THE PROBLEM OF COMBATING BIOFILMS FORMED BY BACTERIA ON VARIOUS SURFACES, EQUIPMENT AND MEDICAL DEVICES

Research aimed at studying the effects of chemical disinfectants on biofilms formed by Gram-positive and Gram-negative bacteria is carried out jointly with the Laboratory of Anatomy of microorganisms in Institute of Epidemiology and Microbiology named after N.F.Gamaley.

The following processes were studied:

  • The effects of tertiary alkyl amines (for example, OPTIMAX) on mature biofilms of clinical isolates of E. coli and St. aureus.

  • The effect of amines on exopolysaccharide matrix of biofilms and bacterial cells therein.

BACKGROUND

Biological film – is a thin bacterial layer adherent on organic or non-organic surfaces, consisting of microorganisms and their – synthesized by them muco-polymer (exopolysaccharide) matrix. Factor of the stability of biofilms is a muco-polymer layer produced immediately after adhesion and including lipopolysaccharide, proteoglycans, glycoproteins, exopolysaccharides similar to peptidoglycan cell wall.

Biofilms of different microorganisms have a similar principle of the structure. Conjunction is carried out due to hyper synthesis of exo-cellular matrix, the main components of which are polysaccharides. The exopolysaccharide matrix (EPS) also consists of proteins, lipids, nucleic acids (DNA and RNA). Polysaccharides have protective effects and prevent the penetration of antimicrobial agents into an organized structure. Bacteria in biofilms are in close cell-to-cell communication. Colonies of Gram-positive and Gram-negative bacteria of biofilms, interacting with the stimulus, produce signaling molecules that regulate the activity of the colony - autoinducers. With increasing density of colony, concentration of autoinducers increases; reaching a certain concentration, autoinducers bind to their receptors on the membrane surface surrounding the bacteria (the quorum), internal bacterial signaling pathways are activated, under the influence of which expression of certain genes changes is changed.

An example of protective functions of the polymeric film is the survival of Salmonella in the chlorination, which proves the stability of the biofilm to the present method of disinfection. Hypochlorite ions also reduce their activity through inactivation of biopolymers in the surface layers of bacterial film. The protective property of biofilms is shown in respect to quaternary ammonium compounds (QAC) against biofilms E. coli, Pseudomonas, Listeria and S. aureus, even in high concentrations: negatively charged polysaccharides BPM can bind positively charged molecules QAC and thereby protect the biofilm from destruction. Such a mechanism causes the resistance of microbial biofilms to biguanide and disinfectants based on phenolic compounds. Widely used disinfectants based on formaldehyde, glutaryl aldehyde and ortho-phthalaldehyde proved to be ineffective too.

Evaluation of antimicrobial activity of disinfectants is held by traditional microbiological methods, which resulted in the ability of micro-organisms to grow in nutrient media after exposure to microbicides. The use of traditional microbiological methods for assessing microbicidal activity against biofilms formed by bacteria in adverse conditions is inefficient because growth properties of bacteria in the biofilms are changed. No growth of bacteria from biofilms after treatment by disinfectants cannot be considered as the criterion of loss of their viability. Resistance of microorganisms in the biofilm is also due to the protective properties of exo-cellular polysaccharide matrix, the presence of which is the main feature of the formed biofilm. It is not possible to assess the effect of the disinfectant on exo-cellular biofilm matrix with help of only microbiological methods.

Usage of microscopic techniques significantly enhances opportunities of the objective estimation of microbicidal action of drugs on microorganisms, since the use of these methods visualizes process of oriented action of molecules of biocidal substances on the structural components of microorganisms and biofilms. According to the degree of disorganization of certain structural components of microorganisms we can judge their viability.

The study demonstrated that the drug OPTIMAX on the basis of tertiary alkyl amines is effective against biofilms of bacteria and the following conclusions and recommendations are made:

  • A target for attack of tertiary alkylamines molecules – is phosphate group of biological molecules: proteins, phospholipids, DNA and RNA, that is, as a result of the drug’s exposure leads to total destruction of membrane component COP (gram-negative bacteria), cytoplasmic membrane, cytoplasmic proteins, ribosomes, plasmids, and the nucleoid.

  • There were also found significant changes in the peptidoglycan.

  • Under the influence of OPTIMAX bacteria flocculation occurs with the formation of multicellular conglomerates, significant destruction of the cell wall and the cytoplasmic membrane, cytoplasm disorganization and destruction of the ribonucleoprotein complex (nucleoid, ribosomes, plasmids - extrachromosomal factors responsible for the development of resistance).

  • This mechanism of biocidal effect limits the development of bacterial resistance.

  • Destruction of bacteria within the biofilms shows that disinfectant OPTIMAX overcomes exo-cellular matrix (protective barrier), that is an important indicator of biocidal activity of tertiary alkylamine.

  • Identified changes in the structure of the exopolysaccharide matrix and destruction of bacterial cells inside allow us to consider the drug OPTIMAX as a drug for the effective control of biofilms.