ABSTRACT
BACKGROUND: Recent advancements in next-generation sequencing (NGS) technology have ushered in significant improvements in sequencing speed and data throughput, thereby enabling the simultaneous analysis of a greater number of samples within a single sequencing run. This technology has proven particularly valuable in the context of microbial community profiling, offering a powerful tool for characterizing the microbial composition at the species level within a given sample. This profiling process typically involves the sequencing of 16S ribosomal RNA (rRNA) gene fragments. By scaling up the analysis to accommodate a substantial number of samples, sometimes as many as 2,000, it becomes possible to achieve cost-efficiency and minimize the introduction of potential batch effects. Our study was designed with the primary objective of devising an approach capable of facilitating the comprehensive analysis of 1,711 samples sourced from diverse origins, including oropharyngeal swabs, mouth cavity swabs, dental swabs, and human fecal samples. This analysis was based on data obtained from 16S rRNA metagenomic sequencing conducted on the Illumina MiSeq and HiSeq sequencing platforms. RESULTS: We have designed a custom set of 10-base pair indices specifically tailored for the preparation of libraries from amplicons derived from the V3-V4 region of the 16S rRNA gene. These indices are instrumental in the analysis of the microbial composition in clinical samples through sequencing on the Illumina MiSeq and HiSeq platforms. The utilization of our custom index set enables the consolidation of a significant number of libraries, enabling the efficient sequencing of these libraries in a single run. CONCLUSIONS: The unique array of 10-base pair indices that we have developed, in conjunction with our sequencing methodology, will prove highly valuable to laboratories engaged in sequencing on Illumina platforms or utilizing Illumina-compatible kits.
Subject(s)
Culture , High-Throughput Nucleotide Sequencing , Humans , RNA, Ribosomal, 16S/genetics , Feces , LaboratoriesABSTRACT
AIM: To identify features of the taxonomic composition of the oropharyngeal microbiota of COVID-19 patients with different disease severity. MATERIALS AND METHODS: The study group included 156 patients hospitalized with confirmed diagnosis of COVID-19 in the clinical medical center of Yevdokimov Moscow State University of Medicine and Dentistry between April and June 2021. There were 77 patients with mild pneumonia according to CT (CT1) and 79 patients with moderate to severe pneumonia (CT2 and CT3). Oropharyngeal swabs were taken when the patient was admitted to the hospital. Total DNA was isolated from the samples, then V3V4 regions of the 16s rRNA gene were amplified, followed by sequencing using Illumina HiSeq 2500 platform. DADA2 algorithm was used to obtain amplicon sequence variants (ASV). RESULTS: When comparing the microbial composition of the oropharynx of the patients with different forms of pneumonia, we have identified ASVs associated with the development of both mild and severe pneumonia outside hospital treatment. Based on the results obtained, ASVs associated with a lower degree of lung damage belong predominantly to the class of Gram-negative Firmicutes (Negativicutes), to various classes of Proteobacteria, as well as to the order Fusobacteria. In turn, ASVs associated with a greater degree of lung damage belong predominantly to Gram-positive classes of Firmicutes Bacilli and Clostridia. While being hospitalized, patients with severe pneumonia demonstrated negative disease dynamics during treatment significantly more often. CONCLUSION: We have observed differences in the taxonomic composition of the oropharyngeal microbiota in patients with different forms of pneumonia developed outside hospital treatment against COVID-19. Such differences might be due to the presumed barrier function of the oropharyngeal microbiota, which reduces the risk of virus titer increase.
Subject(s)
COVID-19 , Microbiota , Humans , RNA, Ribosomal, 16S/genetics , Oropharynx/microbiology , LungABSTRACT
Anti-PD-1 immunotherapy has a large impact on cancer treatment but the rate of positive treatment outcomes is 40-45% and depends on many factors. One of the factors affecting the outcome of immunotherapy is the gut microbiota composition. This effect has been demonstrated both in model objects and in clinical patients groups. However, in order to identify clear causal relationships between microbiota and anti-PD1 immunotherapy response, it is necessary to expand the number of patients and experimental samples. This work presents an analysis of metagenomic data obtained using whole-genome sequencing of stool samples from melanoma patients (n=45) with different responses to anti-PD1 therapy. The analysis of the differential representation of microbial species has shown a difference in the composition of the microbiota between the experimental groups. Results of this study indicate existence of a strong link between the composition of the gut microbiota and the outcome of anti-PD1 therapy. Expansion of similar research may help develop additional predictive tools for the outcome of anti-PD1 cancer immunotherapy, as well as increase its effectiveness.
