Abstract
The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of bla CTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5α transformed with bla CTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC50 value (6 nM), high affinity (K i = 0.017 µM) and better acylation efficiency (k +2/K′ = 0.44 µM−1s−1). It forms an acyl-enzyme covalent complex, which is quite stable (k +3 = 0.0057 s−1). Since increasing resistance has been reported against conventional β-lactam antibiotic-inhibitor combinations, we aspire to design a non-β-lactam core containing β-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC50 (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K i = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-β-lactam containing β-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.
Highlights
Antibiotic resistance in Gram-negative bacteria is a major health concern
Antibiotic Susceptibility Testing Based on MIC Analysis The MICs of the b-lactam antibiotics alone or in combination with different inhibitors were determined on E. coli DH5a harbouring blaCTX-M-15 gene from Enterobacter cloacae isolate (EC-15) of clinical background and the results are presented in table 1
The efficacy of antibiotic-inhibitor combination was studied on DH5a transformed with blaCTX-M-15 gene (Table 1)
Summary
Antibiotic resistance in Gram-negative bacteria is a major health concern. It is principally observed due to the emergence of b-lactamase producers, which leads to the resistance against blactam antibiotic [1]. Plasmid-encoded ESBLs of the CTX-M type are increasingly being reported worldwide in Gram-negative bacteria and account for most of the ESBL types found in the Enterobacteriaceae [4,5]. They form a rapidly growing family that comprises more than 100 variants (http://www.lahey.org/studies) and are divided into five groups according to amino acid sequence identity, with different groups being prevalent in different countries [6,7]. The widespread dissemination of CTX-M-15 has a significant impact on the treatment of hospital- and community-acquired infections caused by E. coli and other enteric bacilli [13,14,15]
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