Our aim was to study the nucleotide sequences of 9 previously undescribed strains of B. fragilis collected from patients with intra-abdominal diseases at city hospitals in Nur-Sultan, Kazakhstan.
ABSTRACT
Our aim was to study the nucleotide sequences of 9 previously undescribed strains of B. fragilis collected from patients with intra-abdominal diseases at city hospitals in Nur-Sultan, Kazakhstan.
ANNOUNCEMENT
Bacteroides fragilis is a commensal bacterium that is found in the intestines of most people and that can become an opportunistic pathogen. Amounting to only about 0.1 to 0.5% of the total bacterial mass of the intestine, B. fragilis is the most frequently isolated anaerobe from clinical samples obtained from deep intra-abdominal abscesses, purulent skin infections, soft tissue infections (1, 2), diarrhea, and colorectal cancer (3).
B. fragilis strain no. 4 to 12 were isolated from clinical samples of patients diagnosed with acute peritonitis receiving treatment at City Hospitals No. 1 and No. 2 and Regional General Hospital No. 2. Informed consent and questionnaires were approved by the local ethics committee of the RSE National Center for Biotechnology of the Ministry of Education and Science of the Republic of Kazakhstan (extract from protocol no. 4 of 29 August 2017).
Samples were collected from the drainage wounds by use of swabs with subsequent immersion of probes into tubes containing Amies medium. After that, samples were cultivated on Bacteroides bile esculin agar (BBE; Conda) at 37°C for 72 h under anaerobic conditions (an aerostat, gas pack). Isolates were identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Bremen, Germany). Bacterial colonies growing on solid medium were removed with a sterile plastic tip and resuspended in 1,000 μl of Tris-EDTA (TE) buffer. Total DNA was extracted from all strains using the cetyltrimethylammonium bromide (CTAB) method (4). Then, 150 ng of the total genomic DNA from each isolate of B. fragilis was used for sequencing. Library preparation and Illumina MiSeq sequencing were performed using the Nextera DNA Flex library prep kit and a MiSeq reagent kit v3 with 300-bp paired-end reads (600 cycles) according to the manufacturer’s instructions. Quality assessment of the sequencing data (in FASTQ format) was done using FastQC v0.11.15 (5), followed by trimming of adapters and low-quality bases with a Phred quality score of less than 20 using Trimmomatic (6). Genomes were assembled using the SPAdes assembler v3.13.2 using a k-mer length of 127 with the “--careful” mode (7). Comparative phylogenetic analysis was performed with the CSI Phylogeny v1.4 tool from whole-genome sequences using the following parameters: minimum depth, 80×; minimum relative depth, 100%; minimum distance between single-nucleotide polymorphisms (SNPs), 1,000 bp; minimum SNP quality, 500; minimum read mapping quality, 500; and minimums Z-score, 3.29 (8). We discarded sequences with low quality, i.e., ambiguous bases. Figure 1 was created using iTOL v4 (9). B. fragilis strain NCTC 9343 (GenBank accession no. NC_003228.3) was used as a reference sequence. Genome annotation was performed using PGAP v4.11 (10). Default parameters were used for all software.
FIG 1.
Phylogram of available Bacteroides fragilis genomes based on the concatenated alignment of the high-quality SNPs using CSI Phylogeny v1.4. The following available genomes were retrieved from GenBank (ncbi.nlm.nih.gov/genome/): Bacteroides fragilis strain JIM10 (GenBank accession no. CM004507.1), Bacteroides fragilis strain YCH46 (NC_006347.1), Bacteroides fragilis strain CCUG4856T (CP036555), Bacteroides fragilis strain Q1F2 (NZ_CP018937.1), Bacteroides fragilis strain AM17-19 (NZ_QRJX00000000.1), Bacteroides fragilis strain BOB25 (CP011073.1), Bacteroides fragilis strain OF05-11AC (NZ_QSWE00000000.1), and Bacteroides fragilis strain GUT04 (CP043610.1). The reference genome, Bacteroides fragilis NCTC 9343 (NC_003228.3), is indicated in bold.
Data from the whole-genome sequencing of the 9 clinical B. fragilis strains are presented in Table 1.
TABLE 1.
Genome characteristics of 9 B. fragilis strains
| Strain no. | Strain name | Genome size (bp) | Genome coverage (×) | No. of contigs | Total no. of reads | Avg read length (bp) | N50 (bp) | G+C content (%) | Accession no./SRA no. |
|---|---|---|---|---|---|---|---|---|---|
| 4 | Bfr-4 | 4,920,846 | 20.0 | 128 | 1,724,380 | 192 | 69, 701 | 43.41 | JACEFH000000000/SRX9403175 |
| 5 | Bfr-5 | 5,219,927 | 38.565 | 77 | 1,555,229 | 221 | 181, 129 | 43.36 | JACENG000000000/SRX9403182 |
| 6 | Bfr-6 | 5,259,406 | 49.394 | 41 | 1,398,871 | 231 | 378, 644 | 43.33 | JACFSS000000000/SRX9404153 |
| 7 | Bfr-7 | 5,175,039 | 19.59 | 28 | 736,748 | 273 | 379, 532 | 43.25 | JACFST000000000/SRX9404154 |
| 8 | Bfr-8 | 5,243,270 | 64.17 | 40 | 1,258,909 | 242 | 311, 507 | 43.24 | JACFSU000000000/SRX9404155 |
| 9 | Bfr-9 | 5,125,231 | 29.757 | 53 | 1,501,107 | 218 | 290, 748 | 43.45 | JACFSV000000000/SRX9404156 |
| 10 | Bfr-10 | 5,242,965 | 31.158 | 36 | 1,511,363 | 226 | 397, 964 | 43.12 | JACFSW000000000/SRX9404157 |
| 11 | Bfr-11 | 5,236,934 | 33.68 | 32 | 1,386,003 | 245 | 407, 555 | 43.4 | JACFSX000000000/SRX9404158 |
| 12 | Bfr-12 | 5,321,320 | 31.143 | 92 | 1,272,842 | 229 | 182, 180 | 43.39 | JACFSY000000000/SRX9404159 |
Results of the phylogenetic analysis are shown in Fig. 1. B. fragilis Bfr-10 is located farther from all the strains studied.
Data availability.
The 9 whole-genome shotgun projects have been deposited at DDBJ/ENA/GenBank under the accession numbers listed in Table 1.
ACKNOWLEDGMENTS
We thank the I. Azizov Institute of Antimicrobial Chemotherapy, Smolensk State Medical University (Smolensk, Russia), for scientific support.
The study was supported by grant funding of the Committee of Science of the Ministry of Education and Science of the Republic of Kazakhstan within the framework of the project “Study and Assessment of Sensitivity of Bacteroides fragilis to Broad-Spectrum Antibiotics in Patients with Intra-abdominal Anaerobic Infections” (AP05132131).
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Associated Data
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Data Availability Statement
The 9 whole-genome shotgun projects have been deposited at DDBJ/ENA/GenBank under the accession numbers listed in Table 1.