Outcomes of the multicenter monitoring of the causative agent of invasive listeriosis in the metropolis

Introduction. Invasive listeriosis is a rare disease posing a threat to high-risk groups and often leading to a fatal outcome. Its causative agent is Listeria monocytogenes, a ubiquitous saprophyte that has turned into an important foodborne pathogen with the growing industry of semi-cooked and ready-to-eat products. The aim of the study is the characterization of L. monocytogenes isolates in the Moscow region and identification of possible causes of susceptibility to infection Materials and methods. The multicenter monitoring of L. monocytogenes was conducted in the Moscow metropolitan area, using bacteriological and genomic methods for description of the pathogen, medical history collection and detailed analysis of patient case summaries. Results. In the cohorts of patients with perinatal listeriosis (PL) and meningitis-septicemia (MS), invasive listeriosis had a year-round occurrence with slight upswings in MarchApril and JulyNovember. During the COVID-19 pandemic, in the MS group, the minimum age of patients decreased to 31 years and the proportion of deaths increased 1.57-fold compared to 20182019. During the pandemic, an increase in the diversity of L. monocytogenes genotypes was observed, along with changes in the spectrum of pathogen genotypes throughout the pandemic stages. During the monitoring, a total of 73 L. monocytogenes clinical isolates belonging to 24 genotypes were described. Seven genotypes belonged to the first phylogenetic lineage (PLI); 14 genotypes belonged to PLII. The PL cohort had the highest proportion of PLI genotypes (52%). In the MS cohort, the group of men had the widest diversity of genotypes, 6 of which were identical to genotypes of food isolates. In the analysed set of isolates, 12 new profiles of internalin genes were identified and described. The whole genome sequencing detected the presence of plasmids in 9 of 58 genomes of clinical isolates. The comparison of core genomes revealed an epidemic relationship between isolates of the same genotype for ST4, ST21, and ST425. Conclusion. The performed study presents a detailed description of the diversity and virulence of L. monocytogenes circulating in the Moscow metropolitan area, thus providing information for timely diagnosis and treatment of invasive listeriosis

Genomic features of resistant <i>Klebsiella pneumonia</i>, isolated from the bloodstream and cerebrospinal fluid of pediatric hospital patients

Introduction. Carbapenemase-producing Klebsiella pneumoniae (CP-Kp), which are international high-risk clones, have become a problem of utmost importance. CP-Kps, adapting to the hospital environment, evolve into convergent pathotypes. Such variants combine traits of two genetic lineages: multidrug resistant (MDR) and hypervirulent. The pathotypes, along with MDR K. pneumoniae, pose an exceptional threat to young patients during systemic infection. The objective of this study is the detailed molecular genetic analysis of MDR isolates of K. pneumoniae detected during the monitoring of resistant Gram-negative bacteria at the National Medical Research Center for Children’s Health in 2014–2021. Materials and methods. Whole-genome sequencing with a subsequent bioinformatics analysis of eight MDR isolates from the bloodstream and cerebrospinal fluid. Results. MDR isolates belonged to 4 sublineages (SL): SL307, SL395, SL29 and SL1198. In the genomes of 6 pangrug-resistant (PDR) isolates, genes associated with resistance to all categories of antibiotics recommended for Enterobacteriaceae therapy were identified. Plasmids were present in all genomes. In 6 isolates, plasmids contained heavy metal ion resistance operons in addition to antibiotic resistance genes. Prophages within the plasmids were also involved in the transfer of resistance genes. The ST395 isolate from the cerebrospinal fluid belonged to the convergent pathotype in terms of resistance and virulence. Comparison of genomes within SLs revealed recombination events in the K- and O-locus regions and the Yersiniabactin operon. Conclusion. Thus, in a sample of resistant K. pneumoniae isolated from bloodstream and cerebrospinal fluid, 6 PDR isolates were detected, one of which belongs to the convergent pathotype ST395

Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes

Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A “lacking biofilm production” (LBP) strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR) gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg) has a key role in biofilm formation. The relative location (i.e., by being separated by another gene) of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents

On Burkholderiales order microorganisms and cystic fibrosis in Russia

Abstract Background Microbes infecting cystic fibrosis patients’ respiratory tract are important in determining patients’ functional status. Representatives of Burkholderiales order are the most dangerous. The goal of our investigation was to reveal the diversity of Burkholderiales, define of their proportion in the microbiome of various parts of respiratory tract and determine the pathogenicity of the main representatives. Results In more than 500 cystic fibrosis patients, representing all Federal Regions of Russia, 34.0% were infected by Burkholderia cepacia complex (Bcc), 21.0% by Achromobacter spp. and 12.0% by Lautropia mirabilis. B. cenocepacia was the most numerous species among the Bcc (93.0%), and A. ruhlandii was the most numerous among Achromobacter spp. (58.0%). The most abundant genotype in Bcc was sequence type (ST) 709, and in Achromobacter spp. it was ST36. These STs constitute Russian epidemic strains. Whole genome sequencing of strains A. ruhlandii SCCH3:Ach33–1365 ST36 and B. cenocepacia GIMC4560:Bcn122 ST709 revealed huge resistomes and many virulence factors, which may explain the difficulties in eradicating these strains. An experience of less dangerous B. cenocepcia ST710 elimination was described. Massively parallel sequencing of 16S rDNA amplicons, including V1-V4 hypervariable regions, was used to definite “healthy” microbiome characteristics. Analysis of maxillary sinus lavage of 7 patients revealed infection with Proteobacteria of the same ST as pathogens from sputum, suggesting that the maxillary sinus is a source of infection in cystic fibrosis patients. Conclusions Characterization of the Russian epidemic bacterial strains in the sputum and sinuses of cystic fibrosis patients have better defined the importance of Burkholderiales bacteria. This information may aid in the development of effective approaches for treatment of this disease

<i>Listeria monocytogenes</i> ST37 Distribution in the Moscow Region and Properties of Clinical and Foodborne Isolates

Listerias of the phylogenetic lineage II (PLII) are common in the European environment and are hypovirulent. Despite this, they caused more than a third of the sporadic cases of listeriosis and multi-country foodborne outbreaks. L. monocytogenes ST37 is one of them. During the COVID-19 pandemic, ST37 appeared in clinical cases and ranked second in occurrence among food isolates in the Moscow region. The aim of this study was to describe the genomic features of ST37 isolates from different sources. All clinical cases of ST37 were in the cohort of male patients (age, 48–81 years) with meningitis–septicemia manifestation and COVID-19 or Influenza in the anamnesis. The core genomes of the fish isolates were closely related. The clinical and meat isolates revealed a large diversity. Prophages (2–4/genome) were the source of the unique genes. Two clinical isolates displayed pseudolysogeny, and excided prophages were A006-like. In the absence of plasmids, the assortment of virulence factors and resistance determinants in the chromosome corresponded to the hypovirulent characteristics. However, all clinical isolates caused severe disease, with deaths in four cases. Thus, these studies allow us to speculate that a previous viral infection increases human susceptibility to listeriosis

The Characteristics of Ubiquitous and Unique Leptospira Strains from the Collection of Russian Centre for Leptospirosis

Background and Aim. Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity of Leptospira in natural and anthropurgic foci was not clearly defined. Methods. The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS). Results. The most wide spread and prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmental Leptospira strain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species. Conclusions. Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions

The Variability of the Order Burkholderiales Representatives in the Healthcare Units

Background and Aim. The order Burkholderiales became more abundant in the healthcare units since the late 1970s; it is especially dangerous for intensive care unit patients and patients with chronic lung diseases. The goal of this investigation was to reveal the real variability of the order Burkholderiales representatives and to estimate their phylogenetic relationships. Methods. 16S rDNA and genes of the Burkholderia cenocepacia complex (Bcc) Multi Locus Sequence Typing (MLST) scheme were used for the bacteria detection. Results. A huge diversity of genome size and organization was revealed in the order Burkholderiales that may prove the adaptability of this taxon’s representatives. The following variability of the Burkholderiales in Russian healthcare units has been revealed: Burkholderiaceae (Burkholderia, Pandoraea, and Lautropia), Alcaligenaceae (Achromobacter), and Comamonadaceae (Variovorax). The Burkholderia genus was the most diverse and was represented by 5 species and 16 sequence types (ST). ST709 and 728 were transmissible and often encountered in cystic fibrosis patients and in hospitals. A. xylosoxidans was estimated by 15 genotypes. The strains of first and second ones were the most numerous. Conclusions. Phylogenetic position of the genus Lautropia with smaller genome is ambiguous. The Bcc MLST scheme is applicable for all Burkholderiales representatives for resolving the epidemiological problems

Porin from Marine Bacterium Marinomonas primoryensis KMM 3633T: Isolation, Physico-Chemical Properties, and Functional Activity

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 &deg;C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin