Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization

Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from "pneumococcus-like" oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S. pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S. mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S. pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S. pneumoniae clinical isolates and from 20 atypical organisms classified as S. mitis or S. oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S. pneumoniae infections.     

recA-based PCR assay for accurate differentiation of Streptococcus pneumoniae from other viridans streptococci

Proper identification of Streptococcus pneumoniae by conventional methods remains problematic. The discriminatory power of the 16S rRNA gene, which can be considered the "gold standard" for molecular identification, is too low to differentiate S. pneumoniae from closely related species such as Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis in the routine clinical laboratory. A 313-bp part of recA was selected on the basis of variability within the S. mitis group, showing <95.8% interspecies homology. In addition, 6 signature nucleotides specific for S. pneumoniae were identified within the 313-bp recA fragment. We show that recA analysis is a useful tool for proper identification to species level within the S. mitis group, in particular, for pneumococci.     

Molecular characterization of disease-associated streptococci of the mitis group that are optochin susceptible

Eight optochin-susceptible (Opt(s)) alpha-hemolytic (viridans) streptococcus isolates were characterized at the molecular level. These isolates showed phenotypic characteristics typical of both viridans streptococci and Streptococcus pneumoniae. Comparison of the sequence of housekeeping genes from these isolates with those of S. pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae suggested that the Opt(s) isolates corresponded to streptococci of the mitis group. Besides, the Opt(s) streptococci were negative by a Gen-Probe AccuProbe pneumococcus test and hybridized with specific pneumococcal probes (lytA and ply) but also with ant, a gene not present in most S. pneumoniae strains. Moreover, the isolates were insoluble in 1% sodium deoxycholate but completely dissolved in 0.1% deoxycholate. Sequence analysis of the lytA gene revealed that the Opt(s) streptococci carried lytA alleles characteristic of those present in nonpneumococcal streptococci of the mitis group. The determination of the partial nucleotide sequence embracing the atp operon encoding the F(o)F(1) H(+)-ATPase indicated that the optochin susceptibility of the isolates was due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae by horizontal gene transfer.     

Characteristic signatures of the lytA gene provide a basis for rapid and reliable diagnosis of Streptococcus pneumoniae infections

The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two Streptococcus pseudopneumoniae strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the Streptococcus pneumoniae strains harbored typical lytA alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical lytA alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the lytA gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true S. pneumoniae strains and represents an improved diagnostic tool for the study of pneumococcal carriage.     

Identification of the psaA gene, coding for pneumococcal surface adhesin A, in viridans group streptococci other than Streptococcus pneumoniae

The gene encoding the pneumococcal surface adhesin A (PsaA) protein has been identified in three different viridans group streptococcal species. Comparative studies of the psaA gene identified in different pneumococcal isolates by sequencing PCR products showed a high degree of conservation among these strains. PsaA is encoded by an open reading frame of 930 bp. The analysis of this fragment in Streptococcus mitis, Streptococcus oralis, and Streptococcus anginosus strains revealed a sequence identity of 95, 94, and 90%, respectively, to the corresponding open reading frame of the previously reported Streptococcus pneumoniae serotype 6B strain. Our results confirm that psaA is present and detectable in heterologous bacterial species. The possible implications of these results for the suitability and potential use of PsaA in the identification and diagnosis of pneumococcal diseases are discussed.     

Differentiation of Streptococcus pneumoniae from Nonpneumococcal Streptococci of the Streptococcus mitis Group by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

The differentiation of species within the Streptococcus mitis group has posed a problem in the routine diagnostic microbiology laboratory for some time. It also constitutes a major weakness of recently introduced matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) fingerprinting systems. As the phylogenetic resolution of the spectral similarity measures employed by these systems is insufficient to reliably distinguish between the most closely related members of the group, the major pathogen Streptococcus pneumoniae is frequently misidentified. In this study, a comparative analysis of MALDI-TOF spectra of several species from the S. mitis group has been performed in order to identify single peaks that could be used to improve mass spectrometry-based identification of the respective species. A characteristic peak profile could be identified that unambiguously distinguished the 14 S. pneumoniae isolates studied from 33 nonpneumococcal isolates of the S. mitis group. In addition, specific peak combinations could be assigned to other members of the group. The findings of this study suggest that it is possible to distinguish different species of the S. mitis group by close analysis of their mass peak profiles.     

Biosynthesis of teichoic acids in Streptococcus pneumoniae and closely related species: lessons from genomes

The cell wall of Streptococcus pneumoniae contains an unusually complex wall teichoic acid (WTA), which has identical repeating units as the membrane-anchored lipoteichoic acid (LTA). Both polymers share a common cytoplasmic pathway of precursor synthesis, but several TA enzymes have remained elusive. Bioinformatic analysis of the genome of various pneumococcal strains, including choline-independent mutant strains, has allowed us to identify the missing TA genes. We present here the deduced complete pathways of WTA and LTA synthesis in S. pneumoniae and point to the variations occurring in different pneumococcal strains and in closely related species such as Streptococcus oralis and Streptococcus mitis.     

