The development of molecular methods and increasing knowledge of
S. pneumoniae virulence determinants together with a better understanding of genetic diversity of pneumococci and other Mitis streptococci led to proposing several targets for PCR and real-time PCR in order to improve species identification among Mitis streptococci, especially concerning their questionable representatives. As PCR-based approaches do not require cultured microorganisms, they find also an application in the direct detection of pneumococci in clinical materials [
55,
56], including polymicrobial samples [
57‐
60]. Thus, the right selection of appropriate target gene(s) is of great importance. Ideally, such a target should be exclusively and ubiquitously present among
S. pneumoniae and absent or sufficiently different in other Mitis streptococci to allow the clear discrimination of these groups.
A number of proposed genetic targets included
S. pneumoniae virulence-associated genes. The
ply gene, specifying the extracellular toxin pneumolysin, was one of the first DNA targets proposed to be used for the detection of pneumococci in clinical samples [
61,
62]. However, its utility for identification of
S. pneumoniae turned out to be limited [
63] due to the presence of the
ply gene also in other Mitis streptococci [
26,
64‐
66]. The
lytA gene, which encodes LytA, the major pneumococcal autolysin responsible for the solubilization of pneumococci in bile [
67] was another gene proposed for detection purposes [
68]. However, counterparts of
lytA were found in other Mitis streptococci [
64,
65]. Observed sequence differences between
lytA alleles specific for pneumococci and other Mitis streptococci, the presence of a characteristic 6-bp deletion close to the
lytA 3′ end in Mitis-specific variants of the gene in particular were the basis of a PCR-restriction fragment length polymorphism (PCR-RFLP) test [
69] and a real-time PCR targeting this region [
55]. While these assays are considered to have a very good species specificity for
S. pneumoniae [
38], variants typical for
S. pneumoniae in isolates from the Mitis group, as well as a Mitis-specific allele in pneumococcus, were occasionally observed [
24,
70]. The
psaA gene, encoding pneumococcal surface antigen A (PsaA), which constitutes a manganese ABC-type transporter is commonly found among
S. pneumoniae and was proposed as a target for PCR-based detection [
71], showing good performance on collections of
S. pneumoniae and other Mitis streptococci [
55,
72,
73]. However, also this gene turned out to be present among some isolates of Mitis streptococci [
26,
66,
74,
75]. Yet another proposed virulence-associated target, the
cpsA (
wzg) gene, which is typically the first gene in the
cps operon, responsible for the biosynthesis of capsular polysaccharide [
76], is absent among some non-serotypable pneumococci, as discussed above (see
Immunological methods). In addition, the
cpsA alleles of pneumococcal serotypes 25A, 25F and 38 are too divergent from typical pneumococcal
cpsA genes [
77] to yield positive results in this PCR, and such problems might also occur in the case of some novel serotypes. To additionally complicate identification issues, functional
cps loci, including the conserved genes
cpsABCD, were observed in a number of species of Mitis streptococci [
47‐
49], suggesting a low utility of the
cpsA target for discrimination between
S. pneumoniae and other Mitis group representatives. Moreover, the similarity of some
cps loci from the Mitis group streptococci to their counterparts in
S. pneumoniae challenges the use of multiplex PCR for direct detection of pneumococcal serotypes, e.g. in carriage specimens [
78]. Two genes,
piaA and
piaB encoding components of an ABC transport system essential for iron uptake, were described as specific for
S. pneumoniae but these genes are absent in some pneumococcal isolates, which limits the sensitivity of the test [
79‐
81]. Moreover,
piaA was detected in some
S. mitis and
S. pseudopneumoniae [
66] and recently
S. pseudopneumoniae isolates positive for
piaB have been reported [
70].
Certain other genes, such as housekeeping genes and genes of unknown function were also proposed as targets for
S. pneumoniae detection and identification. Sequence differences between variants of the
recA gene, occurring in
S. pneumoniae and other Mitis streptococci allowed designing a species-specific PCR [
82], which could discriminate also
S. pseudopneumoniae [
83]. Although sequencing of the 16S rRNA gene, considered as the “gold standard” for identification of many bacterial species, has a limited utility for Mitis streptococci due to high similarity of 16S rRNA genes in this group [
32], a cytosine at nucleotide position 203 in the 16S rRNA genes is considered specific for pneumococci, and sequence variants in this region of the gene can be distinguished using PCR-RFLP of the 16S rRNA genes [
84], although the presence of mixed bases at this position was found for some
S. pseudopneumoniae [
26]. On the basis of genomic subtractive hybridization two loci of unknown function, Spn9802 and Spn9828 were proposed to be specific for
S. pneumoniae [
85,
86]. Later studies demonstrated that a PCR targeting spn9802 apart from
S. pneumoniae also detects
S. pseudopneumoniae but not
S. mitis and
S. oralis and thus can be used to distinguish these two groups [
87]. Comparative genomic analyses of
S. pneumoniae and
S. pseudopneumoniae led to the identification of markers specific for these two species, respectively SPN0001/SP2020, encoding a GtnR-family transcriptional regulator, and SPS0002, specifying an osmosensitive potassium channel histidine kinase/response regulator KdpDE [
88]. The specificity of both markers was close to 100%, with SPN0001/SP2020 found in a few non-pneumococcal strains and SPS0002 occasionally present in
S. pneumoniae [
70,
88].
Currently, it appears that a combination of a few assays, such as PCR-RFLP or real-time PCR specific for the 6-bp deletion in the 3′ end of
lytA, together with detection of SPN0001/SP2020 [
70], possibly supplemented with analysis of the presence of
piaA/piaB could give more reliable results than a single-gene detection. Defining only one, “perfect” target, present among all
S. pneumoniae strains, sufficiently conserved to design universal PCR primers and at the same time not found (or at least in a very divergent form) among any other Mitis group streptococci may be very difficult to achieve, if possible at all. This situation is associated with the common genetic ancestry of the Mitis group, relatively frequent HGT among streptococcal species, facilitated by their natural competence, together with a gene loss, resulting in populations with highly diverse gene content [
89,
90]. In consequence, the core genome (genes common for each genome of a species) of
S. pneumoniae is much smaller than the pangenome (a complete set of genes of a species) and many of these genes are shared with other Mitis streptococci, including determinants of factors playing the role in interactions with the host allowing a successful survival [
91‐
94]. The recent genomic analyses of the Mitis group streptococci are discussed in more detail below (see
Genomic studies in the identification of Mitis streptococci).