Invasive Streptococcus oralis Expressing Serotype 3 Pneumococcal Capsule, Japan.

We report 2 adult cases of invasive disease in Japan caused by Streptococcus oralis that expressed the serotype 3 pneumococcal capsule and formed mucoid colonies. Whole-genome sequencing revealed that the identical serotype 3 pneumococcal capsule locus and hyl fragment were recombined into the genomes of 2 distinct S. oralis strains.

Streptococcus oralis is a viridans streptococcus that is divided into 3 subspecies S. oralis subsp. oralis, dentisani, and tigurinus (). Differentiation between these subspecies and other α-hemolytic streptococci, including S. pneumoniae, remains difficult because they share similar biochemical properties. S. oralis inhabits the oral cavity and can cause severe infections in persons with immunodeficiency (). Antimicrobial drug resistance and capsule expression studies have demonstrated that gene transfer can occur from oral Streptococcus spp. to S. pneumoniae (–). Most oral Streptococcus spp. have a pneumococcus-like capsule locus and produce capsular polysaccharides ().

We report 2 cases of invasive streptococcal disease in older adults in Japan (Table). Case 1 occurred in a 69-year-old man with gastric cancer; case 2 occurred in a 78-year-old man with bacteremic meningitis who had no underlying disease. Both patients were successfully treated with antimicrobial agents. The bacterial isolates (ASP0312-Sp from case 1 and SP2752 from case 2) contained α-hemolytic bacteria that formed characteristic mucoid colonies on blood agar (Table). Quellung reactions were strongly positive for pool R or pneumococcal serotype 3 antisera (Statens Serum Institut, https://en.ssi.dk), suggesting that the isolates were S. pneumoniae serotype 3. However, both isolates were optochin-resistant and bile-insoluble. Moreover, multilocus sequence typing (MLST) showed that the sequences of all 7 alleles of ASP0312-Sp and 5 alleles of SP2752 differed from those registered in the MLST database (https://pubmlst.org) (Table). For SP2752, the allele numbers were 341 for gdh and 406 for spi. Furthermore, we observed nucleotide differences between ASP0312-Sp and SP2752 in aroE (31 different bp), gdh (34 bp), gki (25 bp), recP (25 bp), spi (14 bp), xpt (47 bp), and ddl (15 bp), which indicated that the strains were distinct. These results suggested that the 2 strains were nonpneumococcal Streptococcus spp.

Table

Characteristics of invasive Streptococcus oralis expressing serotype 3 pneumococcal capsule from 2 adult patients, Japan*

Case	Onset date	Isolate ID	Source	Positive Quellung reaction	No. different bases
					aroE	gdh	gki	recP	spi	xpt	ddl
1	January 2015	ASP0312-Sp	Blood	Pool R, serotype 3	61	30	44	32	4	41	37
2	April 2014	SP2752	Blood, CSF	Pool R, serotype 3	54	–†	40	33	–†	47	36

*Each indicated allele from S. oralis isolates ASP0312-Sp and SP2752 was compared with corresponding alleles from S. pneumoniae that have been registered in the MLST database (https://pubmlst.org). The number of different bases represents the number of polymorphisms between the isolates and the S. pneumoniae allele with the greatest homology. Pool R and pneumococcal serotype 3 antisera (Statens Serum Institut, https://en.ssi.dk). CSF, cerebrospinal fluid; ID, identification; MLST, multilocus sequence typing; –, no different bases found. †The allele numbers were 341 for gdh and 406 for spi.

For species identification, we performed phylogenetic analyses of whole-genome sequences (Appendix). Homologous core gene clustering showed that ASP0312-Sp and SP2752 belonged to the S. oralis clade (Figure); they were distant from one another, which was consistent with the MLST results.

Figure

Phylogenetic analysis of invasive Streptococcus oralis expressing serotype 3 pneumococcal capsule from 2 adult patients, Japan. Asterisks and orange shading indicate genomes from isolates ASP0312-Sp and SP2752 identified in this study. Homologous core gene clusters of 71 strains from 3 Streptococcus oralis subsp., 2 S. pneumoniae, 5 S. mitis, 5 S. infantis, and 3 S. pseudopneumoniae were downloaded from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov) and compared with the ASP0312-Sp and SP2752 genomes. Branch lengths represent the genetic distance. Scale bar indicates nucleotide substitutions per site.

To investigate recombination events, we compared the sequences surrounding the capsule loci of ASP0312-Sp and SP2752 with those of S. oralis subsp. tigurinus osk_001 and S. pneumoniae serotype 3 OXC141 (Appendix Figure). For ASP0312-Sp, the sequence corresponding to the downstream region of nsik up to the 5′ terminus of the gene encoding the cell wall binding repeat protein in osk_001 was replaced by a fragment of ≈30 kb from pneumococcus. For SP2752, the sequence encoding an ATPase up to the 5′ terminus of the gene encoding the cell wall binding repeat protein in osk_001 was replaced by a fragment of ≈16 kb from pneumococcus. The capsule sequences of ASP0312-Sp and SP2752 were 100% identical to the corresponding sequences located from 303730 to 312820 bp in HU-OH (GenBank accession no. AP018937.1), a serotype 3 pneumococcal strain that was isolated in Japan ().

We performed homology searches of 36 known pneumococcal virulence genes because multifragment recombination has been demonstrated during the capsular transformation process in pneumococcal populations (). In ASP0312-Sp and SP2752, the hyl gene, which encodes hyaluronate lyase (), was located distantly from the capsule locus and shared 96% identity with that of S. pneumoniae. We did not detect homologs of the other 35 genes for either isolate.

A recent study reported that acapsular pneumococcus became virulent after transformation with the capsule gene from SK95, which is an oral S. mitis strain (). This previous study demonstrated a cross-species transformation from a commensal streptococcal species to pneumococcus (). Our results complement this report, although the direction of transformation in our study was reversed. Our analyses of 2 human patients with invasive disease caused by S. oralis provided evidence of cross-species gene transfer from pneumococcus to a commensal streptococcal species. Acquisition of capsule and hyl genes might have increased pathogenicity (,) and contributed to progression of invasive disease in these 2 cases.

In conclusion, because of discrepancies between phenotypic and biochemical analyses, we used MLST and whole-genome sequencing to identify streptococcal species in these 2 patients. Our study indicates a potential pitfall for identifying and serotyping pneumococci that can occur if the bacteria are not isolated. Thus, when α-hemolytic streptococci are isolated from a sterile site, clinicians should request molecular analyses to identify the causative species, regardless of the mucoid phenotype.

Appendix

Additional information for invasive Streptococcus oralis expressing serotype 3 pneumococcal capsule, Japan.