Subject(s)
Gastrointestinal Microbiome , Antibodies , Data Analysis , Humans , Immunotherapy , Metagenome , Programmed Cell Death 1 ReceptorABSTRACT
We report a draft genome sequence of Mycobacterium tuberculosis strain B9741 belonging to Beijing B0/W lineage isolated from a HIV patient from Siberia, Russia. This clinical isolate showed MDR phenotype and resistance to isoniazid, rifampin, streptomycin and pyrazinamide. We analyzed SNPs associated with virulence and resistance. The draft genome sequence and annotation have been deposited at GenBank under the accession NZ_LVJJ00000000.
ABSTRACT
Gamma-amino butyric acid (GABA) is an active biogenic substance synthesized in plants, fungi, vertebrate animals and bacteria. Lactic acid bacteria are considered the main producers of GABA among bacteria. GABA-producing lactobacilli are isolated from food products such as cheese, yogurt, sourdough, etc. and are the source of bioactive properties assigned to those foods. The ability of human-derived lactobacilli and bifidobacteria to synthesize GABA remains poorly characterized. In this paper, we screened our collection of 135 human-derived Lactobacillus and Bifidobacterium strains for their ability to produce GABA from its precursor monosodium glutamate. Fifty eight strains were able to produce GABA. The most efficient GABA-producers were Bifidobacterium strains (up to 6 g/L). Time profiles of cell growth and GABA production as well as the influence of pyridoxal phosphate on GABA production were studied for L. plantarum 90sk, L. brevis 15f, B. adolescentis 150 and B. angulatum GT102. DNA of these strains was sequenced; the gadB and gadC genes were identified. The presence of these genes was analyzed in 14 metagenomes of healthy individuals. The genes were found in the following genera of bacteria: Bacteroidetes (Bacteroides, Parabacteroides, Alistipes, Odoribacter, Prevotella), Proteobacterium (Esherichia), Firmicutes (Enterococcus), Actinobacteria (Bifidobacterium). These data indicate that gad genes as well as the ability to produce GABA are widely distributed among lactobacilli and bifidobacteria (mainly in L. plantarum, L. brevis, B. adolescentis, B. angulatum, B. dentium) and other gut-derived bacterial species. Perhaps, GABA is involved in the interaction of gut microbiota with the macroorganism and the ability to synthesize GABA may be an important feature in the selection of bacterial strains - psychobiotics.
Subject(s)
Bacterial Proteins/genetics , Bifidobacterium/genetics , Gastrointestinal Microbiome/genetics , Glutamate Decarboxylase/genetics , Lactobacillus/genetics , Membrane Proteins/genetics , gamma-Aminobutyric Acid/biosynthesis , Bacterial Proteins/metabolism , Bacteroidetes/drug effects , Bacteroidetes/genetics , Bacteroidetes/isolation & purification , Bacteroidetes/metabolism , Bifidobacterium/drug effects , Bifidobacterium/isolation & purification , Bifidobacterium/metabolism , DNA, Bacterial/genetics , Firmicutes/drug effects , Firmicutes/genetics , Firmicutes/isolation & purification , Firmicutes/metabolism , Gastrointestinal Microbiome/drug effects , Gastrointestinal Tract/microbiology , Gene Expression , Glutamate Decarboxylase/metabolism , Humans , Lactobacillus/drug effects , Lactobacillus/isolation & purification , Lactobacillus/metabolism , Membrane Proteins/metabolism , Metagenome , Proteobacteria/drug effects , Proteobacteria/genetics , Proteobacteria/isolation & purification , Proteobacteria/metabolism , Pyridoxal Phosphate/metabolism , Pyridoxal Phosphate/pharmacology , Sodium Glutamate/metabolism , Sodium Glutamate/pharmacologyABSTRACT
