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.

Background/Objectives: Carbapenem-resistant Enterobacterales (CRE) are a global health threat due to their high morbidity and mortality rates and limited treatment options. This study examines the plasmid-mediated transmission of virulence and antibiotic resistance determinants in carbapenem-resistant Klebsiella pneumoniae (Kpn) and Escherichia coli (E. coli) isolated from Russian hospitals. Methods: We performed short- and long-read whole-genome sequencing of 53 clinical isolates (48 Kpn and 5 E. coli) attributed to 15 genetic lineages and collected from 21 hospitals across nine Russian cities between 2016 and 2022. Results: The plasmid analysis identified 18 clusters that showed high concordance with replicon typing, with all clusters having a major replicon type. The majority of plasmids in the IncHI1B(pNDM-MAR)/IncFIB(pNDM-Mar)-like cluster (79.16%) carried both antibiotic resistance genes (e.g., bla_NDM-1 and bla_OXA-48) and virulence factors (VFs) such as siderophore genes. We hypothesized that hybrid plasmids could play a critical role in the dissemination of antibiotic resistance genes and VFs. Comparative analyses with global plasmid databases revealed high-risk lineages of hybrid plasmids that are predominantly spread throughout Russia at present. Conclusions: Our findings underscore the importance of monitoring plasmid backbones for clinical management, surveillance, and infection control activities.

Background: The dissemination of mobile colistin resistance (mcr) genes is a serious healthcare threat because polymyxins represent “last-line” therapeutics for multi-drug-resistant Gram-negative pathogens. This study aimed to assess the prevalence of colistin resistance and mcr genes and characteristics of clinical Escherichia coli (Eco) and Klebsiella pneumoniae (Kpn) isolates and plasmids carrying these genes in Russia. Methods: A total of 4324 Eco and 4530 Kpn collected in the frame of sentinel surveillance in 2013–2018 were tested for susceptibility to colistin and other antibiotics using the broth microdilution method. mcr genes were screened by real-time PCR. Phylogeny, genomic features and plasmids of mcr-positive isolates were assessed using whole-genome sequencing and subsequent bioinformatic analysis. Results: Colistin resistance was detected in 2.24% Eco and 9.3% Kpn. Twenty-two (0.51%) Eco and two (0.04%) Kpn from distant sites carried mcr-1.1. Most mcr-positive isolates co-harbored ESBLs and other resistance determinants to various antibiotic classes. The mcr-positive Eco belonged to 16 MLST types, with ST359 being most common; Kpn belonged to ST307 and ST23. mcr-1.1 was carried mainly in IncI2 (n = 18) and IncX4 (n = 5) plasmids highly similar to those identified previously in human, animal and environmental isolates. Conclusion: This study demonstrated a dissemination of “typical” mcr-bearing plasmids among diverse Eco and Kpn genotypes and across a wide geographic area in Russia. Given the frequent association of mcr with other resistance determinants and potential clinical impact, the continual surveillance of this threat is warranted.

Background/Objectives: Providencia stuartii is intrinsically resistant to several antibiotic classes, and acquisition of bla_NDM further restricts treatment options. This study aimed to characterize NDM-producing P. stuartii isolates from a small hospital cluster in Russia and to place them within the global genomic context. Methods: Four isolates recovered between June and July 2023 from a single hospital were analyzed using Illumina and Oxford Nanopore sequencing to assess genetic relatedness and plasmid content. Results: The isolates showed identical extensively drug-resistant profiles and were closely related genomically. All carried nearly identical IncC plasmids harboring multiple antimicrobial resistance genes, including bla_NDM. Comparative analysis indicated that these genomes clustered with recent European isolates but differed in the bla_NDM allele and its genomic location. Highly similar IncC plasmids were also found in our previous Klebsiella pneumoniae dataset, demonstrating that this plasmid backbone occurs in multiple bacterial species in the region. Conclusions: The study highlights the role of IncC plasmids in carbapenemase dissemination and underscores the value of genomic surveillance integrating chromosomal and plasmid analyses to track extensively drug-resistant pathogens.

