the phylogenetic results led us to question the existence of six CTX-M groups, and not five as stated until now, as highlighted in Figure 1  These data support the hypothesis of the existence of subgroups within the groups. This unequivocal distancing influences the intragroup identity, thus affecting the similarity percentage of the amino acids shown in Figure 2. As a result, the identity in the CTX-M-1 and CTX-M-9 groups increases approximately from 90% and 92%, respectively, to 97%. In fact, and in agreement with Bonnet [28], it is verified that the CTX-M-9 and CTX-M-1 groups are the most distant ones, in evolutionary terms, being the CTX-M-9 group closer to the common ancestor, and consequently, the CTX-M-1 group further away.
In parallel, it is found that there are 106 enzymes belonging to the CTX-M-1 group, 27 enzymes to the CTX-M-2, three enzymes to the CTX-M-8 group, 63 enzymes to the CTX-M-9, and 14 to the CTX-M-25. Ultimately, these results follow, once again, with complete agreement with the previous phylogenetic grouping proposed by Bonnet [28]. Moreover, it is also noted that, in these results, the CTX-M-151 group consists solely of that ESBL itself. The presence of 13 more amino acids in this enzyme sequence further highlights the singularity of CTX-M-151 among the remaining groups and re-enforces the speculations of a new group among all CTX-M groups described.

Molecular and Structural Study
As previously described, the phylogeny of CTX-M enzymes showed that they share a certain protein identity, supporting the fact that there are critical amino acids that are conserved between the different groups. In addition, these amino acids represent structural requirements of β-lactamases CTX-M, necessary for the presence of their typical profiles (phenotypic as well as and biochemical).
The intercluster molecular and structural study was performed by multiple alignment. The sequence alignment, in Figure 3, demonstrated 291 amino acids in clusters CTX-M-1, Then, in the CTX-M-25 group, there are up to 50 distinct amino acid substitutions, 12 of which are specific to CTX-M-25. Of these, only seven correspond to the chemical alteration of the amino acid (Gly22Ala, Ala123Gln, Thr192Ala, Lys200Asp, Thr212Met, Asp242Gly2, and Pro262Ser).
In addition, the results obtained by comparing the protein sequences confirms that the most divergent group is CTX-M-2. However, the one with the highest number of amino acid substitutions that involves the amino acid chemical combination is the CTX-M-9 group. Therefore, these data corroborate what was previously mentioned regarding the evolutionary proximity of the groups.
For CTX-M-151, the phylogenetic analysis found no other member to this new group; therefore, no diagram was created. CTX-M-151 presents 163 substitutions, when compared with CTX-M-1, representing more than 50% of amino acid substitutions, despite the 3D structural similarities (Figure 3).

CTX-M-151 as a New Lactamases Group
Considering previous speculations regarding this enzyme as a new CTX-M's group, we took upon the task of phylogeny assessment, as well as molecular structure analysis. Results provided a distinct profile, with 13 more amino acids than the other CTX-M groups and with a total of 163 substitutions when compared to CTX-M-1. Looking into the literature, a recent study conducted by Ghiglione et al. provided further insights regarding this β-lactamase, particularly its pharmacological profile. According to the authors, CTX-M-151 presents higher efficiency hydrolyzing ceftriaxone than ceftazidime (3rd generation of cephalosporins) with 6000-fold higher k cat /K m (specificity of an enzyme to a substrate) values when compared with the remaining CTX-Ms groups. Moreover, inhibition by Avibactam showed a deacylation rate (K off ) up to 14-fold higher than the other class A β-lactamases. Although further experimental studies should be conducted to validate the bioinformatics analysis, these results corroborate our proposal to classify CTX-M-151 as a distinct sixth group among the β-lactamase enzymes [22].

Conclusions
Since their first appearance, CTX-M enzymes were considered a fast-growing group among class A hydrolytic enzymes against beta-lactams. This class of enzymes, until now divided into five groups, present common aspects regarding antimicrobial profile, such as their ability to easily hydrolyze cefotaxime but poorly ceftazidime [42,43]. Still, in terms of epidemiological dissemination, their mechanism is not yet fully understood. Perhaps it may be related to the fact that CTX-M enzymes exhibit a striking plasticity, with many allelic variants belonging to several sublineages.
To date, five well-defined groups among the CTX-M family have been accepted since the work in 2004 of Bonnet [28]. In the present work, we proposed six groups, with the inclusion of CTX-M-151 as a new group, a continually fast growing group of resistance enzymes. Besides our results, experimental data found in literature further corroborate the need to update the current classification of five CTX-M groups, since CTX-M-151 shows a distinct drug hydrolysis profile when compared with the other CTX-Ms.
On a more global view, the need for continuous research regarding CTX-Ms becomes evident. As the incidence of infections and mortality numbers in clinical practice increases, the resistance profile of these enzymes proves to mutate efficiently over time. Hence, with the rising of new variants with distinct therapeutic responses, the need to deepen the knowledge of β-lactamases biological patterns are urgent [44]. This paper aims to alert for the evidences of a new CTX-M, highlighting the need for further studies regarding this topic.