Key features of pneumococcal isolates recovered in Central and Northwestern Russia in 2011-2018 determined through whole-genome sequencing.

Invasive pneumococcal disease remains one of the leading causes of morbidity and mortality worldwide. In Russia, 13- valent pneumococcal conjugate vaccine (PCV13) was introduced into the childhood immunization programme nationwide in 2014. As part of the Global Pneumococcal Sequencing Project (GPS), we used genome data to characterize 179 pneumococcal isolates collected from Russia in 2011-2018 to investigate the circulating pneumococcal strains using a standardized genomic definition of pneumococcal lineages (global pneumococcal sequence clusters, GPSCs), prevalent serotypes and antimicrobial resistance profiles.We observed high serotype and lineage diversity among the 179 isolates recovered from cerebrospinal fluid (n=77), nasopharyngeal swabs (n=99) and other non-sterile site swabs (n=3). Overall, 60 GPSCs were identified, including 48 clonal complexes (CCs) and 14 singletons, and expressed 42 serotypes (including non-typable). Among PCV13 serotypes, 19F, 6B and 23F were the top three serotypes while 11A, 15B/C and 8 were the top three among non-PCV13 serotypes in the collection. Two lineages (GPSC6 and GPSC47) expressed both PCV13 and non-PCV13 serotypes that caused invasive disease, and were penicillin- and multidrug-resistant (MDR), highlighting their potential to adapt and continue to cause infections under vaccine and antibiotic selective pressure. PCV13 serotypes comprised 92 % (11/12) of the CSF isolates from the children aged below 5 years; however, the prevalence of PCV13 serotype isolates dropped to 53 % (31/58) among the nasopharyngeal isolates. Our analysis showed that 59 % (105/179) of the isolates were predicted to be non-susceptible to at least one class of antibiotics and 26 % (46/179) were MDR. Four MDR lineages (GPSC1, GPSC6, GPSC10 and GPSC47) accounted for 65 % (30/46) of the MDR isolates and expressed PCV13 serotypes (93 %, 28/30).This study provides evidence of high genetic and serotype diversity contributed by a mix of globally spreading and regionally circulating lineages in Russia. The observations suggest that the PCV13 vaccine could be important in reducing both invasive disease and antimicrobial resistance. We also identify potential lineages (GPSC6 and GPSC47) that may evade the vaccine.

Characteristics of Stenotrophomonas maltophilia isolates from cystic fibrosis patients in Russia.

Stenotrophomonas maltophilia is a common opportunistic microorganism and an important respiratory pathogen in cystic fibrosis (CF). The aim of this study was to determine antimicrobial resistance phenotypes, sequence-types (ST) and genetic determinants of antibiotic resistance in S. maltophilia strains recovered from CF patients in Russia. S. maltophilia isolates recovered from 170 CF patients were analyzed. Minimum inhibitory concentrations of antibacterial agents were determined using Sensititre Gram Negative GNX2F plates and the results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) criteria. Whole-genome sequencing (WGS) was performed on MGISEQ-2000 platform. SPAdes software, Galaxy, ResFinder, Integrall and PubMLST were used for analysis of WGS data. S. maltophilia strains were identified from 24/170 (14%) CF patients. In total, 25 isolates were detected, two strains were isolated from the same patient. The isolates belonged to 17 different STs, including 5 new STs; ST4 was the most prevalent ST. Resistance to ceftazidime was observed in 60% of strains, to ticarcillin-clavulanate - in 32%, to levofloxacin - in 24%, to trimethoprim/sulfamethoxazole - in 12% of strains. All isolates were susceptible to minocycline. All ST4 isolates were resistant or intermediate to ceftazidime and ticarcillin-clavulanate. In two isolates, the sul1 gene was detected. In one isolate, sul1 was part of a class 1 integron. The detected integron also contained the blaGES-7 and aac(6')-Ib-cr genes. The ST4 sequence-type was the most prevalent ST among S. maltophilia strains recovered from CF patients in Russia. Antibiotic resistance genes, including sul1, blaGES-7, aac(6')-Ib-cr, were detected in single strains.

Identification of bacterial antibiotic resistance genes in next-generation sequencing data (review of literature).

