Abstract
Three PCR techniques (amplification of the psaA, ply, and lytA genes) and a commercial kit (AccuProbe [GenProbe, San Diego, Calif.], based on hybridization with the 16S rRNA gene), all four of which claimed to be specific for Streptococcus pneumoniae, were used to identify 49 alpha-hemolytic streptococcal isolates suspected of being pneumococci. The definite phenotypic identification of these organisms as S. pneumoniae was difficult when optochin susceptibility and the presence of a capsule were taken as markers. Furthermore, RsaI digestion of the amplified 16S rRNA gene was applied. All 49 strains were optochin resistant. Eleven of these were encapsulated and were identified as pneumococci by all tests. Twenty of the 38 unencapsulated strains were unambiguously identified as nonpneumococci by all tests. The identities of another 18 unencapsulated strains remained inconclusive due to highly variable reactions for all phenotypic and genotypic techniques applied. The AccuProbe test was positive for seven strains for which the results of the other tests were inconclusive. RsaI restriction of the amplified 16S rRNA gene confirmed the AccuProbe result for all strains, while the result of the psaA-specific PCR was in concordance with encapsulation for all strains. The results presented here indicate that identification problems continue to exist for some strains, despite the application of genotypic and phenotypic tests in combination. We found the psaA-specific PCR to be the genotypic technique best suited for the identification of genuine pneumococci and optochin-resistant pneumococci.
Optochin susceptibility and encapsulation are the phenotypic characteristics that are the most frequently used to differentiate between Streptococcus pneumoniae and other streptococci (22). However, optochin-resistant S. pneumoniae strains are being isolated more frequently (2, 29) and are probably largely overlooked, since in many laboratories primary isolation of pneumococci on culture medium relies on optochin susceptibility itself. The occasional occurrence of encapsulated S. mitis and S. oralis strains and the fact that nontypeable, unencapsulated pneumococci have been reported to comprise 2% of the isolates from normally sterile sites (3) and up to 20% of the conjunctival isolates (12) further complicate the identification of pneumococci. Commercial systems like the API 20S and Vitek2 systems occasionally fail to identify pneumococcal isolates or identify other streptococci as pneumococci (4). Thus, even though the phenotypic identification of typical pneumococci is unambiguous, the existence of optochin-resistant isolates may increasingly cause problems in clinical bacteriological laboratories.
In the last decade, new gene amplification methods, based on the detection of pneumococcal virulence factors, have been developed to identify pneumococcal strains (18, 23, 24, 34) and to detect pneumococci directly from clinical samples (8, 13, 14, 19, 31-33, 35, 36). Hybridization methods (10, 30) have also been used for the identification of pneumococci. A commercial hybridization method (AccuProbe; GenProbe, San Diego, Calif.) that is based on the complementarity of a DNA probe with the 16S rRNA of S. pneumoniae is increasingly being used (6) in clinical laboratories.
Our aim was to evaluate recently described genotypic methods for their abilities to identify pneumococci. We tested the applicabilities of the commercial AccuProbe test and of enzymatic amplification of three different genes, those encoding pneumolysin (ply) (31), pneumococcal surface antigen A (psaA) (24), and autolysin (lytA) (23), for the identification of 49 optochin-resistant pneumococcus-like strains that were sent to the Reference Laboratory for Pneumococcus (KTL), National Public Health Institute, Oulu, Finland, during the last few years. In addition, RsaI restriction digestion of the amplified 16S rRNA gene (rDNA) (amplified rDNA restriction analysis [ARDRA]) was carried out (5, 16).
MATERIALS AND METHODS
Bacterial strains.
