Background: Acinetobacter species other than A. baumannii are becoming increasingly more important as opportunistic pathogens for humans. The primary aim of this study was to assess the prevalence, species distribution, antimicrobial resistance patterns, and carbapenemase gene content of clinical Acinetobacter non-baumannii (Anb) isolates that were collected as part of a sentinel surveillance program of bacterial infections in hospitalized patients. The secondary aim was to evaluate the performance of MALDI-TOF MS systems for the species-level identification of Anb isolates. Methods: Clinical bacterial isolates were collected from multiple sites across Russia and Kazakhstan in 2016–2022. Species identification was performed by means of MALDI-TOF MS, with the Autobio and Bruker systems used in parallel. The PCR detection of the species-specific bla_OXA-51-like gene was used as a means of differentiating A. baumannii from Anb species, and the partial sequencing of the rpoB gene was used as a reference method for Anb species identification. The susceptibility of isolates to antibiotics (amikacin, cefepime, ciprofloxacin, colistin, gentamicin, imipenem, meropenem, sulbactam, tigecycline, tobramycin, and trimethoprim–sulfamethoxazole) was determined using the broth microdilution method. The presence of the most common in Acinetobacter-acquired carbapenemase genes (bla_OXA-23-like, bla_{OXA-24/40-like}, bla_OXA-58-like, bla_NDM, bla_IMP, and bla_VIM) was assessed using real-time PCR. Results: In total, 234 isolates were identified as belonging to 14 Anb species. These comprised 6.2% of Acinetobacter spp. and 0.7% of all bacterial isolates from the observations. Among the Anb species, the most abundant were A. pittii (42.7%), A. nosocomialis (13.7%), the A. calcoaceticus/oleivorans group (9.0%), A. bereziniae (7.7%), and A. geminorum (6.0%). Notably, two environmental species, A. oleivorans and A. courvalinii, were found for the first time in the clinical samples of patients with urinary tract infections. The prevalence of resistance to different antibiotics in Anb species varied from <4% (meropenem and colistin) to 11.2% (gentamicin). Most isolates were susceptible to all antibiotics; however, sporadic isolates of A. bereziniae, A. johnsonii, A. nosocomialis, A. oleivorans, A. pittii, and A. ursingii were resistant to carbapenems. A. bereziniae was more frequently resistant to sulbactam, aminoglycosides, trimethoprim–sulfamethoxazole, and tigecycline than the other species. Four (1.7%) isolates of A. bereziniae, A. johnsonii, A. pittii were found to carry carbapenemase genes (bla_OXA-58-like and bla_NDM, either alone or in combination). The overall accuracy rates of the species-level identification of Anb isolates with the Autobio and Bruker systems were 80.8% and 88.5%, with misidentifications occurring in 5 and 3 species, respectively. Conclusions: This study provides important new insights into the methods of identification, occurrence, species distribution, and antibiotic resistance traits of clinical Anb isolates.

