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.

[Application of visible longwave radiation for inactivation of microorganisms]. / Primenenie dlinnovolnovogo izlucheniya vidimogo spektra dlya inaktivatsii mikroorganizmov.

PURPOSE: To perform a comparative assessment of the bactericidal and fungicidal effects of various parts of the radiation spectrum (Ultraviolet A, red, green and blue). MATERIAL AND METHODS: The study included strains of the most clinically significant microorganisms, which are the most common causes of purulent keratitis - S. aureus, P. aeruginosa and fungi C. albicans. After populating the surface of Petri dishes uniformly with microorganisms of each culture, on four out of the five specimens the central zone of the surface with a diameter of 1 cm was irradiated with light of different spectrum - from ultraviolet to red, with a total radiation energy density of 5.4 J/cm2. One specimen remained as the control subject. After irradiation, scanning electron microscopy with lanthanides contrasting (SEMLC) was used to evaluate the total metabolic activity, the activity of the efflux systems and the morphological characteristics of the microorganisms. RESULTS: The damaging effect of visible spectrum light and UVA radiation on S. aureus, P. aeruginosa and C. albicans cultures was proved by SEMLC. Green spectrum emission with a wavelength of 500 nm had the highest antimicrobial activity. It was manifested by a decrease in the overall level of metabolic activity (from 40-63 c.u. to 26-37 c.u. (S. aureus (p<0.01), P. aeruginosa (p<0.01) and C. albicans (p<0.05)), as well as a 2-fold increase in the proportion of S. aureus cells with active efflux systems. CONCLUSION: SEMLC allows evaluation of parameters of the microorganisms` state: morphological (form and size) and functional (general metabolic activity, activation of efflux systems). Investigation of S. aureus, P. aeruginosa and C. albicans cultures using SEMLC demonstrated the antimicrobial activity of green spectrum radiation of 500 nm wavelength. This will serve as a basis for further research and development of a method of treating infectious keratitis using green light.

[Integron diversity in blavim-2-carrying carbapenem-resistant clinical Pseudomonas aeruginosa isolates.]

The growing prevalence of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa in nosocomial pathogen populations has been attributed to their clonal spread, and/or horizontal transfer of MBL determinants in mobile genetic elements, including integrons. To characterize the genetic background of the beta-lactamase VIM-2 encoding gene in the population of carbapenemresistant (Carba-R) P. aeruginosa clinical isolates.The detection of class 1 integrons was performed by PCR. Typing of the class 1 integrons containing the blaVIM gene cassette was performed by the PCR-restriction fragment length polymorphism (RFLP) approach followed by sequencing of variable regions of class 1 integrons. Five types of the blaVIM-2-carrying integrons were identified: ST654-isolates accounting for more than 50% of the Carba-R population harbored In56; ST235-isolates contained In559 (26% Carba-R isolates); ST111-isolates (19% Carba-R isolates) were characterized by carrying In59-like integron; two ST235-isolates harbored In59 and In249 each. Except In56, carrying the only blaVIM-2-gene cassette, all other identified integron types harbored the genes of resistance to trimethoprim and/or aminoglycosides. No new types of integrons were identified in the P. aeruginosa clinical isolates. The observed correlation of the integron type with specific STs indicates a clonal dissemination of significant resistance determinant producers - ST111, ST654 and ST235 epidemic lines. The features of the integron variable regions can be used for the epidemiological characterization of clinical P. aeruginosa isolates.

[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.

[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 possibilities, problems and perspectives of mass-spectrometry in medical microbiology: publications review].

The publications review deals with analysis of possibilities, shortcomings and perspectives of mass-spectrometry - important applied technology that is headlong introduced in medical microbiology. The main achievement of mass-spectrometry is fast and valid identification of most species of microbes actual for medicine both in pure growths and biologic samples. There are only single taxonomic which representatives are identified unreliably in case of using of mass-spectrometry. The examples are given concerning methods of intraspecific identification of clinically/epidemiologically significant strains and evaluation of their characteristics, including virulence and resistance. The perspectives of mass-spectrometry are related to implementation into microbiological practice the modes of evaluation of virulence, resistance and identification of microorganisms in mixed cultures (including biological samples) and absence of standard criteria in evaluation of antibiotic resistance of microbes.