Abstract
A blaCTX-M-15 gene is one of the most prevalent resistant marker found in member of enterobacteriaceae. It encodes cefotaxime hydrolysing β-lactamase-15 (CTX-M-15) causing resistance against beta lactam antibiotics. Since single antibiotic therapy fails to control infection caused by multidrug resistance strain, therefore combination therapy was came into practice as an effective treatment. We have first time explained the mechanism where two antibiotics of different classes work against resistant strains. Binding parameters obtained by spectroscopic approach showed significant interaction and complex formation between drugs and CTX-M-15 enzyme with decreased ksv and kq values. CD analysis showed altered conformation and significant changes in alpha helical content of CTX-M-15 enzyme on interaction with streptomycin in combination with cephalosporin. Steady state kinetics revealed decrease in hydrolytic efficiency of enzyme to about 27% by cooperative binding behavior upon sequential treatment of enzyme with streptomycin and cefotaxime. Therefore, the study concludes that combination therapy against CTX-M-15 producing strain with Cefotaxime/Streptomycin in 1:10 molar ratio, decreases CTX-M-15 efficiency significantly because of the fact that streptomycin induced structural changes in CTX-M-15 hence cefotaxime was not properly bound on its active site for hydrolysis rather available for the target to inhibit bacterial cells.
Highlights
Β-lactamases are the group of enzymes that cleave amide bond in beta lactam rings of beta lactam antibiotics rendering them harmless to bacteria
It has been reported that bacteria expressing Extended spectrum beta lactamases (ESBLs) are resistant toward various β-lactam antibiotic groups such as Penicillins, different generations of Cephalosporins, Aztreonam and to various antibiotic /inhibitor combinations (Faheem et al, 2013).The widespread dissemination of CTX-M-15 by E. coli and other enteric bacilli has a significant impact on hospital and community-acquired infections (Bush, 2010b; Chen et al, 2014)
Agarose gel of double digestion showed two bands of 4.8 kb corresponding to pQE-2 vector and 800 bp corresponding to blaCTX-M-15 gene (Figure S1).Purity of the protein obtained after dialysis in phosphate buffer saline (PBS) was estimated to be more than 97% as determined by single band of 31 kDa on SDS-PAGE (Figure S2)
Summary
Β-lactamases are the group of enzymes that cleave amide bond in beta lactam rings of beta lactam antibiotics rendering them harmless to bacteria Production of these enzymes is the predominant cause of gram negative bacterial resistance against β-lactam antibiotics which has become a major health concern (Bonnet, 2004; Bush, 2010a). In Enterobacteriaceae, ESBLs (Extended spectrum beta lactamases) encoding CTX-M type markers on plasmid, are reported worldwide (Coque et al, 2008; Hawkey and Jones, 2009). It hydrolyzes the oxyimino-cephalosporin and cefotaxime with about 1000-fold higher catalytic efficiency than other class A β-lactamases (Bauernfeind et al, 1992; Bonnet, 2004). We have already reported earlier that the synergistic effect of cefoxitin with streptomycin and cefotaxime proved an effective combination against multidrug resistant bacterial strains (Hasan et al, 2013)
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