The following reference strains were used: S. pneumoniae LMG 14545T, LMG 15155, LMG 16738, GUH S91 01273, GUH H91 04493, GUH 93 08 1310, GUH 93 0 3230, GUH 93 09 1111, GUH 98 10 1630, GUH 98 10 3326, and GUH 98 10 3367; S. mitis LMG 14557T, LMG 14553, LMG 14552, GUH 94 03 0728, GUH 94 04 0401, GUH 97 03 2943, GUH 98 05 5898, GUH 98 07 1207, GUH 98 09 0066, KTL 101, KTL 102, and KTL 103; S. oralis LMG 14532T, LMG 14533, LMG 14534, GUH 94 08 5574, GUH 98 05 5050, and GUH 98 10 1512; and S. sanguinis LMG 14656 and LMG 14657. Table 1 lists the 49 optochin-resistant alpha-hemolytic streptococcal isolates included in this study. These strains were originally identified as pneumococci or suspected of being pneumococci on the basis of the results of the optochin susceptibility test and evaluation of their colony morphologies, performed in routine clinical laboratories. They were sent to the reference laboratory (KTL) for identification and serotyping because they were isolated from blood or cerebrospinal fluid or because they were not fully susceptible to penicillin. They were included in this study because they were found to be optochin resistant in the reference laboratory.
TABLE 1.
Phenotypic and genotypic identification results for 49 optochin-resistant alpha-hemolytic pneumococcus-like Streptococcus spp.
| Group and study isolate no. | Original isolate no. | AccuProbe result | Capsule formation | PCR result
|
ARDRA patterna | Bile solubility | ||
|---|---|---|---|---|---|---|---|---|
| psaA | lytA | ply | ||||||
| Group I | ||||||||
| STR119 | KTL 004 | + | + | + | + | + | p | + |
| STR120 | KTL 005 | + | + | + | + | + | ND | + |
| STR125 | KTL 013 | + | + | + | + | + | p | + |
| STR127 | KTL 017 | + | + | + | + | + | ND | + |
| STR141 | KTL 043 | + | + | + | + | + | p | + |
| STR144 | KTL 051 | + | + | + | + | + | ND | + |
| STR147 | KTL 056 | + | + | + | + | + | ND | + |
| STR149 | KTL 063 | + | + | + | + | + | p | + |
| STR155a | KTL 076 | + | + | + | + | + | ND | + |
| STR163 | KTL 091 | + | + | + | + | + | ND | + |
| STR164 | KTL 093 | + | + | + | + | + | ND | + |
| Group IIa | ||||||||
| STR157 | KTL 079 | + | − | − | wb | + | p | − |
| STR148b | KTL 062b | + | − | − | w | + | p | − |
| STR150 | KTL 065 | + | − | − | w | + | p | − |
| STR162 | KTL 089 | + | − | − | − | + | p | − |
| STR165 | KTL 096 | + | − | − | w | + | p | + |
| STR142b | KTL 046b | + | − | − | − | + | p | + |
| STR146b | KTL 055b | + | wc | − | − | − | p | − |
| Group IIb | ||||||||
| STR122 | KTL 007 | − | − | − | − | + | m | − |
| STR133 | KTL 028 | − | − | − | − | + | ND | − |
| STR126b | KTL 016b | − | − | − | w | + | m | − |
| STR129 | KTL 020 | − | − | − | w | + | m | − |
| STR130 | KTL 021 | − | − | − | − | + | ND | + |
| STR137 | KTL 035 | − | − | − | w | + | m | + |
| STR152 | KTL 069 | − | − | − | w | + | m | + |
| STR153 | KTL 072 | − | − | − | − | + | m | + |
| STR166b | KTL 099b | − | − | − | − | + | m | + |
| STR131 | KTL 022 | − | − | − | w | − | m | + |
| STR151 | KTL 068 | − | − | − | − | − | ND | + |
| Group IIc | ||||||||
| STR118 | KTL 003 | − | − | − | − | − | ND | − |
| STR121 | KTL 006 | − | − | − | − | − | ND | − |
| STR136 | KTL 034 | − | − | − | − | − | ND | − |
| STR138 | KTL 038 | − | − | − | − | − | ND | − |
| STR145 | KTL 054 | − | − | − | − | − | ND | − |
| STR154 | KTL 073 | − | − | − | − | − | ND | − |
| STR123 | KTL 008 | − | − | − | − | − | ND | − |
