An improved and extended dual-index multiplexed 16S rRNA sequencing for the Illumina HiSeq and MiSeq platform.

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.

[Study of the resistome of human microbial communities using a targeted panel of antibiotic resistance genes in COVID-19 patients].

AIM: To study overall drug resistance genes (resistome) in the human gut microbiome and the changes in these genes during COVID-19 in-hospital therapy. MATERIALS AND METHODS: A single-center retrospective cohort study was conducted. Only cases with laboratory-confirmed SARS-CoV-2 RNA using polymerase chain reaction in oro-/nasopharyngeal swab samples were subject to analysis. The patients with a documented history of or current comorbidities of the hepatobiliary system, malignant neoplasms of any localization, systemic and autoimmune diseases, as well as pregnant women were excluded. Feces were collected from all study subjects for subsequent metagenomic sequencing. The final cohort was divided into two groups depending on the disease severity: mild (group 1) and severe (group 2). Within group 2, five subgroups were formed, depending on the use of antibacterial drugs (ABD): group 2A (receiving ABD), group 2AC (receiving ABD before hospitalization), group 2AD (receiving ABD during hospitalization), group 2AE (receiving ABD during and before hospitalization), group 2B (not receiving ABD). RESULTS: The median number of antibiotic resistance (ABR) genes (cumulative at all time points) was significantly higher in the group of patients treated with ABD: 81.0 (95% CI 73.8-84.5) vs. 51.0 (95% CI 31.1-68.4). In the group of patients treated with ABD (2A), the average number of multidrug resistance genes (efflux systems) was significantly higher than in controls (group 2B): 47.0 (95% CI 46.0-51.2) vs. 21.5 (95% CI 7.0-43.9). Patients with severe coronavirus infection tended to have a higher median number of ABR genes but without statistical significance. Patients in the severe COVID-19 group who did not receive ABD before and during hospitalization also had more resistance genes than the patients in the comparison group. CONCLUSION: This study demonstrated that fewer ABR genes were identified in the group with a milder disease than in the group with a more severe disease associated with more ABR genes, with the following five being the most common: SULI, MSRC, ACRE, EFMA, SAT.

[Composition of oropharyngeal microbiota in patients with COVID-19 of different pneumonia severity].

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.

[Bioinformatics analysis of antimicrobial resistance genes and prophages colocalized in human gut metagenomes]. / Bioinformaticheskii analiz kolokalizatsii genov rezistentnosti i profagov v metagenomakh kishechnika cheloveka.

The constant increase of antibiotic-resistant strains of bacteria is caused by extensive uses of antibiotics in medicine and animal breeding. It was suggested that the gut microbiota serves as a reservoir for antibiotics resistance genes that can be carried from symbiotic bacteria to pathogenic ones, in particular, as a result of transduction. In the current study, we have searched for antibiotics resistance genes that are located inside prophages in human gut microbiota using PHASTER prophage predicting tool and CARD antibiotics resistance database. After analysing metagenomic assemblies of eight samples of antibiotic treated patients, lsaE, mdfA and cpxR/cpxA genes were identified inside prophages. The abovementioned genes confer resistance to antimicrobial peptides, pleuromutilin, lincomycins, streptogramins and multidrug resistance. Three (0.46%) of 659 putative prophages predicted in metagenomic assemblies contained antibiotics resistance genes in their sequences.