The spread of antibiotic-resistant human bacterial pathogens is a serious threat to modern medicine. Antibiotic susceptibility testing is essential for treatment regimens optimization and preventing dissemination of antibiotic resistance. Therefore, development of antibiotic susceptibility testing methods is a priority challenge of laboratory medicine. The aim of this review is to analyze the capabilities of the bioinformatics tools for bacterial whole genome sequence data processing. The PubMed database, Russian scientific electronic library eLIBRARY, information networks of World health organization and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) were used during the analysis. In this review, the platforms for whole genome sequencing, which are suitable for detection of bacterial genetic resistance determinants, are described. The classic step of genetic resistance determinants searching is an alignment between the query nucleotide/protein sequence and the subject (database) nucleotide/protein sequence, which is performed using the nucleotide and protein sequence databases. The most commonly used databases are Resfinder, CARD, Bacterial Antimicrobial Resistance Reference Gene Database. The results of the resistance determinants searching in genome assemblies is more correct in comparison to results of the searching in contigs. The new resistance genes searching bioinformatics tools, such as neural networks and machine learning, are discussed in the review. After critical appraisal of the current antibiotic resistance databases we designed a protocol for predicting antibiotic resistance using whole genome sequence data. The designed protocol can be used as a basis of the algorithm for qualitative and quantitative antimicrobial susceptibility testing based on whole genome sequence data.

Genome features and antibiotic resistance of Pseudomonas aeruginosa strains isolated in patients with cystic fibrosis in the Russian Federation.

Cystic fibrosis (CF) is a common genetic disease, manifested by airway obstruction and chronic respiratory infection. The most prevalent infectious agent in airways of CF patients is Pseudomonas aeruginosa. This study aimed to determine sequence-types, antimicrobial resistance phenotypes and genes defining adaptive antibiotic resistance in P. aeruginosa isolates recovered from CF patients in Russia. In total, 84 P. aeruginosa strains from 64 CF patients were analyzed. Susceptibility to antibiotics was determined by disk diffusion test. Whole-genome sequencing (WGS) was performed on MGISEQ-2000 platform. SPAdes software, Galaxy, ResFinder, PubMLST were used for analysis of WGS data. Examined P. aeruginosa isolates belonged to 53 different sequence-types (STs), including 6 new STs. High-risk epidemic clone ST235 (10%) and clonal CF P. aeruginosa strains ST17, ST242, ST274 (7%) were detected. Non-susceptibility to ticarcillin-clavulanate, cefepime, imipenem was observed in 63%, 12% and 25% of isolates, respectively; to tobramycin - in 24%, to amikacin - in 35%; to ciprofloxacin, levofloxacin - in 35% and 57% of strains, respectively. Multidrug-resistant phenotype was detected in 18% of isolates. In examined strains, genes of beta-lactamases VIM-2 (5 ST235 strains), VEB-1 (two ST2592 strains), GES-1 (1 ST235 strain), PER-1 (1 ST235 strain) were found. Ciprofloxacin-modifying enzyme CrpP gene was detected in 67% of isolates, aminoglycoside-modifying enzymes AAD, ANT, AAC genes - in 7%, 4%, 12% of strains, respectively. P. aeruginosa isolates from CF patients in Russia demonstrate a high clonal diversity, which is similar to other P. aeruginosa infections. The isolates of high-risk clone and clonal CF P. aeruginosa strains are detected.

Noncontiguous finished genome sequence of Megasphaera sp. ASD88, isolated from faeces of a child with autism spectrum disorder.

We report here a draft genome sequence of Megasphaera sp. ASD88, a strain from the intestinal microbiota of a child with autism spectrum disorder, representing a previously undescribed species of the genus Megasphaera. The assembled sequence consists of 88 scaffolds, and the total size is 2.59 Mb.

[Comparative Genotyping of Staphylococcus aureus Strains Isolated from Skin Lesions, Nasal Cavities, and Feces of Children with Atopic Dermatitis].