| STR128 | KTL 019 | − | − | − | − | − | m | − |
| STR132 | KTL 023 | − | − | − | − | − | ND | − |
| STR134 | KTL 029 | − | − | − | − | − | ND | − |
| STR139 | KTL 039 | − | − | − | − | − | m | − |
| STR158 | KTL 081 | − | − | − | − | − | ND | − |
| STR159 | KTL 083 | − | − | − | − | − | ND | − |
| STR156 | KTL 077 | − | − | − | − | − | m | − |
| STR117 | KTL 002 | − | − | − | − | − | ND | − |
| STR140 | KTL 041 | − | − | − | − | − | ND | − |
| STR160 | KTL 085 | − | − | − | − | − | ND | − |
| STR161 | KTL 087 | − | − | − | − | − | ND | − |
| STR135 | KTL 030 | − | w | − | − | − | m | − |
| STR143 | KTL 050 | − | w | − | − | − | m | − |
ARDRA, PCR-based amplification of the 16S rRNA-gene followed by RsaI digestion, agarose electrophoresis, and ethidium bromide staining. m, S. mitis-specific pattern, p, S. pneumoniae-specific pattern, ND, not determined.
Weak band after agarose gel electrophoresis and ethidium bromide staining.
Weak capsule reaction, with the strain considered unencapsulated.
Phenotypic assays.
Optochin resistance testing, the tube bile solubility test, and the capsular swelling test (the Quellung reaction) were performed as described by Kaijalainen et al. (17). Briefly, after four commercially available tests for optochin susceptibility were compared, it was decided to use the result obtained with the Biodisk (Biodisk PDM Diagnostic Disks, Solna, Sweden) as the result of record. Bile solubility was tested by the method described by Facklam and Washington (9). The Quellung reaction test was performed as described by Austrian (1).
AccuProbe test.
DNA hybridization with S. pneumoniae-specific 16S rRNA sequences was performed by using the AccuProbe S. pneumoniae culture identification test (GenProbe) according to the instructions of the manufacturer (17).
PCR assays.
Amplification of the pneumolysin (ply) gene fragment was performed as described by Rintamäki et al. (31). Briefly, one pneumococcal colony was suspended in 100 μl of the PCR mixture, and primers WO506 (5′-biotin-CCCACTCTTCTTGCGGTTGA) and WO507 (5′-TGAGCCGTTATTTTTTCATACTG) amplified a 209-bp region of the pneumolysin gene.
Amplification was done with the following PCR program: predenaturation for 10 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C; and postextension for 6 min at 72°C. The amplification products were identified by agarose gel electrophoresis with ethidium bromide staining and were confirmed by solution hybridization with a europium-labeled probe.
For amplification of the pneumococcal surface antigen A (psaA) gene and the autolysin (lytA) gene, DNA was extracted by alkaline lysis, which was carried out by suspending one colony in 20 μl of 0.25% sodium dodecyl sulfate-0.05 N NaOH and heating of the mixture at 95°C for 15 min, followed by a final dilution with 180 μl of distilled water.
Part of the psaA gene (838 bp) was amplified with 1.6 μM each primers psaA1 (5′-CTT TCT GCA ATC ATT CTT G) and psaA2 (5′-GCC TTC TTT ACC TTG TTC TGC), designed by Morrison et al. (24). Amplification was performed in a reaction mixture of 10 μl containing 5 μl of PCR Master Mix (Promega Benelux, Leiden, The Netherlands) and 1 μl of the DNA extract in a GeneAmp PCR System 9600 instrument (Applied Biosystems, Foster City, Calif.) by using the following cycling parameters: 95°C for 5 min, followed by 30 cycles of 30 s at 94°C, 30 s at 52°C, and 2 min at 72°C, with a final extension for 8 min at 72°C.