Background: Stenotrophomonas spp. are increasingly recognized opportunistic pathogens characterized by intrinsic multidrug resistance. Despite recent taxonomic reclassification and the delineation of new species within the S. maltophilia complex (Smc), routine diagnostics often misidentify these species, and their distinctive antibiotic resistance traits and genetic makeup remain underexplored. We aimed to characterize the phylogenomic diversity, genotypic and phenotypic antibiotic resistance traits, virulence markers and horizontally transferrable elements in a large national cohort of clinical Stenotrophomonas isolates. Methods: We analyzed 323 clinical isolates from the Russian sentinel surveillance program (2002–2021). Analysis included MALDI-TOF MS identification, broth microdilution susceptibility testing to six agents [aztreonam-avibactam, colistin, levofloxacin, minocycline, tigecycline, and trimethoprim-sulfamethoxazole (TMP-SMX)], and long-read Nanopore WGS with hybrid polishing. We performed average nucleotide identity (ANI)-based classification, core- and accessory-genome comparisons, and comprehensive profiling of antibiotic resistance genes (ARGs), virulence factors (VFs), plasmids, and prophages. Results: While MALDI-TOF MS identified all isolates as S. maltophilia , ANI (≥95% threshold) and core-genome phylogeny revealed 17 distinct species: 52% S. maltophilia sensu stricto, 36.8% across eight other ICNP-named Smc species, and 11.2% within eight “novel” genomic lineages identified elsewhere. Pan-genome comprised 24,815 genes (9.6% core genes), with accessory genes showing robust species-level clustering in UMAP. TMP-SMX resistance was prevalent (40.2%) among all species, though only 13.1% of resistant isolates harbored sul genes. Intrinsic β-lactamase genes ( bla _L1 - and bla _L2 -like) were ubiquitous but exhibited high sequence variability (up to 20.6% and 25.1%) and evidence of inter-species exchange. Phosphoethanolamine transferase genes ( mcr -5- and mcr -8-like) were identified as intrinsic chromosomal components. Acquired ARGs were primarily associated with chromosomal integrative conjugative and mobilizable elements (ICEs/IMEs). Plasmids (2.5 to >334 kb) were identified in 8% of isolates across 10 species, including a cluster of eight nearly identical replicons distributed among five species despite lacking canonical mobilization genes. Prophages were ubiquitous (median: 4 per genome), dominated by Caudoviricetes and Inoviridae . Several VFs linked to motility, capsule formation, and invasion/toxicity were universally present across the genus. Conclusion: Our findings demonstrate that current routine diagnostics fail to resolve the significant taxonomic diversity within clinical Stenotrophomonas isolates, leading to widespread species misidentification. Integrating the genomic and phenotypic insights from this study into routine surveillance is essential to overcome these diagnostic limitations and to modernize the clinical management of infections caused by this formidable genus.

Background. Currently, it is customary to distinguish two pathotypes of Klebsiella pneumoniae — classical and hypervirulent, which have the ability to cause community-acquired infections in healthy people. It has been shown that an increase in virulence is associated with the acquisition of additional genetic material — a plasmid carrying a cluster of aerobactin genes. Aim. To assess the prevalence of the aforementioned virulent plasmids around the globe and in Russia in particular, as well as to identify their key genetic features. Materials and methods. Plasmid sequences were downloaded from PLSDB and BV-BRC databases, annotated with the Abricate and Kleborate programs; cluster analysis was performed using the mge-cluster program, and phylogenetic analysis was performed using the Parsnp program. Results. 296 plasmid sequences isolated from 23 countries from clinical isolates of K.pneumoniae between 2006 and 2021 with a peak in 2019 were analyzed, with more than half of the plasmids coming from China. More than 30 sequence types were identified, among which ST11 and ST23 were predominant. Replicon genes of the IncFIB group were identified in almost all plasmids studied. The pre dominant type of aerobactin in the studied sequences was the first type (iuc1); sequences with iuc3 and iuc5 were also identified. Salmochelin synthesis genes were identified in only 37.1% of sequences; the yersiniabactin cluster was identified in two plasmids from China. 32.1% of plasmids carried resistance genes, of which 7.4% carried extended-spectrum beta-lactase genes and 5% contained carbapenemase genes. Nine clusters of sequences were obtained; almost all plasmids from Russia were assigned to one cluster and were NDM-positive. Together with plasmids from other European countries (Great Britain, Norway, Czech Republic), they formed a separate branch on the phylogenetic tree. Conclusion. Virulent plasmids carrying the aerobactin synthesis gene cluster are distributed globaly, and almost a third of them also carry antibiotic resistance genes.