Group B Streptococcus (GBS) is an asymptomatic colonizer of the female reproductive tract but can cause maternal and neonatal infections and adverse pregnancy outcomes. Here, we closed the genome sequence of strain CJB111, a neonatal GBS clinical isolate from a case of late-onset bacteremia without focus (Houston, TX; 1990).
ABSTRACT
Group B Streptococcus (GBS) is an asymptomatic colonizer of the female reproductive tract but can cause maternal and neonatal infections and adverse pregnancy outcomes. Here, we closed the genome sequence of strain CJB111, a neonatal GBS clinical isolate from a case of late-onset bacteremia without focus (Houston, TX; 1990).
ANNOUNCEMENT
The Gram-positive beta-hemolytic bacterium Streptococcus agalactiae (group B Streptococcus [GBS]) asymptomatically colonizes the gastrointestinal and female genital tracts of healthy adults but can cause neonatal infections (pneumonia, bacteremia, meningitis [1]) and adverse pregnancy outcomes (2). Serotype V GBS isolates are emerging among adults and infants (3–6), and serotype V isolate CJB111 exhibits hypervirulence and vaginal persistence in murine models of GBS infection and colonization (7, 8).
CJB111 (ATCC BAA23) was isolated by Carol J. Baker from the blood of a female infant with late-onset sepsis on 18 July 1990, grown in Todd Hewitt broth (THB), and stored in glycerol at –90°C. The patient received intravenous ampicillin and gentamicin 1 day post onset of illness. Upon GBS isolation, therapy was switched on day 3 to intravenous penicillin (10-day treatment total). Following clean cerebrospinal fluid tests, she was diagnosed with bacteremia without focus at age 55 days and discharged without apparent sequelae. While CJB111’s sequence is currently available in 155 contigs (GenBank accession no. AAJQ01000000), a closed genome sequence may ease future genomic analyses.
CJB111 was grown statically overnight at 37°C in THB, genomic DNA was purified (Gentra PureGene Yeast/Bact kit), and concentration and quality were confirmed by NanoDrop spectroscopy. The Microbial Genome Sequencing Center (MiGS; Pittsburgh, PA) performed short- and long-read sequencing (Illumina and Oxford Nanopore technologies [ONT], respectively) and de novo assembly. Default parameters were used except where otherwise noted. Short reads were obtained using the Illumina Nextera kit and NextSeq 550 platform (9). For ONT sequencing, libraries were prepared using kit SQK-LSK109 to the manufacturer’s specifications (no DNA size selection/shearing), sequencing was performed on a MinION R9 flow cell, and base calling was performed using Guppy v4.2.2 (GPU mode) (10). Illumina paired-end reads (2 × 150 bp) and ONT long reads were provided as fastq files (Illumina: 7,410,044 reads, 989,364,400 bases, 472× coverage; ONT: 175,394 reads, 650,701,562 bases, 310× coverage, N50 value of 4,577 bp). bcl2fastq v2.20.0.422 was used for demultiplexing, quality control, and trimming of the Illumina reads (11) and Porechop v0.2.4 for quality trimming and removing adapters for ONT sequencing (12). Hybrid assembly via Unicycler v0.4.8 with a verbosity value of 2 (13) yielded six contigs, which were further assembled into three nonoverlapping contigs upon mapping to CJB111 contigs (AAJQ01000000) in Geneious v11.1.5 (14). The genome sequence was closed via PCR using primers flanking the nonoverlapping contigs (Table 1) and Phusion high-fidelity polymerase/buffer (New England Biolabs) under the following cycling conditions on a Bio-Rad T100 thermal cycler: 98°C, 2-min hot start; 34 cycles (98°C, 10seconds; Tm °C, 20seconds; 72°C, 30- second extension/kb); and 72°C, 10-min extension. Purified PCR amplicons (Qiagen) were Sanger sequenced using Applied Biosystems 3730/3500xl genetic analyzers, yielding 2× sequencing in both directions. Contigs were assembled de novo, overlapping ends were trimmed, and the genome sequence was rotated manually in Geneious v11.1.5 to start with dnaA.
TABLE 1.
