Long-Read Whole Genome Sequencing Elucidates the Mechanisms of Amikacin Resistance in Multidrug-Resistant Klebsiella pneumoniae Isolates Obtained from COVID-19 Patients.

Klebsiella pneumoniae is a Gram-negative, encapsulated, non-motile bacterium, which represents a global challenge to public health as one of the major causes of healthcare-associated infections worldwide. In the recent decade, the World Health Organization (WHO) noticed a critically increasing rate of carbapenem-resistant K. pneumoniae occurrence in hospitals. The situation with extended-spectrum beta-lactamase (ESBL) producing bacteria further worsened during the COVID-19 pandemic, due to an increasing number of patients in intensive care units (ICU) and extensive, while often inappropriate, use of antibiotics including carbapenems. In order to elucidate the ways and mechanisms of antibiotic resistance spreading within the K. pneumoniae population, whole genome sequencing (WGS) seems to be a promising approach, and long-read sequencing is especially useful for the investigation of mobile genetic elements carrying antibiotic resistance genes, such as plasmids. We have performed short- and long read sequencing of three carbapenem-resistant K. pneumoniae isolates obtained from COVID-19 patients in a dedicated ICU of a multipurpose medical center, which belonged to the same clone according to cgMLST analysis, in order to understand the differences in their resistance profiles. We have revealed the presence of a small plasmid carrying aph(3')-VIa gene providing resistance to amikacin in one of these isolates, which corresponded perfectly to its phenotypic resistance profile. We believe that the results obtained will facilitate further elucidating of antibiotic resistance mechanisms for this important pathogen, and highlight the need for continuous genomic epidemiology surveillance of clinical K. pneumoniae isolates.

Genomic Characterization of Clinical Acinetobacter baumannii Isolates Obtained from COVID-19 Patients in Russia.

The coronavirus disease 2019 (COVID-19) pandemic has already affected all realms of public healthcare and, in particular, has led to increasing use of various antibiotics to treat possible bacterial coinfections even in cases for which such infections were not confirmed clinically. This could lead to an increase in the fraction and severity of multidrug-resistant bacterial isolates in healthcare facilities, especially in intensive care units (ICU). However, detailed epidemiological investigations, possibly including whole genome sequencing (WGS), are required to confirm the increase in antibiotic resistance and changes, if any, in the population and clonal structures of bacterial pathogens. In this study, we performed a comprehensive genomic and phenotypic characterization of selected multidrug-resistant A. baumannii isolates obtained from the patients of a dedicated COVID-19 ICU in Moscow, Russia. Hybrid short- and long-read sequencing allowed us to obtain complete profiles of genomic antimicrobial resistance and virulence determinants, as well as to reveal the plasmid structure. We demonstrated the genomic similarity in terms of cgMLST profiles of the isolates studied with a clone previously identified in the same facility. We believe that the data provided will contribute to better understanding the changes imposed by the COVID-19 pandemic on the population structure and the antimicrobial resistance of bacterial pathogens in healthcare facilities.

COVID-19-Associated Pulmonary Aspergillosis in Russia.

We studied the risk factors, etiology, clinical features and the effectiveness of therapy of COVID-19-associated pulmonary aspergillosis (CAPA) in adult patients. In this retrospective study, we included 45 patients with proven (7%) and probable (93%) CAPA. The ECMM/ISHAM, 2020 criteria were used to diagnose CAPA. A case-control study was conducted to study the risk factors of CAPA; the control group included 90 adult COVID-19 patients without IA. In CAPA patients, the main underlying diseases were diabetes mellitus (33%), and hematological and oncological diseases (31%). The probability of CAPA developing significantly increased with lymphocytopenia >10 days (OR = 8.156 (3.056-21.771), p = 0.001), decompensated diabetes mellitus (29% vs. 7%, (OR = 5.688 (1.991-16.246), p = 0.001)), use of glucocorticosteroids (GCS) in prednisolone-equivalent dose > 60 mg/day (OR = 4.493 (1.896-10.647), p = 0.001) and monoclonal antibodies to IL-1ß and IL-6 (OR = 2.880 (1.272-6.518), p = 0.01). The main area of localization of CAPA was the lungs (100%). The clinical features of CAPA were fever (98% vs. 85%, p = 0.007), cough (89% vs. 72%, p = 0.002) and hemoptysis (36% vs. 3%, p = 0.0001). Overall, 71% of patients were in intensive care units (ICU) (median-15.5 (5-60) days), mechanical ventilation was used in 52% of cases, and acute respiratory distress syndrome (ARDS) occurred at a rate of 31%. The lung CT scan features of CAPA were bilateral (93%) lung tissue consolidation (89% vs. 59%, p = 0.004) and destruction (47% vs. 1%, p = 0.00001), and hydrothorax (26% vs. 11%, p = 0.03). The main pathogens were A. fumigatus (44%) and A. niger (31%). The overall survival rate after 12 weeks was 47.2%.

