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.

Characteristics of Stenotrophomonas maltophilia isolates from cystic fibrosis patients in Russia.

Stenotrophomonas maltophilia is a common opportunistic microorganism and an important respiratory pathogen in cystic fibrosis (CF). The aim of this study was to determine antimicrobial resistance phenotypes, sequence-types (ST) and genetic determinants of antibiotic resistance in S. maltophilia strains recovered from CF patients in Russia. S. maltophilia isolates recovered from 170 CF patients were analyzed. Minimum inhibitory concentrations of antibacterial agents were determined using Sensititre Gram Negative GNX2F plates and the results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) criteria. Whole-genome sequencing (WGS) was performed on MGISEQ-2000 platform. SPAdes software, Galaxy, ResFinder, Integrall and PubMLST were used for analysis of WGS data. S. maltophilia strains were identified from 24/170 (14%) CF patients. In total, 25 isolates were detected, two strains were isolated from the same patient. The isolates belonged to 17 different STs, including 5 new STs; ST4 was the most prevalent ST. Resistance to ceftazidime was observed in 60% of strains, to ticarcillin-clavulanate - in 32%, to levofloxacin - in 24%, to trimethoprim/sulfamethoxazole - in 12% of strains. All isolates were susceptible to minocycline. All ST4 isolates were resistant or intermediate to ceftazidime and ticarcillin-clavulanate. In two isolates, the sul1 gene was detected. In one isolate, sul1 was part of a class 1 integron. The detected integron also contained the blaGES-7 and aac(6')-Ib-cr genes. The ST4 sequence-type was the most prevalent ST among S. maltophilia strains recovered from CF patients in Russia. Antibiotic resistance genes, including sul1, blaGES-7, aac(6')-Ib-cr, were detected in single strains.

Genome features and antibiotic resistance of Pseudomonas aeruginosa strains isolated in patients with cystic fibrosis in the Russian Federation.

Cystic fibrosis (CF) is a common genetic disease, manifested by airway obstruction and chronic respiratory infection. The most prevalent infectious agent in airways of CF patients is Pseudomonas aeruginosa. This study aimed to determine sequence-types, antimicrobial resistance phenotypes and genes defining adaptive antibiotic resistance in P. aeruginosa isolates recovered from CF patients in Russia. In total, 84 P. aeruginosa strains from 64 CF patients were analyzed. Susceptibility to antibiotics was determined by disk diffusion test. Whole-genome sequencing (WGS) was performed on MGISEQ-2000 platform. SPAdes software, Galaxy, ResFinder, PubMLST were used for analysis of WGS data. Examined P. aeruginosa isolates belonged to 53 different sequence-types (STs), including 6 new STs. High-risk epidemic clone ST235 (10%) and clonal CF P. aeruginosa strains ST17, ST242, ST274 (7%) were detected. Non-susceptibility to ticarcillin-clavulanate, cefepime, imipenem was observed in 63%, 12% and 25% of isolates, respectively; to tobramycin - in 24%, to amikacin - in 35%; to ciprofloxacin, levofloxacin - in 35% and 57% of strains, respectively. Multidrug-resistant phenotype was detected in 18% of isolates. In examined strains, genes of beta-lactamases VIM-2 (5 ST235 strains), VEB-1 (two ST2592 strains), GES-1 (1 ST235 strain), PER-1 (1 ST235 strain) were found. Ciprofloxacin-modifying enzyme CrpP gene was detected in 67% of isolates, aminoglycoside-modifying enzymes AAD, ANT, AAC genes - in 7%, 4%, 12% of strains, respectively. P. aeruginosa isolates from CF patients in Russia demonstrate a high clonal diversity, which is similar to other P. aeruginosa infections. The isolates of high-risk clone and clonal CF P. aeruginosa strains are detected.

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.

[The mass-spectrometric evaluation of сarbapenemase activity of Pseudomonas aeruginosa.]

The Pseudomonas aeruginosa is among number of leading opportunistic pathogens. The evaluation of sensitivity of hospital isolates of P. aeruginosa to antibiotics is an important stage in the struggle with Pseudomonas sepsis pathology. The purpose of study is to confirm diagnostic efficiency of mass spectrometry approach in evaluation of сarbapenemase activity in clinical isolates of P. aeruginosa. The study was targeted to detection of сarbapenemases in 50 clinical isolates of P. aeruginosa, non-sensitive to сarbapenemas (control group - 9 isolates of P. aeruginosa sensitive to сarbapenemas). The comparative analysis was implemented concerning the results obtained using laser desorption ionization time-of-flight mass spectrometry and using such common techniques as phenotype detection of presence of metallo-beta-lactamase using E-tests and detection of presence of genes of carbapenemases (VIM, IMP, NDM) using polymerase chain reaction in real-time. The metallo-beta-lactamase activity was established in 14 (28%) out of 50 non-sensitive to сarbapenemas strains. All of them had genes of carbapenemases VIM-type. No IMP and NDM genes were detected in any strain. The VIM genes were detected only in metallo-beta-lactamase positive strains and metallo-beta-lactamase activity was registered only in carriers of VIM genes. According data of MALDI-TOF, all metallo-beta-lactamase and VIM positive strains demonstrated increased capacity of hydrolyzing meropenem. The percentage of hydrolysis under testing of the given strains made up to from 7.6 to 59.3. The absence of carbapenemase activity was demonstrated by 36 (72%) out of 50 strains non-sensitive to сarbapenemases with percentage of hydrolysis from 0 to 4. None of 9 control isolates sensitive to сarbapenemases had metallo-beta-lactamase activity, carried analyzed genes of сarbapenemas and hydrolyzed meropenem. The MALDI-TOF mass spectrometry is a perspective technique to be applied in practice of clinical microbiology for detect isolates of P. aeruginosa, producing сarbapenemases.

