Influence of gentamicin on enterococci biofilm formation

Abstract

Introduction. Today, it is well established that almost 80% of all infectious diseases are caused by microorganisms that exist in the form of biofilms. Microorganisms in the biofilms acquire signs of increased resistance to antibiotics, disinfectants and other aggressive environmental factors, complicate the course of infectious diseases and play an important role in their chronicity.Formation of biofilms by hospital strains of bacteria poses a serious threat to the practical medicine. Enterococci, foremost Enterococcus faecium and Enterococcus faecalis, are the third most common cause of hospital infections, most of which involve the use of permanent medical equipment. Internal hospital infections gain particular importance in intensive care units and in surgical hospitals, since the formation of biofilms is the cause of severe catheter and fan associated infections, sepsis, pneumonia and endocarditis. It should be noted that ineffective antibiotic therapy of infections, accompanied by the formation of biofilms, also leads to significant economic losses.The aim of the work was to study the effects of gentamicin and gentamicin in combination with a penetrator on the processes of enterococci biofilm formation.Materials and methods. The objects of the study included 3 strains of bacteria genus Enterococcus, obtained from the bacteria museum of the Mechnikov Institute of Microbiology and Immunology National Academy of Medical Sciences of Ukraine: E. faecalis ATCC 29212, E. faecalis ІМІ (Х) 49 р, E. faecium ІМІ (Х) 80.The biofilms modelling was performed in 4-section polystyrene Petri dishes. To study the influence of compounds on biofilm formation, a photometric method was used. The optical density (OD) of eluates from enterococci biofilms, stained with crystal violet, was measured with the SF-56L spectrophotometer at a wavelength of 590 nm. Statistical processing of the obtained data was carried out by means of nonparametric statistical methods using Microsoft Excel 2007 and STATISTICA 6.0 programs. The validity of the differences between the two related samples was assessed by the Wilcoxon test and the Sign test. The effect of the compound was evaluated using biofilm inhibitionindex (BII), which was calculated according to the formula: [(OD positive control - OD tested) / OD positive control] × 100%. Reduction of the OD value by more than 25% in the experiment relative to OD positive control was considered as a positive effect.Results and discussions. Analysis of the results allowed to conclude that gentamicin at concentration of 8 mcg/ml is capable of preventing the formation of biofilms taken in experiments with enterococci. Further increase in the concentration of gentamicin to 64 mcg/ml did not lead to an increase in its activity relative to biofilm formation. When applying gentamicin at concentrations of 8 mcg/ml, 16 mcg/ml, 32 mcg/ml and 64 mcg/ml, the index of inhibition equaled 53,8%, 56,6%, 49,9% and 49,6% respectively. A higher inhibitory effect of gentamicin was identified for the formation of E. faecium biofilms than for E. faecalis ones. Thus, when applying gentamicin at concentrations of 8 mcg/ml, 16 mcg/ml, 32 mcg/ml and 64 mcg/ml for E. faecalis biofilm formation, the inhibition index was equal to 45,5 %, 46,7 %, 49,6 % and 48,5 %, for E. faecium – 54,8 %, 53,5 %, 65,3 % and 60,2 % respectively.One way of facilitating the transportation of biocides through the extracellular matrix of biofilms to the target of action may be the use of so-called penetrators. Polyethylene glycol was used as a penetrator (PNT) in our studies. Inhibition index analysis showed a statistically significant increase in the suppressing effect of the combination of gentamicin and PNT on the enterococci biofilm formation compared to the effect of gentamicin without PNT (р<0,05). The inhibition index of gentamicin at concentartions of 8 mcg/ml, 16 mcg/ml, 32 mcg/ml and 64 mcg/ml with PNT amounted to 73,5 %, 74,4 %, 77,2 % and 78,2 % respectively. Also, a higher suppressing effect of gentamicin with penetrator on enterococci biofilm formation was found for E. faecium compared to E. faecalis. Thus, the results obtained suggest that polyethylene glycol can increase the penetration of gentamicin through the biofilm glycocalyx. Conclusion. Gentamicin at concentrations of 8 mcg/ml, 16 mcg/ml, 32 mcg/ml and 64 mcg/ml shows an inhibitory effect on enterococci biofilm formation (inhibition index varied from 49,6 % to 56,6 %). Inhibitory effect of gentamicin at concentrations of 8 mcg/ml, 16 mcg/ml, 32 mcg/ml and 64 mcg/ml on enterococci biofilm formation is enhanced under the influence of polyethylene glycol (inhibition index – from 73,5 % to 78,2 %).