Primers used in this study
| PCRs flanking nonoverlapping contigs | Primer namea | Sequence (5′–3′) | PCR Tm (°C) | PCR product length (bp) | PCR extension time (min:s) |
|---|---|---|---|---|---|
| PCR 1 | 5121 | TATCAATAACGATAGTATGCCCAGCG | 65 | 4,712 | 2:21 |
| 3121 | TTCCAATAGGTCTTGATAGTGAGGTG | ||||
| PCR 2 | 5122 | GTTTGTTGCAGTCGTCGTTATCTC | 63 | 3,392 | 1:42 |
| 3122 | CGTCGGAATTAAATCTTGGAATACC | ||||
| PCR 3 | 5123 | GGCATCAGGAATGATCTGATTTACAC | 65 | 2,064 | 1:02 |
| 3123 | TGCCTCCCATTGGATTACTGTATAC | ||||
| PCR 4 | 5124 | GACTCGATAGGGTATATGGTGCC | 65 | 5,126 | 2:34 |
| 3124 | GGTTCGATTGCGTTACTGCG | ||||
| Sequencing primers | |||||
| PCR 1 | 5131 | GTGACATAGATTGGAATAGGGTTAGC | |||
| 5132 | TATTCTCAGTGTCTGTGTACTACTGC | ||||
| 5133 | AAATCTTGGCAGACAGTGGTTATC | ||||
| 5134 | CAACAGGAGGAACCTGTAGAAGTTC | ||||
| 5135 | TACAATCCATCTCTGGAATTCAC | ||||
| 3131 | GTGACATAGATTGGAATAGGGTTAGC | ||||
| 3132 | ATAATAAGGTGTCAGACAAACTCGC | ||||
| 3133 | GGTTCGTCATTTATGAATGGTGTAC | ||||
| 3134 | TTGACTATGGTTATGCTTTCAGG | ||||
| 3135 | TTCTCAACCTTGATTCTCTCTTTGG | ||||
| 3136 | GTGCCGTTTCAAAGGTCGCT | ||||
| 3137 | CCGGGCTCGCTCCATATAGATAAG | ||||
| PCR 2 | 5136 | TATGCTCTCATAGGTAACACCACC | |||
| 5137 | AACGATCACCTAAATTAGTACCTGC | ||||
| 5138 | TCTATCTTGTTCCTGTTTCCTTG | ||||
| 5139 | TTTAGGTTAGAAAGGAGATACTGCC | ||||
| 5140 | TACTTCAAATGGTATGCAAGCTATGG | ||||
| 3138 | GCTGAACAAGCTGCTGTTATTGC | ||||
| 3139 | TTTAGTTGAGGATGCTTATCGAG | ||||
| 3140 | AGTTATCTGTCTATAAGGAATGTCG | ||||
| 3141 | AAGCTATGGTTGAAGCTGTTG | ||||
| PCR 3 | 5141 | TTAAATTAACTCCTGAAGTACTCCG | |||
| 3142 | AGGTAATTTCCATTTCTCACCTGAAG | ||||
| 3143 | TTTCGGCGACAATTCATTGAACTGAG | ||||
| PCR 4 | 5125 | GTAACTAGTTATCTCTAGCCTGTAGC | |||
| 5126 | CACGAAAGCAACTTAATCCGTCG | ||||
| 5127 | CCCTTGACTACATAAGTACTAACCC | ||||
| 5128 | CTGTTAATAAATCAGCTCCATGAGC | ||||
| 5129 | TTTCCCTTGCATTTCCCATAGACC | ||||
| 5130 | GCCTATCCAATTATTCGTTTGGAG | ||||
| 3125 | TTTACCTCTGTTGCATCCACAATC | ||||
| 3126 | GCAAAGCAATTGTATTCCGTCTT | ||||
| 3127 | AAAGTGTCGTTACCAACTCTGAAG | ||||
| 3128 | AAATTATGAATCAGGCATGCTCCTGG | ||||
| 3129 | AATAAAGCCTGAAACCAGTTCAGAG | ||||
| 3130 | CATCACTCTGGCCTCTATTATTT |
Primer names beginning with 5 indicate forward primers. Primer names beginning with 3 indicate reverse primers.
The CJB111 sequence was deposited at GenBank as one circular contig (2,093,987 bp; GC content, 35.52%). BUSCO_v1 and CheckM v1.0.18 confirmed the genome completeness (15, 16). GenBank annotated the CJB111 genome sequence using PGAP v4.13 (17).
Data availability.
The CJB111 sequence is available in GenBank under accession no. CP063198. The raw sequence reads are accessible under Sequence Read Archive accession no. SRX9273111, SRX9273112, and SRX9273113; BioProject accession no. PRJNA663970; and BioSample accession no. SAMN16191206.
ACKNOWLEDGMENTS
This work was funded by NIH/NIAID F32 AI143203 to B.L.S., NIH/NINDS R01 NS116716 to K.S.D., and NIH/NIAID R01 AI141479 to B.A.D.
We acknowledge Dan Snyder and the Microbial Genome Sequencing Center (Pittsburgh, PA) for Illumina and Nanopore sequencing and Mark Farrell and the Barbara Davis Center Molecular Biology Core at University of Colorado-Anschutz Medical Campus (Aurora, CO) for Sanger sequencing.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The CJB111 sequence is available in GenBank under accession no. CP063198. The raw sequence reads are accessible under Sequence Read Archive accession no. SRX9273111, SRX9273112, and SRX9273113; BioProject accession no. PRJNA663970; and BioSample accession no. SAMN16191206.