Complete genome sequences of six Lactobacillus iners strains isolated from Kenyan women

TL;DR

This paper reports the full genome sequences of six Lactobacillus iners strains from Kenyan women, contributing to understanding their role in vaginal health.

Contribution

The study provides the first complete genome sequences of L. iners strains isolated from Kenyan women.

Findings

Six complete L. iners genomes were sequenced from cervicovaginal secretions in Nairobi.

The genomes may help clarify the role of L. iners in vaginal health and disease.

Abstract

Lactobacillus iners is one of the most common members of the vaginal microbiome, with a controversial role in vaginal health. Here, we present the complete genome sequences of six L. iners strains isolated from cervicovaginal secretions from women in Nairobi, Kenya.

Tables

TABLE 1. Genome sequencing, assembly, and annotation statistics per straina

Strain	GenBank accession	Raw reads	Length (bp)	No. of reads	Avg read quality	Read length N50	Assembly coverage	GC content (%)	CDS	tRNA	rRNA	ANI
NVM020S14	CP194987.1	SRX29568733	1,354,189	52,819	13.8	4,861	53×	33.5	1,235	70	18	98.8
NVM025S01	CP173714.1	SRX29568738	1,359,327	86,416	15.2	3,901	79×	33.5	1,271	70	18	98.4
NVM041S27	CP194988.1	SRX29568734	1,319,338	351,366	21.8	1,298	203×	33.5	1,196	70	18	98.8
NVM076S08	CP194989.1	SRX29568735	1,305,175	419,916	20.8	1,132	260×	33.0	1,182	69	15	98.6
NVM196S05	CP194990.1	SRX29568736	1,340,659	12,535	12.9	6,476	70×	33.0	1,217	69	15	98.9
NVM210S01	CP194991.1	SRX29568737	1,353,401	607,684	20.8	932	285×	33.0	1,236	69	15	98.9

Full text

ANNOUNCEMENT

Most vaginal microbiomes are predominantly colonized by a single Lactobacillus species, such as Lactobacillus iners, which is estimated to be the most common vaginal bacterium globally (1, 2). Whether L. iners colonization protects against bacterial vaginosis is contested (1, 3, 4). Characterization of L. iners biology and the diversity of strains is needed to elucidate the role of L. iners in vaginal health. Here, we present the sequences of six L. iners strains isolated from Kenyan women.

Female sex workers in Nairobi, Kenya, were enrolled through the Sex Worker’s Outreach Program clinics. All strains presented here were isolated from L. iners-dominated vaginal microbiomes (determined by 16S rRNA gene sequencing), and each strain is from a different participant. Bacteria were isolated as previously described (5); in brief, frozen pellets of cervicovaginal secretions, collected using SoftCup (Evofem, San Diego, CA, USA), were plated on New York City III agar (6) and incubated anaerobically at 37°C for 48 h. All bacterial isolation and culture were performed in an anaerobic chamber (AS-580, Anaerobe Systems, Morgan Hill, CA, USA) using a gas mixture of 10% CO_2_, 10% H_2_, balance N_2_ (Linde, Mississauga, ON, Canada). L. iners isolates were identified using colony PCR for the inerolysin gene (5′-TACTAAGCCTGCACAAGCTG-3′; 5′-TGCATCAAATACATCACCTGG-3′).

For sequencing, 4 mL of Serrador’s Lactobacillus-adapted Iscove’s medium (5) was inoculated with an L. iners colony and incubated anaerobically at 37°C for 42 h. gDNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA, A1120), with the following modifications: 1.5 mL of culture was used, and for strains with low DNA yields, two preparations were combined during re-hydration. gDNA libraries were prepared using the Rapid Sequencing DNA V14 Barcoding kit (SQK-RBK114.24, Oxford Nanopore Technologies, Oxford, UK) using manufacturer’s instructions. Size selection was not performed. gDNA libraries were subjected to long-read DNA sequencing on a MinION Mk1B using Kit 14 chemistry (R10.4.1, Oxford Nanopore Technologies, Oxford, UK). All tools were run with default settings unless otherwise stated. Base-calling was performed using Dorado in duplex mode (v0.8.3, using the SUP model). Reads were demultiplexed and adapter trimmed using Dorado (v0.8.3, demux mode). Read quality and length were computed using NanoComp (v1.25.3) (7) (Table 1). Error correction was not performed, and no reads were removed during quality control.

Genome assembly was performed with Flye (v2.9.5) (8, 9). Assembly coverage ranged from 53× to 285× across all six genomes, each a single circular contig (Table 1). Repeat graphs were manually visualized using Bandage (v0.9.0), confirming edge-to-edge coverage in genomes except NVM196S05 (12). NVM196S05 overlap trimming was done manually by mapping reads back to the assembly with minimap2 (v2.26-r1175) (13). Genomes were reoriented to begin at dnaA. The genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (v6.10) (10). Completeness and percent contamination were determined using CheckM (v1.2.3) (14). The average genome length was 1,338,681.5 bp with an average GC content of 33.25%. Each genome has an average nucleotide identity greater than 95% with Lactobacillus iners strain C0322A1, determined using FastANI (v1.33), confirming taxonomic assignment (11) (Table 1).