bla CTX-M-152, a Novel Variant of CTX-M-group-25, Identified in a Study Performed on the Prevalence of Multidrug Resistance among Natural Inhabitants of River Yamuna, India

Abstract

Natural environment influenced by anthropogenic activities creates selective pressure for acquisition and spread of resistance genes. In this study, we determined the prevalence of Extended Spectrum β-Lactamases producing gram negative bacteria from the River Yamuna, India, and report the identification and characterization of a novel CTX-M gene variant bla CTX-M-152 . Of the total 230 non-duplicate isolates obtained from collected water samples, 40 isolates were found positive for ESBL production through Inhibitor-Potentiation Disc Diffusion test. Based on their resistance profile, 3% were found exhibiting pandrug resistance (PDR), 47% extensively drug resistance (XDR), and remaining 50% showing multidrug resistant (MDR). Following screening and antimicrobial profiling, characterization of ESBLs (bla TEM and bla CTX-M ), and mercury tolerance determinants (merP, merT, and merB) were performed. In addition to abundance of bla TEM-116 (57.5%) and bla CTX-M-15 (37.5%), bacteria were also found to harbor other variants of ESBLs like bla CTX-M-71 (5%), bla CTX-M-3 (7.5%), bla CTX-M-32 (2.5%), bla CTX-M-152 (7.5%), bla CTX-M-55 (2.5%), along with some non-ESBLs; bla TEM-1 (25%) and bla OXY (5%). Additionally, co-occurrence of mercury tolerance genes were observed among 40% of isolates. In silico studies of the new variant, bla CTX-M-152 were conducted through modeling for the generation of structure followed by docking to determine its catalytic profile. CTX-M-152 was found to be an out-member of CTX-M-group-25 due to Q26H, T154A, G89D, P99S, and D146G substitutions. Five residues Ser70, Asn132, Ser237, Gly238, and Arg273 were found responsible for positioning of cefotaxime into the active site through seven H-bonds with binding energy of -7.6 Kcal/mol. Despite small active site, co-operative interactions of Ser237 and Arg276 were found actively contributing to its high catalytic efficiency. To the best of our knowledge, this is the first report of bla CTX-M-152 of CTX-M-group-25 from Indian subcontinent. Taking a note of bacteria harboring such high proportion of multidrug and mercury resistance determinants, their presence in natural water resources employed for human consumption increases the chances of potential risk to human health. Hence, deeper insights into mechanisms pertaining to resistance development are required to frame out strategies to tackle the situation and prevent acquisition and dissemination of resistance determinants so as to combat the escalating burden of infectious diseases.

Keywords: ESBL; antibiotics; mechanisms of resistance; polluted environment; resistance genes.

Figure 1

Percentage of resistant isolates against the β-lactam and non β-lactam classes of antibiotics.

Figure 2

Tree diagram showing similarity among the CTX-M lineage enzymes and clustering of different CTX-M group members. The tree was constructed with the MEGA6 software. The variant CTX-M-152 is highlighted red and variants identified in this study are marked by asterisk.

Figure 3

Superimposed image of CTX-M-152(blue) and CTX-M-9 (green). The conserved element number 1 (S70-T71-S72-K73), conserved element number 2 (S130-D131-N132), conserved element number 3 (K234-T235-G236) and conserved element number 4 (E166-P167-T168-L169-N170) are labeled to show the active site.

Figure 4

Key polar interactions observed between enzyme CTX-M-152 (A) or CTX-M-9 (B) and Cefotaxime. The broken lines represent hydrogen bonds and interacting amino acid residues are labeled. The cefotaxime is shown in stick form; oxygen atoms are red, nitrogen atoms in blue, carbon atoms white, and sulfur atoms in yellow.

Figure 5

Active site of CTX-M-152 (A) and CTX-M-9 (B) with substrate cefotaxime. Residues around the binding pocket of substrate are depicted in transparent cloud form. The cefotaxime is presented in stick form; oxygen atoms are red, nitrogen atoms in blue, carbon atoms white, and sulfur atoms in yellow.