One micromolar each of primers lytAF (5′-ACG CAA TCT AGC AGA TGA AGC) and lytAR (5′-TGT TTG GTT GGT TAT TCG TGC), designed by McAvin et al. (23), were used to amplify a 101-bp fragment of the lytA gene. The cycling parameters for the lytA gene were 95°C for 5 min, followed by 35 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C, with a final extension at 72°C for 8 min.
The presence of psaA and lytA amplification products was checked by electrophoresis on 2% agarose gels stained with ethidium bromide.
ARDRA.
ARDRA, i.e., amplification of the 16S rRNA gene and subsequent restriction digestion with RsaI followed by agarose electrophoresis, was carried out as described previously (5, 16).
RESULTS
A set of three phenotypic and five genotypic identification techniques was carried out with a total of 78 Streptococcus mitis group strains, comprising 12 S. mitis, 6 S. oralis, and 11 encapsulated optochin-sensitive S. pneumoniae strains, and with 49 optochin-resistant streptococci of uncertain identities. A complete list of the optochin-resistant strains and their reactions are given in Table 1.
The phenotypic identification techniques used were optochin susceptibility, bile solubility, and encapsulation. The genotypic identification methods used were amplification of the pneumolysin gene (ply), the autolysin gene (lytA), and the pneumococcal surface antigen A gene (psaA); hybridization with 16S rRNA (the AccuProbe test); and RsaI restriction of the amplified 16S rRNA gene (ARDRA).
For the group of phenotypically well-identified S. mitis, S. oralis, and S. pneumoniae strains, the results of all phenotypic and genotypic techniques were in agreement with published data. S. mitis and S. oralis were optochin resistant, unencapsulated, bile insoluble, and negative by the AccuProbe test; gave no amplification with ply-, lytA-, and psaA-specific primers; and revealed the mitis group-specific RsaI ARDRA pattern (restriction fragments of 114, 146, 262, 355, and 625 bp). The S. pneumoniae strains, however, had opposite characteristics and a different RsaI restriction pattern, composed of fragments of 114, 146, 335, and 887 bp. The latter pattern was also observed for the two S. sanguinis isolates tested.
For a better understanding, the 49 optochin-resistant pneumococcus-like strains were split into a group consisting of the 11 encapsulated strains (group I) and a group consisting of the remaining 38 strains, of which 35 were nonencapsulated and 3 had a weak Quellung reaction (group II). The group II strains were further subdivided into groups IIa, IIb, and IIc, according to their reactions by the different genotypic tests.
For the group I isolates (optochin resistant and encapsulated), the results obtained by all other techniques were positive, pointing to an unequivocal identification as S. pneumoniae.
The seven optochin-resistant, nonencapsulated isolates of group IIa were positive by the AccuProbe test and had an ARDRA pattern typical for S. pneumoniae (ARDRA positive), although five isolates were negative by all three phenotypic assays and two were only bile soluble. Six of the seven strains were also positive for ply, and four of seven of the isolates had a weakly positive lytA amplification signal.
The 11 optochin-resistant, nonencapsulated isolates in group IIb were positive only by the ply-specific PCR and/or bile positive. Four isolates also showed a weak positive reaction by the lytA-specific PCR.
For the 20 optochin-resistant, nonencapsulated isolates of group IIc, all techniques pointed to an identification as nonpneumococci.
In conclusion, all optochin-resistant and encapsulated isolates (group I) were identified as pneumococci by all other phenotypic and genotypic techniques. Twenty of the 38 optochin-resistant, unencapsulated isolates (group IIc) were identified as nonpneumococci by all techniques, but 18 of the 38 optochin-resistant, nonencapsulated isolates (groups IIa and IIb) could not be unequivocally identified, due to the contradicting results of the phenotypic and genotypic tests.
The results of RsaI restriction of the amplified 16S rDNA were always in agreement with the results obtained with the AccuProbe system, and the psaA-specific PCR result was always in agreement with encapsulation.
