Complete Genome Sequence of Invasive MLST-Untypeable Serotype 3 xpt-Deficient Streptococcus pneumoniae Strain B1900, Isolated from the Brain of an Infant Rhesus Monkey (Macaca mulatta) That Died of Meningitis

ABSTRACT

Serotype 3 Streptococcus pneumoniae is associated with major invasive pneumococcal diseases in humans. We report the circularized 2.0-Mbp complete genome sequence of invasive serotype 3 S. pneumoniae strain B1900, untypeable by multilocus sequence typing (MLST). This strain was isolated from the brain of an infant rhesus monkey (Macaca mulatta) that suddenly died of meningitis with no clinical symptoms.

ANNOUNCEMENT

Serotype 3 Streptococcus pneumoniae is evasive from vaccine protection, though it has been included in recent vaccines (1). Its evasiveness is likely linked to heavily mucoid capsular polysaccharide (CPS), which enables it to survive against protective antibodies or prevents antibody production (1, 2). Multilocus sequence typing (MLST) has been the standard method for differentiation of invasive S. pneumoniae isolates from other streptococcal species (3).

A swab sample from the brain of an infant rhesus monkey that suddenly died with no clinical signs was submitted for microbiological evaluation under the animal health monitoring program at a research center in Arkansas. The sample was streaked onto tryptic soy agar (TSA) agar with 5% sheep blood (Remel, San Diego, CA) and incubated at 37°C for 24 h. The plate was covered in pure culture with large heavily mucoid colonies. The bacterium was an alpha-hemolytic Gram-positive diplococcus, identified as S. pneumoniae using the MALDI Biotyper system (Bruker, Billerica, MA). The cause of death was diagnosed as meningitis by attending pathologists. The isolate was serotype 3 with no significant antimicrobial resistance profile, as determined by the Streptococcus Laboratory at the CDC (https://www.cdc.gov/streplab/index.html).

Genomic DNA was isolated from an overnight culture at 37°C on TSA agar with 5% sheep blood using the blood and tissue kit, following the manufacturer’s instructions (Qiagen, Redwood City, CA). The quality and quantity of DNA were determined using a NanoDrop spectrophotometer and a Qubit fluorimeter (Fisher Scientific, Waltham, MA). The same genomic DNA was used for Illumina and Nanopore sequencing with no shearing. A short-read genomic library was constructed using the Nextera XT library prep kit and sequenced using the MiSeq v2 reagent kit (2 × 250 bp) on MiSeq (Illumina, San Diego, CA). For long reads, a genomic library was constructed using the ligation sequencing kit SQK-LAK109 and sequenced on a Nanopore MinION FLO-MIN107 instrument (Oxford Nanopore Technologies, Oxford, UK). Base calling was performed using Guppy v6.4.8 integrated with MinKNOW v22.12.5. Default parameters were used for all software unless otherwise noted. The raw short reads were quality checked and trimmed using Fastq Utilities (Cutadapt v2.2 with Python v3.7.9) in PATRIC, and the raw long reads were quality checked and trimmed using Porechop v0.2.1 and Galore v0.6.7 in Galaxy (4). The Illumina short reads (n = 1,570,670) and Nanopore long reads (n = 4,000; N₅₀ = 5,824 bp) were assembled in a circularized genome with 457× coverage depth using Unicycler v0.4.8 in PATRIC (5). The genome of S. pneumoniae B1900 is 2,024,947 bp long with a G+C content of 39.7%. The P1900 genome, annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.11, has 1,841 coding genes, 191 pseudogenes, 58 tRNAs, 12 rRNAs, and 3 noncoding RNAs (ncRNAs) (6). The closest reference genome is S. pneumoniae R6 (GenBank accession number NC_003098), with an average nucleotide identity (ANI) of 98.9% as estimated using MinHash v2.3 (7). Strain B1900 was nontypeable by MLST due to the absence of the xpt gene, one of seven housekeeping genes used in sequence typing. The xpt gene is required for the synthesis of xanthine phosphoribosyltransferase, to convert xanthine to xanthosine 5′-monophosphate (8). The B1900 genome predicted two other purine phosphoribosyltransferases (hypoxanthine- and guanine-xanthine phosphoribosyltransferases), as reported in the Escherichia coli purine salvage pathway (9). A 4.7-kb chromosomal region containing S. pneumoniae genes required for the production of type 3 capsular polysaccharide was identified (10).

Data availability.

The whole-genome sequence has been deposited at DDBJ/ENA/GenBank under the accession number CP051650. The raw reads have been deposited in the Sequence Read Archive (SRA) under the accession numbers SRR15907666 and SRR15907667.