Detection of Streptococcus pneumoniae DNA in blood cultures by PCR.

We have developed a PCR assay, with primers derived from the autolysin (lyt) gene, for the detection of Streptococcus pneumoniae DNA in blood cultures. The predicted fragment of 247 bp was detected in all strains of pneumococci, embracing 12 different serotypes that were tested. Although DNA extracted from four viridans streptococci spp. Streptococcus oralis, Streptococcus mitis, Streptococcus sanguis, and Streptococcus parasanguis) gave amplification products, these were quite different from the predicted fragment for S. pneumoniae. Application of the assay for diagnosis of septicemia caused by S. pneumoniae showed concordance between PCR and culture results. However, on four occasions PCR was positive in supernatants from both paired culture bottles while pneumococci were cultured from only one. Performing PCR on negative cultures in controlled studies such as vaccine trials may provide a sensitive tool for increasing case detection.     

Crossing the barrier: evolution and spread of a major class of mosaic pbp2x in Streptococcus pneumoniae, S. mitis and S. oralis

A paradigm for Streptococcus interspecies gene transfer is represented by the mosaic pbp genes encoding the target enzymes for beta-lactam antibiotics, the penicillin-binding proteins, in Streptococcus pneumoniae. We investigated a collection of oral streptococci from three continents by comprehensive multi-locus sequence typing analysis in order to trace the origin of a mosaic block belonging to a dominant family of mosaic pbp2x implicated in penicillin resistance of S. pneumoniae. One widespread family of mosaic pbp2x occurred in all three distinct clusters of S. pneumoniae, Streptococcus mitis and Streptococcus oralis, documenting independent inter- and intraspecies recombination events. Moreover, potential ancestor genes of this mosaic block could be identified in two penicillin-susceptible S. mitis strains from South Africa and Spain, facilitating the identification of pbp2x mutations relevant for resistance development.     

Molecular peculiarities of the lytA gene isolated from clinical pneumococcal strains that are bile insoluble

The autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical Streptococcus pneumoniae) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent lytA alleles (pairwise evolutionary distances of about 20%) compared to the lytA alleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to S. pneumoniae. The clinical importance of these findings is underlined by the role of the lytA gene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the Streptococcus mitis group.     

Characterization of nontypeable and atypical Streptococcus pneumoniae pediatric isolates from 1994 to 2010

Streptococcus pneumoniae is a major cause of bacteremia, meningitis, pneumonia, sinusitis, and acute otitis media in children. Although optochin susceptibility, bile solubility, and Quellung testing are the standards for identifying and differentiating pneumococci, there are several reports of nontypeable pneumococci that give inconsistent results with one or more of these tests. We characterized 52 isolates previously labeled as nontypeable pneumococci. Microbiological methods included repeating the Quellung reaction using a new and expanded group of antisera, optochin susceptibility and bile solubility tests, and automated Vitek 2 identification. Molecular methods included PCR detection of ply and psaA genes, multilocus sequence typing (MLST), 16S rRNA gene sequencing, and pyrosequencing. Of the 52 isolates, 38 (73%) were optochin susceptible, were psaA and ply positive, and could be serotyped by the Quellung reaction. The remaining 14 isolates, isolated from patients with otitis media (n = 6), bacteremia (n = 6), meningitis (n = 1), and pneumonia (n = 1), underwent further analysis. Three of these 14 isolates were nontypeable due to autoagglutination but were pneumococci by all tests and represented pneumococcal sequence types previously recognized by MLST. The 11 remaining isolates were optochin resistant, and 6 of these were bile soluble. Three of 11 were both psaA and ply positive and clustered with pneumococci by MLST (2 were bile soluble); 8 lacked psaA (5 ply positive, 4 bile soluble) and likely belonged to other Streptococcus species. In conclusion, few isolates were truly nontypeable by Quellung reaction, and MLST and the presence of psaA proved useful in distinguishing between atypical pneumococci and other streptococcal species.     

Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae

It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulence-factor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.     

Streptococcus tigurinus, a Novel Member of the Streptococcus mitis Group, Causes Invasive Infections

