Detection of Large Numbers of Pneumococcal Virulence Genes in Streptococci of the Mitis Group

Abstract

Seven streptococcal isolates from the mitis group were analyzed for the presence of pneumococcal gene homologues by comparative genomic hybridization studies with microarrays based on open reading frames from the genomes of Streptococcus pneumoniae TIGR4 and R6. The diversity of pneumolysin (ply) and neuraminidase A (nanA) gene sequences was explored in more detail in a collection of 14 S. pseudopneumoniae and 29 mitis group isolates, respectively. The mitis group isolates used in the microarray experiments included a type strain (NCTC 12261), two S. mitis isolates from the nasopharynxes of children, one S. mitis isolate from a case of infective endocarditis, one S. mitis isolate from a dental abscess, and one S. oralis isolate and one S. pseudopneumoniae isolate from the nasopharynxes of children. The results of the microarray study showed that the 5 S. mitis isolates had homologues to between 67 and 82% of pneumococcal virulence genes, S. oralis hybridized to 83% of pneumococcal virulence genes, and S. pseudopneumoniae hybridized to 92% of identified pneumococcal virulence genes. Comparison of the pneumolysin, mitilysin (mly), and newly identified pseudopneumolysin (pply) gene sequences revealed that mly and pply genes are more closely related to each other than either is to ply. In contrast, the nanA gene sequences in the pneumococcus and streptococci from the mitis group are closely clustered together, sharing 99.4 to 99.7% sequence identity with pneumococcal nanA alleles.Streptococcus pneumoniae, also known as the pneumococcus, is an important human pathogen causing high levels of mortality worldwide. Diseases caused include meningitis, pneumonia, and otitis media. S. pneumoniae is a member of the viridans group streptococci (VGS) and is closely related to Streptococcus mitis and Streptococcus oralis, sharing Ͼ99% homology by 16S rRNA gene analysis (13,23). Recently, a subset of "atypical" pneumococci were identified that were distinct from both S. mitis and S. pneumoniae by housekeeping gene analysis (48). It is likely that these isolates belonged to the species Streptococcus pseudopneumoniae, which has since been identified, with isolates appearing phenotypically similar to S. mitis but undistinguishable from S. pneumoniae in genotypic tests (2). As a result of the close relationships between these species, clear identification can be problematic. A recent study showed that definitive species identification can be achieved by phylogenetic analysis of 4 housekeeping genes. Furthermore, although it has been reported that comparison of superoxide dismutase genes (sodA) was sufficient to identify most species, this did not distinguish between S. pneumoniae and S. pseudopneumoniae isolates (19).Despite the close genetic relationships between these species, while S. pneumoniae can be a severe pathogen, S. oralis and S. mitis are usually commensal organisms of mucous membranes. However, on rare occasions, they are able to cause opportunistic disease in immunocompromised patients, particularly those with replacem...

Novel Molecular Method for Identification of Streptococcus pneumoniae Applicable to Clinical Microbiology and 16S rRNA Sequence-Based Microbiome Studies

“…E xact differentiation of the important pathogen Streptococcus pneumoniae and its close commensal relatives is a prerequisite for full appreciation of their clinical significance, pathogenic properties, and epidemiology both in routine clinical microbiology and in research. Although a number of phenotypic traits were previously assumed to be specific markers of S. pneumoniae, recent reports demonstrated that such properties, including optochin susceptibility, capsule production, and bile solubility, are also present in a substantial proportion of isolates of the commensal Streptococcus mitis (2,3,6,19,23,40). Moreover, the realization that clinical isolates of S. pneumoniae, e.g., many isolates from conjunctivitis, do not express a capsule (hence the term "atypical pneumococci") further emphasizes this problem (4, 7).…”

“…This is becoming an increasing problem due to the frequent and automated use of this procedure in clinical microbiology and in the annotation process employed in attempts to map complex microbiotas, such as in the Human Microbiome Project. Likewise, the suggested identification of clinical isolates of S. pneumoniae by detection of genes encoding regulatory proteins of capsule biosynthesis, e.g., cpsA/wzg, or putative virulence proteins such as autolysin (lytA), pneumolysin (ply), the surface protein PspA (pspA) (1, 30) is bound to result in a number of misidentifications due to the presence of homologs of these genes in a proportion of commensal streptococci (19,20,23,27,38,40).The biological explanation for the described problems is the phylogenetic history of these streptococcal species. In spite of striking differences in pathogenic potential, S. pneumoniae and S. mitis, S. pseudopneumoniae, Streptococcus oralis, Streptococcus infantis, and Streptococcus oligofermentans share an immediate common ancestor.…”

Streptococcus pseudopneumoniae Identification by Pherotype: a Method To Assist Understanding of a Potentially Emerging or Overlooked Pathogen

“…Virulence factors that were once thought to be exclusive to the pneumococcus, such as pneumolysin (encoded by ply) and autolysin A (encoded by lytA), have been detected in commensal streptococcal species (18,35,49), compromising their specificity as species identification markers. The pathogenic potential of S. pseudopneumoniae (the pseudopneumococcus) has been demonstrated in a murine model (12) as well as in humans (2,18,23,24,28,40). Rapid, correct identification of this organism in the clinical setting is essential for diagnosis and for understanding its disease potential.…”

“…Optochin sensitivity and bile solubility, the two standard pneumococcal phenotypic identification tests, have proven to be inconclusive for differentiating pneumococci from these atypical strains (3,4,9,10,17,19,20,22,27,29,32,34,38,39,49). Virulence factors that were once thought to be exclusive to the pneumococcus, such as pneumolysin (encoded by ply) and autolysin A (encoded by lytA), have been detected in commensal streptococcal species (18,35,49), compromising their specificity as species identification markers. The pathogenic potential of S. pseudopneumoniae (the pseudopneumococcus) has been demonstrated in a murine model (12) as well as in humans (2,18,23,24,28,40).…”

Direct Detection and Prediction of All Pneumococcal Serogroups by Target Enrichment-Based Next-Generation Sequencing

“…Viridans group streptococci have been found to harbor a large number of genes originally identified as pneumococcal genes (20,21). Our assay incorporated a specific pair of primers that amplified a signature of the streptococcal autolysin gene that differentiated S. pneumoniae from nonpneumococcal isolates.…”