Background: The lesion of skin of the majority atopic dermatitis patients is chronically colonized by bacteria belonging to the species Staphylococcus aureus. Topical antibacterial and anti-inflammatory therapy treatment are often ineffective due to fast recolonization by S. aureus and exacerbation of allergic process. Aims: Our aim was to determine a frequency of S. aureus colonization in skin lesions, mucous membranes of the nasal cavity and intestine of children with atopic dermatitis, to compare the genotypes of Staphylococcus aureus strains isolated from different biotopes of atopic dermatitis patients, and to clarify whether the intestinal and nasal cavities microbiota may act as a source of S. aureus recolonization of skin lesions. Materials and Methods: Bacteriological examination of fecal samples, skin, and nasal swabs was conducted in 38 atopic dermatitis patients. The pure bacterial cultures of S. aureus were identified using API Staph (Biomerieux, France) and Vitek 2 MS (Biomerieux, France). Isolates of S. aureus were subjected to genotyping by analysis of rRNA internal 16S-23S rRNA spacer regions and high resolution melting analysis (HMR) of polymorphic spa X-regions. Results: 99% S. aureus strains were successfully identified using MALDI-TOF mass-spectrometry. S. aureus cultures were isolated from all biotopes in 31,6% of children, from skin and nasal cavities ­ in 42% of cases, from skin and feces ­ in 2,6% of cases, only from skin ­ in 10,5%, from nasal cavities and feces ­ in 2,6%, and only from nasal cavities ­ in 2,6% of cases. In 8% of children, S. aureus was not detected in any of the biotopes. Genotyping of the isolates enabled the detection of 17 different genotypes. A match between the genotypes of skin and nasal strains, and skin and fecal strains was observed in 88% and 61% of the cases respectively. Conclusions: The observed a high-frequency matching genotypes suggests the possibility of migration of S. aureus strains inside biotopes in humans and the absence of specialization to colonization of any of the niches.

[Species Diversity of Bifidobacteria in the Intestinal Microbiota Studied Using MALDI-TOF Mass-Spectrometry].

BACKGROUND: The members of genus Bifidobacterium represent a significant part of intestinal microbiota in adults and predominate in infants. Species repertoire of the intestinal bifidobacteria is known to be subjected to major changes with age; however, many details of this process are still to be elucidated. OBJECTIVE: Our aim was to study the diversity of intestinal bifidobacteria and changes of their qualitative and quantitative composition characteristics during the process of growing up using MALDI-TOF mass-spectrometric analysis ofpure bacterial cultures. METHODS: A cross-sectional study of bifidobacteria in the intestinal microbiota was performed in 93 healthy people of the ages from 1 month to 57 years. Strains were identified using Microflex LT MALDI-TOF MS, the confirmation was performed by 16S rRNA gene fragment sequencing. RESULTS: 93% of isolated bifidobacterial strains were successfully identified using MALDI-TOF mass-spectrometry. At least two of the strains from each species were additionally identified by 16S rRNA gene fragment sequencing, in all of the cases the results were the same. It was shown that the total concentration of bifidobacteria decreases with age (p<0.001) as well as the frequency of isolation of Bifidobacterium bifidum (p=0.020) and Bifidobacterium breve (p<0.001), and the frequency of isolation of Bifidobacterium adolescentis, increases (p<0.001), representing the continuous process of transformation of microbiota. CONCLUSION: The method of MALDI-TOF mass spectrometry demonstrated the ability to perform rapid and reliable identification of bifidobacteria that allowed the study of changes in the quantitative and qualitative characteristics of human microbiota in the process of growing up.

Draft Genome Sequence of Lactobacillus fermentum NB-22.

We announce here a draft genome sequence of Lactobacillus fermentum NB-22, a strain isolated from human vaginal microbiota. The assembled sequence consists of 190 contigs, joined into 137 scaffolds, and the total size is 2.01 Mb.

Draft Genome Sequence of Coprobacter fastidiosus NSB1T.

Coprobacter fastidiosus is a Gram-negative obligate anaerobic bacterium belonging to the phylum Bacteroidetes. In this work, we report the draft genome sequence of C. fastidiosus strain NSB1(T) isolated from human infant feces.

Draft Genome Sequences of Two Pairs of Human Intestinal Bifidobacterium longum subsp. longum Strains, 44B and 1-6B and 35B and 2-2B, Consecutively Isolated from Two Children after a 5-Year Time Period.

We report the genome sequences of four isolates of a human gut symbiont, Bifidobacterium longum. Strains 44B and 35B were isolated from two 1-year-old infants, while 1-6B and 2-2B were isolated from the same children 5 years later. The sequences permit investigations of factors enabling long-term colonization of bifidobacteria.