Inhibitory effect of streptococci on the growth of M. catarrhalis strains and the diversity of putative bacteriocin-like gene loci in the genomes of S. pneumoniae and its relatives.

S. pneumoniae is a facultative human pathogen causing a wide range of infections including the life-threatening pneumoniae or meningitis. It colonizes nasopharynx as well as its closest phylogenetic relatives S. pseudopneumoniae and S. mitis. Both the latter, despite the considerable morphological and phenotypic similarity with the pneumococcus, are considerably less pathogenic for humans and cause infections mainly in the immunocompromized hosts. In this work, we compared the inhibitory effect of S. pneumoniae and its relatives on the growth of Moraxella catarrhalis strains using the culture-based antagonistic test. We observed that the inhibitory effect of S. mitis strains is kept when a hydrogen peroxide produced by cells is inactivated by catalase, and even when the live cells are killed in chloroform vapors, in contrast to the pneumococcus whose inhibiting ability disappeared when the cells die. It was suggested that this effect may be due to the production of bacterial antimicrobial peptides by S. mitis, so we examined the genomes of our strains for the presence of bacteriocin-like peptides encoding genes. We observed that a set of bacteriocin-like genes in the genome of S. mitis is greatly poorer in comparison with S. pneumoniae one; moreover, in one S. mitis strain we found no bacteriocin-like genes. It could mean that there are probably some additional opportunities of S. mitis to inhibit the growth of competing neighbors which are still have to be discovered.

[SPECTRUM OF ANTIBIOTIC RESISTANCE AND PREVALENCE OF OXA-CARBAPENEMASES AMONG ACINETOBACTER BAUMANNII STRAINS, ISOLATED FROM PATIENTS OF SURGICAL AND REANIMATION DEPARTMENTS IN MOSCOW].

AIM: Characterize spectrum of antibiotics resistance of Acinetobacter baumannii strains, isolated from patients of 8 surgical and reanimation departments of 3 medical institution of Moscow, and determine molecular-genetic mechanisms of stability of their carbapenem-resistant forms. MATERIALS AND METHODS: 95 strains of A. baumannii, isolated from patients of reanimation and surgical departments of Moscow in 2012-2014, were studied. Sensitivity of strains to antibiotics was tested phenotypically according to recommendations of EUCAST. The presence of VIM, IMP, OXA-23, OXA-40, OXA-48, OXA-58 and NDM genes in the studied strains was determined by polymerase chain reaction in real time. RESULTS: 86.3% of strains turnedout to be non-sensitive to carbapenems, sensitive--13.7%. 80.0% of strains were non-sensitive to gentamicin, 80.0% of strains--to netilmicin, 94.7% of strains--to ciprofloxacin 2.1%--to colistin. 91.6% of isolates have shown non-sensitivity to members of 2 and more classes of antibiotics, 78.9% of strains--to members of 3 classes. 2 strains were panresistant, 4.2% (4/95) of the isolates were sensitive to all the classes of antibiotics. Metallo-ß-lactamases were not detected. Genes of carbapenemases (OXA-23 and/or OXA-40) were detected in 85.3% (81/95) of strains, characterized phenotypically as non-sensitive to carbapenems. CONCLUSION: The results obtained shown an increase of resistance to carbapenems and multiple resistance in clinically significant strains of A. baumannii. Resistance to carbapenems is associated with OXA-23 and OXA-40 genes. The conclusions allow to justify perspectives of introduction of technologies of molecular-genetic testing of antibiotics resistance.

The mystery of the fourth clone: comparative genomic analysis of four non-typeable Streptococcus pneumoniae strains with different susceptibilities to optochin.

Optochin-resistant pneumococci can be rarely caught in clinical microbiology laboratories because of the routine identification of all such strains as viridans group non-pneumococci. We were lucky to find four non-typeable Streptococcus pneumoniae clones demonstrating the different susceptibilities to optochin: one of them (Spn_13856) was resistant to optochin, while the other three (Spn_1719, Spn_27, and Spn_2298) were susceptible. Whole genome nucleotide sequences of these strains were compared to reveal the differences between the optochin-resistant and optochin-susceptible strains. Two adjacent genes coding maltose O-acetyltransferase and uridine phosphorylase which were presented in the genomes of all optochin-susceptible strains and missed in the optochin-resistant strain were revealed. Non-synonymous substitutions in 14 protein-coding genes were discovered, including the Ala49Ser mutation in the C-subunit of the F0 part of the ATP synthase rotor usually associated with pneumococcal optochin resistance. Modeling of a process of optochin interaction with the F0 part of the ATP synthase rotor indicates that the complex of optochin with "domain C" composed by wild-type C-subunits is more stable than the same complex composed of Ala49Ser mutant C-subunits.