Abstract
Acinetobacter baumannii, an important nosocomial pathogen, is increasingly becoming resistant to antibiotics including recent β-lactam like imipenem. Production of different types of β-lactamases is one of the major resistance mechanisms which bacteria adapt. We recently reported the presence of a β-lactamase, OXA-51, in clinical strains of A. baumannii in ICUs of our hospital. This study is an attempt to understand the structure–function relationship of purified OXA-51 in carbapenem resistance in A. baumannii. The OXA-51 was cloned, expressed in E. coli Bl-21(DE3) and further purified. The in vitro enzyme activity of purified OXA-51 was confirmed by two independent techniques; in-gel assay and spectrophotometric method using nitrocefin. Further in vivo effect of OXA-51 was followed by transmission electron microscopy of bacterium. Biophysical and biochemical investigations of OXA-51 were done using LC-MS/MS, UV–Vis absorption, fluorescence, circular dichroic spectroscopy and isothermal calorimetry. Native OXA-51 was characterized as 30.6 kDa, pI 8.43 with no disulphide bonds and comprising of 30% α-helix, 27% β-sheet. Secondary structure of OXA-51 was significantly unchanged in broad pH (4–10) and temperature (30–60 °C) range with only local alterations at tertiary structural level. Interestingly, enzymatic activity up to 75% was retained under above conditions. Hydrolysis of imipenem by OXA-51 (km,1 μM) was found to be thermodynamically favourable. In the presence of imipenem, morphology of sensitive strain of A. baumannii was drastically changed, while OXA-51-transformed sensitive strain retained the stable coccobacillus shape, which demonstrates that imipenem is able to kill sensitive strain but is unable to do so in OXA-51-transformed strain. Hence the production of pH- and temperature-stable OXA-51 appears to be a major determinant in the resistance mechanisms adopted by A. baumannii in order to evade even the latest β-lactams, imipenem. It can be concluded from the study that OXA-51 plays a vital role in the survival of the pathogen under stress conditions and thus poses a major threat.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.