THE NEW METALL-BETA-LACTAMASE’S INHIBITOR EFFICACY IN A MODEL SYSTEM IN VITRO

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Abstract

The Enterobacteriaceae antibiotics resistance depends on a combination of several mechanisms, such as the beta-lactamases overproduction, the microbial cell reduction outer membrane permeability (usually associated with loss of protein porin), the presence of efflux systems. Particularly noteworthy are the metallo-beta-lactamases (MBL) whose presence causes resistance of gram-negative microorganisms to all beta-lactam antibiotics (in some cases except aztreonam). Currently there are no MBL inhibitors permitted for use in the clinic. The effective inhibitors search for carbapenem-resistant bacteria’ MBL authorized for use in the clinic and reinforcing effects of carbapenems, served as the basis for the present study. The work was carried out in three stages: 1) creating a model system using a standard enzyme reagent metallo-beta-lactamase P. aeruginosa recombinant expressed in E. coli, to evaluate the increasing of minimal inhibitory concentrations (MIC) of carbapenems against previously sensitive Gram-negative microorganisms strains in vitro;

2) evaluation of MBL promising inhibitors in the presence of the same standard enzyme reagent; 3) evaluation of the ability of the identified inhibitors increase the carbapenems effects against clinical isolates of Gram-negative microorganisms producing MBL, in terms of the their MIC and fractional inhibitory concentration index (FIC index). The checkerboard array was modified to evaluate the combined use of carbapenems and potential MBL inhibitor — a drug from the group of bisphosphonates — etidronic acid. Using a standard enzyme reagent metallo-beta-lactamase P. aeruginosa recombinant expressed in E. coli, we created a model system that allows to assess the prospects of new inhibitors MBL gram-negative microorganisms. A dose-dependent effect of increasing the meropenem level MIC from reagent MBL quantity in a model system against previously antibiotic sensitive reference strains of microorganisms was revealed. MBL enzyme inactivation was noted in the presence of even small doses of bisphosphonate, in the tests the appearance of logarithmic phase of P. aeru ginosa ATCC 27853 growth was shown delayed up to 12 hours compared to the control. In this case the maximum dose of etidronic acid 50 000–100 000 μg/ml completely inhibited the MBL, there was no a log phase microbe’s growth due to the effect of meropenem on the reference level of sensitivity (2 μg/ml). The synergistic effect (FIC index < 0.5) of combined meropenem with etidronic acid use was identified against clinical isolates Gram-negative microorganisms resistant to carbapenems and producing MBL, wherein the enhancing action of the antibiotic was more 8–512 times compared with the initial MIC levels.

About the authors

A. G. Afinogenova

St. Petersburg Pasteur Institute, St. Petersburg, Russian Federation
St. Petersburg State University, St. Petersburg, Russian Federation

Author for correspondence.
Email: spbtestcenter@mail.ru

PhD, MD (Biology), Head of LaboratoryTesting Centre, St. Petersburg Pasteur Institute; Professor of Surgical Dentistry Department, St. Petersburg State University, St. Petersburg, Russian Federation;

Russian Federation

T. M. Voroshilova

The Nikiforov All-Russian Center of Emergency and Radiation Medicine, St. Petersburg, Russian Federation

Email: spbtestcenter@mail.ru

Bacteriologist, Head of Bacterial Laboratory, The Nikiforov All-Russian Center of Emergency and Radiation Medicine

Russian Federation

G. E. Afinogenov

St. Petersburg State University, St. Petersburg, Russian Federation

Email: spbtestcenter@mail.ru

PhD, MD (Medicine), Professor of Surgical Dentistry Department

Russian Federation

D. Yu. Maday

St. Petersburg State University, St. Petersburg, Russian Federation

Email: spbtestcenter@mail.ru

PhD, MD (Medicine), Professor, Head of Surgical Dentistry Department

Russian Federation

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