DISCUSSION
Susceptibility to optochin is a cornerstone in the phenotypic identification of pneumococci and is often used in laboratories as the primary and sometimes the only identification method (21). The occurrence of optochin-resistant S. pneumoniae strains, which can result from mutations in the H+-ATPase (11), is not uncommon (2, 10, 21, 25, 26, 28), jeopardizing the detection and correct identification of pneumococci. Other problems with optochin susceptibility testing may be caused by alpha-hemolytic streptococci and aerococci, which show small zones of inhibition around optochin disks (23). Besides optochin susceptibility testing, several problems exist with other phenotypic tests for the identification of pneumococci (1, 7, 10, 23, 25, 27). An extensive comparison of the available biochemical and serological methods was published recently, and that analysis pointed to the high degrees of sensitivity but limited specificities of most of the phenotypic tests (20).
The capsular reaction is sometimes difficult to interpret, as was the case for two AccuProbe test-negative strains and one AccuProbe test-positive strain included in the present study. To simplify the discussion, these three strains were considered unencapsulated, and therefore, in this study only strong capsular swelling was considered indicative of an identification as S. pneumoniae.
This study was set up to evaluate which of some of the recently described genotypic techniques for the identification of pneumococci are best suited for the unambiguous identification of a series of 49 strains that were sent to KTL in recent years for serotyping or because they were “possibly pneumococcal.”
The results of this study can be summarized as follows. The group I and group IIc isolates posed no identification problems. Although the 11 group I isolates are optochin resistant, they were readily recognized as pneumococci by the presence of a capsule, by AccuProbe assay positivity, and by a positive psaA-specific PCR result. Group IIc isolates were readily identified as nonpneumococcal alpha-hemolytic streptococci since they were negative by all phenotypic and genotypic methods applied in this study.
Taking into account all of the tests carried out, one is inclined to consider the group IIa and IIb isolates to be nonpneumococcal alpha-hemolytic streptococci which, however, show one or more characteristics of pneumococci. Most problematic were the positive results by the AccuProbe assay (confirmed by ARDRA) for the seven group IIa isolates. Group IIa isolates showed several other reactions characteristic of those of pneumococci, like positivity by the ply- and lytA-specific PCRs and bile solubility, but all were unencapsulated and psaA-specific PCR negative. The isolates in group IIb were only ply-specific PCR positive and/or bile soluble, and four group IIb isolates also showed a weak positive reaction by the lytA-specific PCR.
This prompted us to consider the specificity of the AccuProbe test, which has been used as a “gold standard” for the identification of pneumococci in several studies (6, 37).
Comparison of all 16S rRNA sequences of the S. mitis group species showed that S. pneumoniae and S. sanguinis lack a RsaI restriction site (GTAC) at Escherichia coli position 635, which is present in S. mitis, S. oralis, and S. parasanguinis. S. sanguinis is a species for which RsaI digestion of the 16S rDNA results in a pattern that is similar to that for S. pneumoniae but that is due to another point mutation. At E. coli position 635, the sequences are GTAG in S. pneumoniae and GTAT in S. sanguinis. We found complete concordance between the results of the AccuProbe test and ARDRA, probably because the AccuProbe test targets the same region as ARDRA with RsaI digestion. This points to the possibility that digestion of amplified 16S rDNA with RsaI could be a valuable and cheaper alternative for differentiation between pneumococci and nonpneumococcal streptococci, although the false-positive results for S. sanguinis isolates must be taken into account.
Although the AccuProbe test and RsaI digestion of 16S rDNA appear to enable identification of all genuine S. pneumoniae isolates, the possibility that closely related nonpneumococcal S. mitis strains have (nearly) identical 16S rDNAs, resulting in false-positive AccuProbe test and ARDRA results, cannot be excluded. On the basis of all data gathered in the present study and in the study of Whatmore et al. (37), we believe that not only the AccuProbe test-negative group IIb strains but also the AccuProbe test-positive group IIa strains are indeed not pneumococci. Mundy et al. (25) also reported that of 115 atypical pneumococci with inconsistent combinations of optochin susceptibilities, tube bile solubilities, and Quellung reactions, 33 reacted by the AccuProbe assay. Of these 33 isolates, only 3 showed a capsule that reacted in the Quellung test. All group IIa strains were isolated from the upper respiratory tract and not from patients with invasive disease. Whatmore et al. (37) conclude, “Our results indicated that many of the isolates identified as pneumococci by AccuProbe could actually represent organisms which are genetically rather divergent from typical pneumococci.”