Cell metabolic reprogramming is one of the cancer hallmarks. Glycolysis activation, along with suppression of oxidative phosphorylation and, to a lower extent, the TCA cycle, occurs in the majority of malignant tumors. A bioinformatics search for the glucose metabolism genes that are differentially expressed in colorectal cancer (CC) was performed using the data of The Cancer Genome Atlas (TCGA) Project. OGDHL for an oxoglutarate dehydrogenase complex subunit, which is involved in the TCA cycle and is indirectly responsible for the induction of apoptosis, was identified as one of the most promising candidates. A quantitative PCR analysis showed, on average, an eightfold downregulation of OGDHL in 50% (15/30) of CC samples. Based on the TCGA data, promoter hypermethylation was assumed to be a major mechanism of OGDHL inactivation. Bisulfite sequencing identified the OGDHL promoter region (+327 ... +767 relative to the transcription start site) that is often methylated in CC samples with downregulated ODGHL expression (80%, 8/10) and is possibly crucial for gene inactivation. Thus, frequent and significant OGDHL downregulation due to hypermethylation of a specific promoter region was demonstrated for CC. The OGDHL promoter methylation pattern was assumed to provide a marker for differential diagnosis of CIMP+ (CpG island methylator phenotype) tumors, which display dense hypermethylation of the promoter region in many genes.
ABSTRACT
We report draft genome sequences of two pyrazinamide (PZA)-resistant isolates, Mycobacterium tuberculosis 13-4152 and 13-2459. Isolate 13-4152 is PZA resistant, though it lacks mutations in known genes of PZA resistance. The comparative analysis of these genomes with those stored in GenBank revealed unique mutations, which may elucidate new mechanisms of PZA resistance.
ABSTRACT
We report a draft genome sequence of Mycobacterium tuberculosis strain E186hv, belonging to the Beijing B0/W lineage and isolated from a patient from Kurgan, Russia. This clinical isolate showed a reduced virulence phenotype unusual for this lineage and resistance to isoniazid, rifampin, ethambutol, pyrazinamide, and ofloxacin. We analyzed single nucleotide polymorphisms (SNPs) associated with virulence.
ABSTRACT
The toxin-antitoxin gene systems (TASs) are present in the genomes of the overwhelming majority of bacteria and archaea. These systems are involved in various cellular regulatory processes (including stress response), and have not been previously investigated in Lactobacilli. We identified 6 putative TASs with toxins belonging to the MazE and RelE superfamilies (PemK1-Ð1Lrh, PemK2-Ð2Lrh, PemK3-RelB2Lrh, RelE1Lrh, RelB3-RelE3Lrh, and YefM-YoeBLrh) in the genomes of annotated strains of Lactobacillus rhamnosus. PCR analyses revealed that all systems were found in the genomes of 15 strains of L. rhamnosus isolated from humans in central Russia. These strains were highly heterogeneous with respect to the presence of TASs, as well as their nucleotide and amino acid sequences. In three cases, the relE1 genes contained IS3 elements. TAS heterogeneity may be used to reveal inter-genus differences between strains. Cloning of the toxin genes of 3 TASs inhibited Escherichia coli growth, thus confirming their functionality. Cell growth arrest caused by expression of the toxin genes could be reverted by the expression of a cognate antitoxins. Transcription of toxin-antitoxin loci in L. rhamnosus was shown by RT-PCR.