CRISPR Element Patterns vs. Pathoadaptability of Clinical Pseudomonas aeruginosa Isolates from a Medical Center in Moscow, Russia.

Pseudomonas aeruginosa is a member of the ESKAPE opportunistic pathogen group, which includes six species of the most dangerous microbes. This pathogen is characterized by the rapid acquisition of antimicrobial resistance, thus causing major healthcare concerns. This study presents a comprehensive analysis of clinical P. aeruginosa isolates based on whole-genome sequencing data. The isolate collection studied was characterized by a variety of clonal lineages with a domination of high-risk epidemic clones and different CRISPR/Cas element patterns. This is the first report on the coexistence of two and even three different types of CRISPR/Cas systems simultaneously in Russian clinical strains of P. aeruginosa. The data include molecular typing and genotypic antibiotic resistance determination, as well as the phylogenetic analysis of the full-length cas gene and anti-CRISPR genes sequences, predicted prophage sequences, and conducted a detailed CRISPR array analysis. The differences between the isolates carrying different types and quantities of CRISPR/Cas systems were investigated. The pattern of virulence factors in P. aeruginosa isolates lacking putative CRISPR/Cas systems significantly differed from that of samples with single or multiple putative CRISPR/Cas systems. We found significant correlations between the numbers of prophage sequences, antibiotic resistance genes, and virulence genes in P. aeruginosa isolates with different patterns of CRISPR/Cas-elements. We believe that the data presented will contribute to further investigations in the field of bacterial pathoadaptability, including antimicrobial resistance and the role of CRISPR/Cas systems in the plasticity of the P. aeruginosa genome.

Diversity of International High-Risk Clones of Acinetobacter baumannii Revealed in a Russian Multidisciplinary Medical Center during 2017-2019.

Acinetobacter baumannii is a dangerous bacterial pathogen possessing the ability to persist on various surfaces, especially in clinical settings, and to rapidly acquire the resistance to a broad spectrum of antibiotics. Thus, the epidemiological surveillance of A. baumannii within a particular hospital, region, and across the world is an important healthcare task that currently usually includes performing whole-genome sequencing (WGS) of representative isolates. During the past years, the dissemination of A. baumannii across the world was mainly driven by the strains belonging to two major groups called the global clones or international clones (ICs) of high risk (IC1 and IC2). However, currently nine ICs are already considered. Although some clones were previously thought to spread in particular regions of the world, in recent years this is usually not the case. In this study, we determined five ICs, as well as three isolates not belonging to the major ICs, in one multidisciplinary medical center within the period 2017-2019. We performed WGS using both short- and long-read sequencing technologies of nine representative clinical A. baumannii isolates, which allowed us to determine the antibiotic resistance and virulence genomic determinants, reveal the CRISPR/Cas systems, and obtain the plasmid structures. The phenotypic and genotypic antibiotic resistance profiles are compared, and the possible ways of isolate and resistance spreading are discussed. We believe that the data obtained will provide a better understanding of the spreading and resistance acquisition of the ICs of A. baumannii and further stress the necessity for continuous genomic epidemiology surveillance of this problem-causing bacterial species.

Genomic and Phenotypic Analysis of Multidrug-Resistant Acinetobacter baumannii Clinical Isolates Carrying Different Types of CRISPR/Cas Systems.