Objective. To study the prevalence and mechanisms of antibiotic resistance, including carbapenemase production, in clinical isolates of Klebsiella pneumoniae and Escherichia coli isolated in different regions of Russia as part of the sentinel multicenter surveillance study in 2020–2021, and to explore the population structure of K. pneumoniae and the impact of “high-risk clones” on antibiotic resistance. Materials and Methods. Consecutive, non-duplicate isolates of K. pneumoniae (n = 2503) and E. coli (n = 2055) isolated from various specimens (blood, cerebrospinal fluid, respiratory samples, urine, wound secretions, etc.) of hospitalized patients with clinical signs of infection in 55 hospitals of 29 cities of Russia were studied. Species identification of isolates was performed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibilities were determined by serial broth microdilution or, in the case of fosfomycin, by agar dilution method, and results were interpreted according to EUCAST v13 MIC breakpoints. Carbapenemase production was determined phenotypically by carbapenem inactivation method (CIM), the presence of genes of the most common serine carbapenemases (KPC, OXA-48) and metallo-β-lactamases (VIM, IMP, NDM) was determined by real-time PCR. K. pneumoniae clinical isolates were genotyped and assigned to the known clonal complexes (CC) and sequence types (ST) using SNP typing and multilocus sequencing typing (MLST) methods. K- and O-serotypes, acquired resistance and virulence genes, and plasmids carrying these genes were characterized using whole-genome sequencing of selected isolates (n = 215). Results. The resistance rates of nosocomial/community-acquired isolates of K. pneumoniae were as follows: amoxicillin-clavulanate – 88.63⁄57.99%, piperacillin-tazobactam – 82.92⁄45.49%, cefotaxime – 87.74⁄56.97%, ceftazidime – 84.76⁄53.07%, cefepime – 81.43⁄49.18%, aztreonam – 1.63⁄53.28%, ceftazidime-avibactam – 30, 88⁄9.22%, ceftolozan-tazobactam – 70.06/31.35%, ertapenem – 72.10⁄28.69%, meropenem – 49.60⁄15.16%, imipenem – 44.54⁄13.73%, gentamicin – 60.82⁄30.33%, amikacin – 42.06/17.21%, ciprofloxacin – 85.10⁄49.39%; trimethoprimsulfamethoxazole – 74.38⁄48.16%, colistin – 5.96⁄2.25%. The resistance of nosocomial/outpatient isolates of E. coli were: ampicillin – 84.93⁄67.67%, amoxicillin-clavulanate – 57.37⁄39.73%, piperacillin-tazobactam – 19.48⁄8.70%, cefotaxime – 63.83⁄34.19%, ceftazidime – 45.32⁄20.34%, cefepime – 35.95⁄16.61%, aztreonam – 51.78⁄26.11%, ceftazidime-avibactam – 5.71⁄0.80%, ceftolozane-tazobactam – 11, 95⁄2.22%, ertapenem – 8.18⁄1.42%, meropenem – 5.17⁄0.53%, imipenem – 4.95⁄0.36%, gentamicin – 24.54⁄13.68%, amikacin – 5.49⁄1.42%, ciprofloxacin – 54, 14⁄32.50%, trimethoprim-sulfamethoxazole – 52.21⁄38.54%, fosfomycin – 2.48⁄1.43%, colistin – 1.60⁄1.07%, tigecycline – 6.35⁄3.11%. The frequency of carbapenemase production among K. pneumoniae nosocomial isolates was 65.32% (OXA-48 – 40.75%, NDM – 30.28%, KPC – 8.74%, OXA-48 + NDM – 10.62%, OXA-48 + KPC – 2.98%, NDM + KPC – 0.45%, OXA-48 + NDM + KPC – 0.20%). More than 70% of nosocomial isolates of K. pneumoniae belonged to only 7 major genetic lineages known as “high-risk international clones”: CC395 – 37.40%, CC23 – 9.59%, CC307 – 8.64%, CC147 – 7.61%, CC15 – 2.95%, CC258 – 2.92%, and CC11 – 2.41%. The population of community-acquired K. pneumoniae was characterized by significantly greater genetic diversity (Simpson diversity index: D = 0.919; 95% CI: 0.904 to 0.933) compared with the population of nosocomial strains (Simpson diversity index: D = 0.815; 95% CI: 0.802 to 0.827). Strains of the “hypervirulent” genetic lineage of K. pneumoniae CC23 were more common in community-acquired infections. Conclusions. The extremely high frequency of resistance to cephalosporins in K. pneumoniae (> 80%) and E. coli (> 60%), as well as the high frequency of combined resistance to aminoglycosides and fluoroquinolones precludes their empirical use for the treatment of serious nosocomial infections caused by these pathogens. K. pneumoniae shows a rapid increase in resistance to carbapenems, mainly due to the spread of carbapenemases of three major groups: OXA-48, NDM and KPC. The overall increase in the frequency of carbapenemase production is accompanied by the growing diversity of carbapenemases, the increasing prevalence of strains producing NDM and KPC enzymes and those co-producing multiple carbapenemases simultaneously. In community-acquired infections, the high prevalence of resistance to cephalosporins in E. coli (> 30%) and K. pneumoniae (> 50%) remains the most important problem.

