Application of Dimethicone to Prevent Culture Media from Drying in Microbiological Diagnostics.

The search for novel modifications of culture media aimed at culture prolongation is a prerequisite for microbiological diagnostic progress. The aim of the study was to assess the possibilities of applying dimethicone (polymethylsiloxane) as a barrier between the agar surface and atmosphere to prevent drying of solid and semisolid culture medium providing the retention of its useful properties. Materials and Methods: We studied the dynamics of water (volume) loss of culture media used in microbiology, and the effect of dimethicone on the process. Dimethicone was arranged in layers on culture medium surface. The effect of dimethicone on growth and generation of fast-growing (Staphylococcus aureus, Escherichia coli, Salmonella enterica Serovar Typhimurium, Burkholderia cenocepacia) and slow-growing (Mycobacterium avium) bacteria was studied, as well as on bacterial mobility (Pseudomonas aeruginosa and Escherichia coli) in semisolid agars. Results: The dynamics of water loss in culture media showed the weight loss in all media without dimethicone (control) in 24 h to be statistically significant (p<0.05); 7-8 days later, they lost 50% of weight, and 14 days later they lost approximately 70%. The weight of media under dimethicone underwent no significant changes during the observation period. Growth index of fast-growing bacteria (S. aureus, E. coli, S. Typhimurium, B. cenocepacia) on control culture media without applying any substance, and on culture media under dimethicone had no significant differences. Visible M. avium growth on chocolate agar in controls was recorded on day 19, under dimethicone - on days 18-19. The number of colonies on culture day 19 under dimethicone tenfold exceeded the control values. The mobility indices of P. aeruginosa and E. coli on semisolid agar under dimethicone 24 h later were significantly higher than under control conditions (p<0.05 in both cases). Conclusion: The study confirmed marked deterioration of culture media properties under prolonged cultivation. The suggested protection technology of culture media growth properties using dimethicone showed beneficial effects.

Computer Program for Detection and Analyzing the Porin-Mediated Antibiotic Resistance of Bacteria.

The aim of this work was to develop a new software tool for identifying gene mutations that determine the porin-mediated resistance to antibiotics in gram-negative bacteria and to demonstrate the functionality of this program by detecting porin-mediated resistance to carbapenems in clinical isolates of Pseudomonas aeruginosa. Materials and Methods: The proposed algorithm is based on searching for a correspondence between the reference and the studied genes. When the sought nucleotide sequence is found in the analyzed genome, it is compared with the reference one and analyzed. The genomic analysis is then verified by comparing between the amino acid sequences encoded by the reference and studied genes. The genes of the susceptible P. aeruginosa ATCC 27853 strain were used as the reference nucleotide sequences encoding for porins (OprD, OpdD, and OpdP) involved in the transport of carbapenems into the bacterial cell. The complete genomes of clinical P. aeruginosa isolates from the PATRIC database 3.6.9 and our own collection were used to test the functionality of the proposed program. The analyzed isolates were phenotypically characterized according to the CLSI standard. The search for carbapenemase genes in the studied genomes of P. aeruginosa was carried out using the ResFinder 4.1. Results: The developed program for detecting the genetic determinants of non-plasmid antibiotic resistance made it possible to identify mutations of various types and significance in the porin genes of P. aeruginosa clinical isolates. These mutations led to modifications of the peptide structure of porin proteins. Single amino acid substitutions prevailed in the OpdD and OpdP porins of carbapenem-susceptible and carbapenem-resistant isolates. In the carbapenem-resistant strains, the gene encoding for OprD porin was found heavily modified, including insertions and/or deletions, which led to premature termination of porin synthesis. In several isolates resistant to meropenem, no mutations were detected in the gene encoding for OprD, which might be associated with alternative mechanisms of resistance to carbapenems. Conclusion: The proposed software product can become an effective tool for deciphering the molecular genetic mechanisms of bacterial chromosomal resistance to antibiotics. Testing the program revealed differences between the occurrences of mutations significant for carbapenem resistance in the oprD, opdD, and opdP genes.

[Bacteria survival strategies in contact with antibiotics.]

Bacteria survival in the conditions of antimicrobial therapy is the global problem of health care. This review highlights the complexity and diversity of mechanisms used by bacteria to neutralize antibiotics. To analyze the problem, the search was made using PubMed database, Russian scientific electronic library eLIBRARY, search system of World Health Organization and European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Based on the analysis of survival strategies in the conditions of antibiotics action we propose new classification of resistant bacteria. Classification criteria include the ability to divide under antibiotics action, the survival strategies application as a species trait, the presence of specialized genes determining the transition to the state with reduced/stopped metabolism. Two main groups are resistant bacteria and bacteria with reduced/stopped metabolism, which survive but do not divide in the presence of antibiotic. The first group includes two subgroups: bacteria with intrinsic and adaptive resistance. The second group includes (1) bacteria with specialized genes responsible for cell transformation to the state with reduced/stopped metabolism, (2) bacteria transforming to the state with reduced/stopped metabolism without involvement of special genes, and (3) cell forms with special morphology - spores, cysts and cyst-like cells. We described the usefulness of proposed classification including improved understanding of the correlation between bacteria survival in the presence of antibiotics and molecular mechanism of cell metabolism inhibition, presence or absence of targets for using molecular-genetic methods of bacteria resistant variant determination, the possibility for development of rational antimicrobial therapy methods.