Acinetobacter baumannii is an opportunistic pathogen being one of the most important causative agents of a wide range of nosocomial infections associated with multidrug resistance and high mortality rate. This study presents a multiparametric and correlation analyses of clinical multidrug-resistant A. baumannii isolates using short- and long-read whole-genome sequencing, which allowed us to reveal specific characteristics of the isolates with different CRISPR/Cas systems. We also compared antibiotic resistance and virulence gene acquisition for the groups of the isolates having functional CRISPR/Cas systems, just CRISPR arrays without cas genes, and without detectable CRISPR spacers. The data include three schemes of molecular typing, phenotypic and genotypic antibiotic resistance determination, as well as phylogenetic analysis of full-length cas gene sequences, predicted prophage sequences and CRISPR array type determination. For the first time the differences between the isolates carrying Type I-F1 and Type I-F2 CRISPR/Cas systems were investigated. A. baumannii isolates with Type I-F1 system were shown to have smaller number of reliably detected CRISPR arrays, and thus they could more easily adapt to environmental conditions through acquisition of antibiotic resistance genes, while Type I-F2 A. baumannii might have stronger "immunity" and use CRISPR/Cas system to block the dissemination of these genes. In addition, virulence factors abaI, abaR, bap and bauA were overrepresented in A. baumannii isolates lacking CRISPR/Cas system. This indicates the role of CRISPR/Cas in fighting against phage infections and preventing horizontal gene transfer. We believe that the data presented will contribute to further investigations in the field of antimicrobial resistance and CRISPR/Cas studies.

Multidrug-Resistant Proteus mirabilis Strain with Cointegrate Plasmid.

Proteus mirabilis is a component of the normal intestinal microflora of humans and animals, but can cause urinary tract infections and even sepsis in hospital settings. In recent years, the number of multidrug-resistant P. mirabilis isolates, including the ones producing extended-spectrum ß-lactamases (ESBLs), is increasing worldwide. However, the number of investigations dedicated to this species, especially, whole-genome sequencing, is much lower in comparison to the members of the ESKAPE pathogens group. This study presents a detailed analysis of clinical multidrug-resistant ESBL-producing P. mirabilis isolate using short- and long-read whole-genome sequencing, which allowed us to reveal possible horizontal gene transfer between Klebsiella pneumoniae and P. mirabilis plasmids and to locate the CRISPR-Cas system in the genome together with its probable phage targets, as well as multiple virulence genes. We believe that the data presented will contribute to the understanding of antibiotic resistance acquisition and virulence mechanisms for this important pathogen.

Molecular Typing, Characterization of Antimicrobial Resistance, Virulence Profiling and Analysis of Whole-Genome Sequence of Clinical Klebsiella pneumoniae Isolates.

Klebsiella pneumoniae is one of the most important pathogens concerned with multidrug resistance in healthcare-associated infections. The treating of infections caused by this bacterium is complicated due to the emergence and rapid spreading of carbapenem-resistant strains, which are associated with high mortality rates. Recently, several hypervirulent and carbapenemase-producing isolates were reported that make the situation even more complicated. In order to better understand the resistance and virulence mechanisms, and, in turn, to develop effective treatment strategies for the infections caused by multidrug-resistant K. pneumoniae, more comprehensive genomic and phenotypic data are required. Here, we present the first detailed molecular epidemiology report based on second and third generation (long-read) sequencing for the clinical isolates of K. pneumoniae in the Russian Federation. The data include three schemes of molecular typing, phenotypic and genotypic antibiotic resistance determination, as well as the virulence and plasmid profiling for 36 K. pneumoniae isolates. We have revealed 2 new multilocus sequence typing (MLST)-based sequence types, 32 multidrug-resistant (MDR) isolates and 5 colistin-resistant isolates in our samples. Three MDR isolates belonged to a very rare ST377 type. The whole genome sequences and additional data obtained will greatly facilitate further investigations in the field of antimicrobial resistance studies.

Current Evidence about Nutrition Support in Cardiac Surgery Patients-What Do We Know?

Nutrition support is increasingly recognized as a clinically relevant aspect of the intensive care treatment of cardiac surgery patients. However, evidence from adequate large-scale studies evaluating its clinical significance for patients’ mid- to long-term outcome remains sparse. Considering nutrition support as a key component in the perioperative treatment of these critically ill patients led us to review and discuss our understanding of the metabolic response to the inflammatory burst induced by cardiac surgery. In addition, we discuss how to identify patients who may benefit from nutrition therapy, when to start nutritional interventions, present evidence about the use of enteral and parenteral nutrition and the potential role of pharmaconutrition in cardiac surgery patients. Although the clinical setting of cardiac surgery provides advantages due to its scheduled insult and predictable inflammatory response, researchers and clinicians face lack of evidence and several limitations in the clinical routine, which are critically considered and discussed in this paper.