Subject(s)
Antitoxins/chemistry , Antitoxins/genetics , Bacterial Proteins/genetics , Lacticaseibacillus rhamnosus/chemistry , Lacticaseibacillus rhamnosus/genetics , Toxins, Biological/chemistry , Toxins, Biological/genetics , Adult , Amino Acid Sequence , Antitoxins/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Base Sequence , Feces/microbiology , Gene Expression Regulation, Bacterial , Humans , Infant , Infant, Newborn , Intestines/microbiology , Polymorphism, Genetic , Russia , Saliva/microbiology , Toxins, Biological/isolation & purificationABSTRACT
AIM: To study strains of bacteria from Lactobacillus genus using combination of microbiological and molecular biological methods in order to define more accurately their systematic position and biochemical characteristics. MATERIALS AND METHODS: Thirteen cultures of Lactobacillus bacteria isolated from stool of healthy persons were studied: L. plantarum CS 396, L. plantarum 8-PA-3, L. plantarum 421-2, L. fermentum 90-TC-4, L. delbrueckii gKNM 101, L. delbrueckii gKNM 526, L. acidophilus Er 317/402 NARINE, L. acidophilus 100 ash, L. acidophilus NK-1, L. acidophilus NNIE, L. acidophilus K3sh24, L. brevis gKNM 23 11, L. casei gKNM 577. Their enzymatic activity relative to 50 sugars was studied using API-50 system. Structure of proximal region of 16S rRNA gene was studied also. RESULTS: According to results of 16S rRNA gene sequence analysis strains were divided on 2 groups: 1) L. casei gKNM 577, L. plantarum 8-PA-3, L. plantarum CS 396, which species belonging corresponded to stated description. Comparison of nucleotide sequence of 16S rRNA gene of group 2 strains with nucleotide sequences database revealed that cultures NK-1, Er315/402 NARINE, 100 ash, NNIE identified early as L. acidophilus belong to species L. helveticus; L. brevis gKNM 23 and L. acidophilus K3sh24--to group L. casei/paracasei, L. delbrueckii gKNM 101 and L. fermentum 90-TC-4--to L. plantarum, L. delbrueckii gKNM 526--to L. fermentum, and L. plantarum 421-2--to L. rhamnosus. CONCLUSION: Obtained data allowed to perform taxonomic reclassification of species belonging of studied probiotic cultures of lactobacilli according to modem level of systematic of bacteria.
Subject(s)
Lactobacillus/classification , Probiotics/classification , Carbohydrate Metabolism , Humans , Lactobacillus/enzymology , Lactobacillus/genetics , Oligonucleotide Array Sequence Analysis , Probiotics/isolation & purification , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/geneticsABSTRACT
Thirteen strains of industrial bacterial cultures of the genus Lactobacillus (from a collection of Gabrichevsky Research Institute of Epidemiology and Microbiology) were studied. These strains were used for decades in Russian Federation for food and drug production, as ferments for lactic acid products, for production of probiotics, biologically active and veterinary preparations. Complex analysis of data on cultures obtained using microbiological and molecular-genetic methods was conducted for the first time. Biochemical characteristics of these cultures were studied and the sequence of the proximal region of 16S ribosomal RNA gene was determined. The employment of the test system API-50CHL was shown to broaden the opportunities of a more accurate biochemical identification of bacteria belonging to the genus Lactobacillus, in comparison with the set ANAEROTEST-23. According to the results obtained in a comparative analysis of nucleotide sequences of 16S rRNA gene, all strains examined show 97-99% homology of the proximal region of this gene with that of the type representatives of studied species. These data allowed taxonomic reclassification of the species position of cultures with consideration of the more advanced level of systematics. Nucleotide sequences of gene fragments of examined lactobacilli strains were recorded in NCBI database (accession numbers of deposits GU560031, GU560032, GU560033, GU560034, GU560035, GU560036, GU560037, GU560038, GU560039, GU560040, GU560041, GU560042, GU560043).
Subject(s)
Lactobacillus/classification , Bacterial Typing Techniques , Food-Processing Industry , Lactobacillus/genetics , Molecular Sequence Data , Phylogeny , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Russia , Sequence Homology, Nucleic AcidABSTRACT
Examination of dental deposits from 45 healthy individuals detected 3 lactobacillus strains showing a high antagonism toward test cultures. The api 50 CH "bio Merieux" test systems were employed to identify strains as Lactobacillus fermentum 39, Lactobacillus rhamnosus 24 and Lactobacillus paracasei 50. The results of analyzing the sequences of the 16S rRNA genes of the test strains confirmed this identification, except for the latter strain. The taxonomic status of the third strain L. rhamnosus 50 was determined by the bioinformative analysis of the nucleotide sequence of the 16S rRNA genes.
Subject(s)
Lactobacillus/classification , Mouth/microbiology , Bacterial Typing Techniques , Base Sequence , Female , Humans , Lactobacillus/genetics , Lactobacillus/isolation & purification , Male , Molecular Sequence Data , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Young AdultABSTRACT
The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960-1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adults and represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97-100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.