Objective. To assess the in vitro activity of ceftazidime-avibactam against clinical Enterobacterales and Pseudomonas aeruginosa isolates in various regions of Russia based on results of local susceptibility testing by disk diffusion method. Materials and Methods. Overall, 160 laboratories located in 61 Russian cities participated in this surveillance during 2018-2020. All consecutive clinical isolates of Enterobacterales and Pseudomonas aeruginosa in each participating laboratory were included in the study. Ceftazidime-avibactam susceptibility testing was done by disc-diffusion method in accordance with current EUCAST recommendations. Susceptibility data for carbapenems and III-IV generation cephalosporins, as well as results of carbapenemases detection, were also reported, if available. All the data were recorded in electronic case report form developed on the OpenClinica online platform (www.openclinica.com). Data analysis and reporting were done using AMRcloud online platform (https://amrcloud.net/). Results. In total, we received information on antimicrobial susceptibility of 22,121 isolates, including 17,456 (78.9%) Enterobacterales and 4,665 (21.1%) P. aeruginosa. Less than 9% of Enterobacterales isolates were resistant to ceftazidime-avibactam. At the same time rates of resistance to ceftazidime, cefotaxime, cefepime, ertapenem, imipenem, and meropenem were 54.1%, 58.9%, 59.4%, 41.4%, 23.9%, and 21.3%. Among Enterobacterales the highest level of resistance to ceftazidime-avibactam was detected in K. pneumoniae (16.5%), lowest – in E. coli (2.1%). Some increase of resistance to ceftazidimeavibactam was noted during the study – from 7.8% in 2018-2019 to 9.6% in 2020 (p = 0.0001). Rate of resistance to ceftazidime-avibactam in P. aeruginosa was 33.1%. At the same time rates of resistance to ceftazidime, cefepime, imipenem, and meropenem were 51.1%, 54.5%, 50%, and 47.3%. During the study there was statistically significant decrease in resistance to ceftazidime-avibactam in P. aeruginosa (p = 0.0001). Resistance rates for all beta-lactams for both Enterobacterales and P. aeruginosa were higher in nosocomial isolates than in community-acquired isolates. Conclusions. Ceftazidime-avibactam demonstrated significantly higher in vitro activity against Enterobacterales and P. aeruginosa Russian clinical isolates comparing with commonly used carbapenems and extended spectrum cephalosporins. Access for all study data available at the AMRcloud online platform (https://amrcloud.net/ru/project/cazavi-1-2/).

Macrolide (MLR) and fluoroquinolone (FQR) resistance in Mycoplasma genitalium (MG) has recently become a major problem worldwide. The available data on the prevalence of MLR and FQR in MG in Russia are limited. In this study, we aimed to evaluate the prevalence and pattern of mutations in 213 MG-positive urogenital swabs from patients in Moscow between March 2021 and March 2022. MLR- and FQR-associated mutations were searched in 23S rRNA as well as in the parC and gyrA genes using Sanger sequencing. The prevalence of MLR was 55/213 (26%), with A2059G and A2058G substitutions being the two most common variants (36/55, 65%, and 19/55, 35%, respectively). FQR detection showed 17% (37/213); two major variants were D84N (20/37, 54%) and S80I (12/37, 32.4%) and three minor variants were S80N (3/37, 8.1%), D84G (1/37, 2.7%), and D84Y (1/37, 2.7%). Fifteen of the fifty-five MLR cases (27%) simultaneously harbored FQR. This study revealed the high frequency of MLR and FQR. We conclude that the improvement of patient examination algorithms and therapeutic approaches should be combined with the routine monitoring of antibiotic resistance based on the sensitivity profiles presented. Such a complex approach will be essential for restraining the development of treatment resistance in MG.