The probable close evolutionary relationship between genuine and atypical pneumococci and the sporadic horizontal transfer of some pneumococcal genes to their closest relatives may explain the existence of nonpneumococcal S. mitis cluster strains with a pneumococcal 16S rDNA sequence and with sporadic pneumococcal genes, and this may be an explanation for the identification problems posed by such atypical pneumococci.
Although we tend to consider most of the atypical pneumococcus isolates of group IIa to be nonpneumococcal isolates, despite a positive AccuProbe test result, it may be prudent to treat these isolates as pneumococcal isolates anyway, since they can harbor some pneumococcal virulence genes. In this study, the presence of several pneumococcal virulence genes in the AccuProbe test-positive nonpneumococcal S. mitis cluster strains was obvious, and Whatmore et al. (37) also described the existence of pathogenic S. mitis strains harboring the virulence determinants pneumolysin and autolysin, which are classically associated with S. pneumoniae. Fortunately, the presence of one or another pneumococcal virulence gene seems to coincide in many cases with a positive AccuProbe test result; therefore, the AccuProbe assay could be continued to be used as the gold standard for the detection of pneumococcal virulence genes.
Although Jado et al. (15) reported that the psaA gene could be amplified from isolates of S. mitis, S. oralis, and S. anginosus with their primers, the primers described by Morrison et al. (24), which enabled amplification of the psaA gene from all 90 serotypes of S. pneumoniae and which were used in this study, appear to be very specific for S. pneumoniae. The psaA gene was amplified with the primers of Morrison et al. (24) only from genuine pneumococci and the optochin-resistant but encapsulated pneumococci of group I. This positivity by the psaA-specific PCR always corresponded to encapsulation.
In conclusion, the results presented here indicate that identification problems continue to exist for some strains, despite the application of combined genotypic and phenotypic tests. On the basis of our observations and those of other researchers, we propose that the following recommendations be put into practice in our laboratories and be considered by other researchers. The optochin sensitivity test, when performed carefully, is the standard method for the identification of pneumococci. An optochin-sensitive alpha-hemolytic Streptococcus is almost always a pneumococcus. For optochin-resistant isolates, the presence or absence of a capsule should be verified by the Quellung reaction or the counterimmunoelectrophoresis method, if, on the basis of the origin (e.g., blood or cerebrospinal fluid) or the colony morphology (e.g., a slimy colony) of the isolate, there is a good reason to suspect the presence of pneumococci. An optochin-resistant, encapsulated bacterium is then identified as a pneumococcus. Since we found in this study that a positive result by the psaA-specific PCR with the primers described by Morrison et al. (24) is in perfect correspondence with encapsulation, this genotypic technique is best suited for the identification of genuine pneumococci and can possibly replace both the optochin sensitivity test and the Quellung reaction. The AccuProbe test was considered to be somewhat less specific than the psaA-specific PCR, but the AccuProbe test result largely coincided with the presence of pneumococcal virulence genes in nonpneumococci, and it therefore retains its value. Digestion of amplified 16S rDNA with RsaI (ARDRA) is a cheap and valuable alternative to the AccuProbe test. Amplification of the pneumolysin gene (ply-specific PCR) and the autolysin gene (lytA-specific PCR) is somewhat less specific than the AccuProbe test and probably leads to overestimation of the number of pneumococcal infections. Even though genotypic methods provide new possibilities for the identification of pneumococci, it is obvious that due to the presence of pneumococcal virulence genes in nonpneumococcal S. mitis isolates, it is not always easy to draw a clear line between pneumococcal and nonpneumococcal isolates.
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