Acinetobacter Non-baumannii Species: Occurrence in Infections in Hospitalized Patients, Identification, and Antibiotic Resistance.

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 blaOXA-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 (blaOXA-23-like, blaOXA-24/40-like, blaOXA-58-like, blaNDM, blaIMP, and blaVIM) 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 (blaOXA-58-like and blaNDM, 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.

Genomic analysis of the international high-risk clonal lineage Klebsiella pneumoniae sequence type 395.

BACKGROUND: Klebsiella pneumoniae, which is frequently associated with hospital- and community-acquired infections, contains multidrug-resistant (MDR), hypervirulent (hv), non-MDR/non-hv as well as convergent representatives. It is known that mostly international high-risk clonal lineages including sequence types (ST) 11, 147, 258, and 307 drive their global spread. ST395, which was first reported in the context of a carbapenemase-associated outbreak in France in 2010, is a less well-characterized, yet emerging clonal lineage. METHODS: We computationally analyzed a large collection of K. pneumoniae ST395 genomes (n = 297) both sequenced in this study and reported previously. By applying multiple bioinformatics tools, we investigated the core-genome phylogeny and evolution of ST395 as well as distribution of accessory genome elements associated with antibiotic resistance and virulence features. RESULTS: Clustering of the core-SNP alignment revealed four major clades with eight smaller subclades. The subclades likely evolved through large chromosomal recombination, which involved different K. pneumoniae donors and affected, inter alia, capsule and lipopolysaccharide antigen biosynthesis regions. Most genomes contained acquired resistance genes to extended-spectrum cephalosporins, carbapenems, and other antibiotic classes carried by multiple plasmid types, and many were positive for hypervirulence markers, including the siderophore aerobactin. The detection of "hybrid" resistance and virulence plasmids suggests the occurrence of the convergent ST395 pathotype. CONCLUSIONS: To the best of our knowledge, this is the first study that investigated a large international collection of K. pneumoniae ST395 genomes and elucidated phylogenetics and detailed genomic characteristics of this emerging high-risk clonal lineage.

Detection of carbapenemase-producing Enterobacterales by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with ertapenem susceptibility-testing disks as source of carbapenem substrate.

MALDI-TOF mass spectrometry has become widely used in clinical microbiology and has proved highly accurate for detection of carbapenemases in Gram-negative bacteria. However, the use of carbapenem-hydrolysis assays in routine diagnostics is hampered by the need for antibiotic substances and for making their fresh solutions each time an assay is conducted. Here, we evaluated the use of commercial antibiotic susceptibility-testing disks as source of ertapenem substrate in MALDI-TOF MS-based assay for detection of carbapenemase-producing Enterobacterales (CPE). The assay was validated on 48 CPE isolates of 8 different species expressing NDM-, VIM-, KPC- and OXA-48-type carbapenemases and exhibiting various levels of resistance to carbapenems (MIC range: 0.25- > 32 mg/l), as well as on 48 carbapenemase-non-producing isolates. The assay conditions were optimized as follows: 10-µl loopful of bacterial colonies was suspended in 150 µl 0.01 M Na-PBS buffer, pH 7.4, a 10 µg ertapenem susceptibility-testing disk was immersed in the suspension and incubated 3 h at 35°C, after which supernatant was obtained by centrifugation and applied on a target plate with alpha-cyano-4-hydroxycinnamic acid matrix. Mass spectra were analyzed between 440 and 560 m/z. Carbapenemase activity was detected in all tested CPE isolates by the appearance of m/z peaks corresponding to ertapenem hydrolysis products: [Mh + H]+:494.2, [Mh + Na]+:516.2, [Mh + 2Na]+:538.2, [Mh/d + H]+:450.2, [Mh/d + Na]+:472.2, and simultaneous decrease or loss of peaks of intact antibiotic: [M + H]+:476.2, [M + Na]+:498.1, [M + 2Na]+:520.1. No hydrolysis peaks or loss of intact ertapenem peaks were observed for carbapenemase-negative strains. We therefore report the development of a sensitive, specific and cost-effective MALDI-TOF MS-based assay for detection of CPE, which makes use of antibiotic disks readily available in most laboratories.

Molecular Epidemiology of mcr-1-Positive Escherichia coli and Klebsiella pneumoniae Isolates: Results from Russian Sentinel Surveillance (2013-2018).

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.

Antimicrobial Resistance and Genomic Characterization of OXA-48- and CTX-M-15-Co-Producing Hypervirulent Klebsiella pneumoniae ST23 Recovered from Nosocomial Outbreak.

Multidrug resistance (MDR) and hypervirulence (hv) have been long considered distinct evolutionary traits for Klebsiella pneumoniae (Kp), a versatile human pathogen. The recent emergence of Kp strains combining these traits poses a serious global threat. In this article, we describe the phenotypic and genomic characteristics of an MDR hvKp isolate, MAR14-456, representative of a nosocomial outbreak in Moscow, Russia, that was recovered from a postoperative wound in a patient who later developed multiple abscesses, fatal sepsis, and septic shock. Broth microdilution testing revealed decreased susceptibility of MAR14-456 to carbapenems (MICs 0.5-2 mg/L) and a high-level resistance to most ß-lactams, ß-lactam-ß-lactamase-inhibitor combinations, and non-ß-lactam antibiotics, except ceftazidime-avibactam, amikacin, tigecycline, and colistin. Whole-genome sequencing using Illumina MiSeq and ONT MinION systems allowed to identify and completely assemble two conjugative resistance plasmids, a typical 'European' epidemic IncL/M plasmid that carries the gene of OXA-48 carbapenemase, and an IncFIIK plasmid that carries the gene of CTX-M-15 ESBL and other resistance genes. MLST profile, capsular, lipopolysaccharide, virulence genes encoded on chromosome and IncHI1B/FIB plasmid, and the presence of apparently functional type I-E* CRISPR-Cas system were all characteristic of hvKp ST23, serotype K1-O1v2. Phylogenetic analysis showed the closest relatedness of MAR14-456 to ST23 isolates from China. This report highlights the threat of multiple resistance acquisition by hvKp strain and its spread as a nosocomial pathogen.