A real-time PCR assay based on probe fluorescence quenching by a primer was developed and validated for detection of macrolide resistance (MR)-associated mutations in the 23S rRNA gene of Mycoplasma genitalium. The assay involves identification of any nucleotide substitutions at positions 2058, 2059, and 2611 of 23S rRNA ( Escherichia coli numbering) by the probe-based melting curve analysis immediately after amplification and was capable of detecting target mutations in clinical specimens and spiked samples with 92% sensitivity and 100% specificity. We applied this new assay to assess the prevalence of MR-associated mutations in 949 nonduplicate urogenital samples positive for M. genitalium by routine diagnostic PCR, which were collected from symptomatic patients in five cities in the European part of Russia during the period 2009–2019. Forty-three (4.92%) samples revealed the presence of MR mutations, and no trend toward an increase in resistance prevalence was observed over the 10-year period of the study. The most commonly detected mutations were A2058G (26/43; 60.47%) and A2059G (13/43; 30.23%), while other mutations were rare: A2058T (3/43; 6.98%) and C2611T (1/43; 2.33%). The data obtained underline the need for regular epidemiological monitoring to ensure effective patient management, rational use of antibiotics, and prevention of further spread of resistance.

Objective. To determine spectrum and prevalence of genetic determinants of macrolide and fluoroquinolone resistance in Mycoplasma genitalium from patient in dermatovenerology clinics of two Russian megalopolises: Moscow and St. Petersburg in 2021–2024. Materials and Methods. A total of 530 M. genitalium DNA-positive specimens (urethral and cervical swabs) obtained from men and women from the STD clinic in St. Petersburg and Сlinical facilities of the Moscow Scientific and Practical Center for Dermatovenerology and Cosmetology of the Moscow Department of Health Care were analyzed. Screening in local laboratories was performed using commercially available kits for isolation and detection of M. genitalium DNA. Primary screening for resistance-associated mutations was carried out using the previously developed protocol of PCR-RT (allows detecting any nucleotide substitutions in the 23S rRNA gene and parC gene) [1]. The results were confirmed using Sanger sequencing [2]. The study results are presented as an open project on the AMRcloud platform (https://amrcloud.net/ru/project/demares/). Results. 185 and 345 M. genitalium-positive samples from St. Petersburg and Moscow, respectively, showed the presence of a specific signal in both the 23S rRNA gene and parC gene in primary screening. The macrolide-resistance mutations was in 29% (54⁄185) of samples from St. Petersburg and in 28% (97⁄345) from Moscow, with a predominance of the A2059G 23S rRNA genotype of M. genitalium in 57% (31⁄54) and 68% (66⁄97), respectively. The second largest proportion were samples in which the nucleotide substitution A→G at position 2058 was detected: 23⁄54 (42%) from St. Petersburg and 30⁄97 (30%) from Moscow. Mutations to fluoroquinolones were detected in the parC gene in 47⁄185 (25%) and 73⁄345 (21%) samples from St. Petersburg and Moscow, respectively. In Moscow, the amino acid substitution S80I prevails 39% (29⁄73), the second most abundant D84N occured in 35% (26⁄73), in St. Petersburg, the similar ratio of S80I was 44% (21⁄47), D84N was 29% (14⁄47). In Moscow, seven samples carried the third most common mutation S80N, which is 9%, in St. Petersburg this figure, was twice as high – 19% (9⁄47). The share of other mutations in the two cities was not more than 1% (D84G, D84Y and S81P). In St. Petersburg, simultaneously with mutations in 23S rRNA, the presence of amino acid substitutions in the ParC QRDR was also confirmed, which represented a case of combined resistance to both groups antibiotics; this level was 1.5 times higher than in Moscow (40% (39⁄97)) and amounted to 61% (33⁄54). The most common combination of mutations was A2059G (23S rRNA) + S80I (parC), which made up to 33% (18⁄54) in St. Petersburg and 25.7% (25⁄97) in Moscow. The second most abundant genotype – A2058G (23S rRNA) + D84N (parC) accounted for 14% (8⁄54) and 10% (10⁄97), respectively. Conclusions. The high level of resistance to macrolides and fluoroquinolones in M. genitalium in two major Russian cities is a significant problem and need for comprehensive resistance testing before prescribing therapy and surveillance programs. The introduction of adapted diagnostic and therapeutic algorithms will reduce the duration of drug administration, increase the effectiveness of therapy and reduce the burden on the health care system. The use of long-term monitoring will improve the policy of antibiotic prescription and reduce the growth of antimicrobial resistance and its spread in Russia.