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Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a major nosocomial pathogen with only a few antimicrobial agents, including colistin, remaining active. However, the emergence of colistin-resistant (Col-R) isolates is compromising the activity of colistin. In this study, a collection of 159 CRKP recovered from three hospitals in Moscow (Russian Federation) was examined. The isolates demonstrated resistance to cephalosporins (100%), ciprofloxacin (92.5%), fosfomycin (90.1%), netilmicin (81.1%), gentamicin (84.3%) and amikacin (49.7%). The rate of colistin resistance (MIC > 2 mg/L by broth microdilution) was 44.7%; moreover, 6.7% of isolates were tigecycline-resistant. Among 18 sequence types (STs) discovered, isolates of five lineages including ST307 (n = 46; 28.9%), ST395 (n = 40; 25.2%), ST377 (n = 17; 10.7%), ST48 (n = 17; 10.7%) and ST23 (n = 16; 10.1%) dominated. Carriage of a bla OXA-48-like carbapenemase gene was detected in 146 CRKP (91.8%); 11 (6.9%) and 2 (1.3%) isolates harboured bla NDM-1 and bla KPC-3 , respectively. Among 71 Col-R isolates, colistin MICs ranged from 4 mg/L to >1024 mg/L (MIC 50/90 , 2/512 mg/L). All Col-R isolates were mcr-1-negative. In 19 (26.8%) Col-R isolates, inactivation of mgrB by insertion sequences IS1A, IS1R, ISKpn14 and ISKpn26 and a novel miniature inverted-repeat transposable element (MITE) Kpn1 was observed. Carriage of MITEKpn1 was restricted to six ST307 isolates and affected mgrB at nucleotide position 75. mgrB deletion was observed in four (5.6%) Col-R isolates. Moreover, PmrA and/or PmrB were altered in three (4.5%) Col-R isolates with wild-type mgrB. Thus, bla OXA-48-like -carrying Col-R ST307 K. pneumoniae is emerging as a dominant clone in Moscow., (Copyright © 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

In recent years, because of carbapenemase spreading in Klebsiella pneumoniae strains, the antibiotic of reserve group, colistin, is increasingly prescribed. In vitro testing of colistin susceptibility in everyday practice has a number of difficulties due to the cationic properties of molecule and weak diffusion into agar. Therefore it is recommended to use the reference Broth Microdilution Method (BMD) for determination of the Minimum Inhibitory Concentration (MIC) for colistin. The purpose of the study was to determine susceptibility to colistin in 119 carbapenem-resistant K. pneumoniae (CRKp) which were isolated from the patients at three hospitals in Moscow in 2012-2016 by the broth microdilution method (BMD) and to compare these data with the ones obtained by epsilometer test (E-test) and VITEK 2 Compact. The proportion of resistant isolates (MIC>2 mg/L) was 52%, 39%, 35% respectively. Both commercial methods demonstrated a high level of the very major error (VME) that was 26% for the E-test method and 34% for the VITEK 2 Compact. The values of categorical agreement and essential agreement (CA, EA) were less than 90%. A single major error (ME) was detected for the VITEK 2 Compact. In conclusion, results of both commercial tests for determination of MIC for colistin showed differences with the results of the reference BMD. It is necessary for clinical laboratories to be aware about this discrepancy and to use E-tests and VITEK 2 Compact with caution to determine colistin susceptibility., Competing Interests: The authors declare no conflict of interest.

Klebsiellapneumoniae is a significant pathogen associated with hospital infections. Its was isolated in intensive care units (ICU) at two pediatric hospitals in Moscow in 2012-2014 from 41% (387/935) of the patients. The rate of carbapenem-nonsusceptibility (Carba-NS) amounted to 25% for imipenem and 27% for meropenem. For further analyses, 67 isolates were selected, including 57 Carba-NS and 10 Carba-susceptible (Carba-S). Among the isolates, 100% was nonsusceptible to the III-IV generation cephalosporins, 50-84% was resistant to aminoglycosides. The rate of nonsusceptibility to ciprofloxacin and phosphomycin exceeded 90%. All the tested Carba-S Kpneumoniae isolates were susceptible to tigecycline, whereas 25% of the Carba-NS isolates was tigecycline-NS. The prevalence of the colistin-NS isolates was the same in Carba-S (20%) and Carba-NS (26%) bacteria. The blamrx_ gene was carried by 100% of the Carba-S isolates, combining with the blaTEM gene in 60% of the isolates. In 89% of the Carba-NS isolates the OXA-48 carbapenemase was detected, which was combined with CTX-M and/or TEM in all but 1 isolate. Thus, over the last decade, the rate of Carba-NS among nosocomial Kpneuynoniae increased and the OXA-48 carbapenemase was shown to be dominating in the mechanism of Carba-NS in the pediatric ICUs 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-β-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.

Nosocomial infections and their rational antibiotic treatment represent a major challenge for the healthcare nowadays. In this context, gramnegative bacteria including Pseudomonas aeruginosa, Acinetobacter baumanii and Enterobacteriaceae spp. are etiologically important and characterized by a significant level of antibiotic resistance. To examine dynamics of the respiratory tract colonization by hospital flora, tracheal aspirates obtained at three time points from 69 children with severe craniocerebral trauma during their stay in ICU were analysed. Colonization was observed on the 4th day of the ICU stay with predomination of K. pneumoniae (45%) and A. baumanii (27-37%). P. aeruginosa was detected after the 8th day of the ICU stay with the isolation rate of 33%. Substantial proportions of P. aeruginosa (61%), A. baumanii (78%) and K. pneumoniae (25%) were resistant to carbapenems. In 65 carbapemen resistant isolates, the presence of carbapenemases was examined using PCRs. OXA-48 carbapenemase was detected in 11 out of 14 (78%) K. pneumoniae isolates. Among the A. baumanii isolates, 30/31 (97%) carried OXA-40 and 1/31 (3%) had OXA-23 carbapenemases. None of the examined A. baumanii and K. pneumoniae isolates produced metallo-betalactamases (MBL). In contrast, all 20 carbapenem resistant P. aeruginosa isolates produced a MBL, and in 12 out of 20 (60%) of theme VIM-2 was detected. Thus, gramnegative nosocomial microflora rapidly colonizes ICU patients and has a high level of resistance to antibiotics, including carbapenems.