We recently described the novel species Streptococcus tigurinus sp. nov. belonging to the Streptococcus mitis group. The type strain AZ_3a(T) of S. tigurinus was originally isolated from a patient with infective endocarditis. According to its phenotypic and molecular characteristics, S. tigurinus is most closely related to Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus oralis, and Streptococcus infantis. Accurate identification of S. tigurinus is facilitated by 16S rRNA gene analysis. We retrospectively analyzed our 16S rRNA gene molecular database, which contains sequences of all clinical samples obtained in our institute since 2003. We detected 17 16S rRNA gene sequences which were assigned to S. tigurinus, including sequences from the 3 S. tigurinus strains described previously. S. tigurinus originated from normally sterile body sites, such as blood, cerebrospinal fluid, or heart valves, of 14 patients and was initially detected by culture or broad-range 16S rRNA gene PCR, followed by sequencing. The 14 patients had serious invasive infections, i.e., infective endocarditis (n = 6), spondylodiscitis (n = 3), bacteremia (n = 2), meningitis (n = 1), prosthetic joint infection (n = 1), and thoracic empyema (n = 1). To evaluate the presence of Streptococcus tigurinus in the endogenous oral microbial flora, we screened saliva specimens of 31 volunteers. After selective growth, alpha-hemolytic growing colonies were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and subsequent molecular methods. S. tigurinus was not identified among 608 strains analyzed. These data indicate that S. tigurinus is not widely distributed in the oral cavity. In conclusion, S. tigurinus is a novel agent of invasive infections, particularly infective endocarditis.     

Evaluation of several biochemical and molecular techniques for identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and their detection in respiratory samples

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae, 2 S. pseudopneumoniae, and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO(2) atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O(2) atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated.     

Genetic relatedness,antibiotic susceptibility,and serotype distribution of Streptococcus pneumoniae responsible for meningitis in Poland, 1997-2001

Streptococcus pneumoniae is the third most frequent etiologic agent of bacterial meningitis in Poland. Therefore, the objective of this study was to characterize S. pneumoniae isolates responsible for meningitis in Poland from 1997 to 2001 by determining their serotypes, antimicrobial susceptibility, and genetic relatedness. Among 36 serotypes and rough isolates, the most common were serotypes 3, 8, 19F, 6B, 4, and 23F, altogether accounting for 45.3% of cases. Penicillin-nonsusceptible pneumococci constituted 14.8% of all isolates. A high heterogeneity of pneumococcal isolates concerning serological features as well as genetic relatedness based on pulsed-field gel electrophoresis analysis was observed. With the exception of some serotypes (e.g., 3, 7F, 19F), isolates of the same serotype do not appear to be more closely related to each other than to isolates of different serotypes.     

Novel molecular method for identification of Streptococcus pneumoniae applicable to clinical microbiology and 16S rRNA sequence-based microbiome studies

The close phylogenetic relationship of the important pathogen Streptococcus pneumoniae and several species of commensal streptococci, particularly Streptococcus mitis and Streptococcus pseudopneumoniae, and the recently demonstrated sharing of genes and phenotypic traits previously considered specific for S. pneumoniae hamper the exact identification of S. pneumoniae. Based on sequence analysis of 16S rRNA genes of a collection of 634 streptococcal strains, identified by multilocus sequence analysis, we detected a cytosine at position 203 present in all 440 strains of S. pneumoniae but replaced by an adenosine residue in all strains representing other species of mitis group streptococci. The S. pneumoniae-specific sequence signature could be demonstrated by sequence analysis or indirectly by restriction endonuclease digestion of a PCR amplicon covering the site. The S. pneumoniae-specific signature offers an inexpensive means for validation of the identity of clinical isolates and should be used as an integrated marker in the annotation procedure employed in 16S rRNA-based molecular studies of complex human microbiotas. This may avoid frequent misidentifications such as those we demonstrate to have occurred in previous reports and in reference sequence databases.     

A successful, diverse disease-associated lineage of nontypeable pneumococci that has lost the capsular biosynthesis locus

Streptococcus pneumoniae strains which fail to produce a polysaccharide capsule are commonly isolated from carriage and disease contexts. Here we use a multilocus approach to distinguish genuine nontypeable pneumococci from closely related nontypeable streptococcal isolates in a data set of 121 untypeable pneumococci from nasopharyngeal swabs and middle ear fluid of Finnish children and demonstrate that 70 of these belong to a pneumococcal lineage which has lost its capsular locus. Strains of this relatively old lineage include sequence types 344, 448, and 449. Comparison with the multilocus sequence typing database shows that strains of this lineage have spread intercontinentally and have been isolated from carriage, mucosal, and invasive disease. Furthermore we note a particular association of this nontypeable lineage with outbreaks of conjunctivitis. The diversification and geographic spread of this lineage suggest that loss of capsule is not inconsistent with long-term persistence and raise questions about the capsule's role in pneumococcal transmission.     

Streptococcus pseudopneumoniae identification by pherotype: a method to assist understanding of a potentially emerging or overlooked pathogen

The recent identification of Streptococcus pseudopneumoniae (pseudopneumococcus) has complicated classification schemes within members of the "mitis" streptococcal group. Accurate differentiation of this species is necessary for understanding its disease potential and identification in clinical settings. This work described the use of the competence-stimulatory peptide ComC sequence for identification of S. pseudopneumoniae. ComC sequences from clinical sources were determined for 17 strains of S. pseudopneumoniae, Streptococcus pneumoniae, and Streptococcus oralis. An additional 58 ComC sequences from a range of sources were included to understand the diversity and suitability of this protein as a diagnostic marker for species identification. We identified three pherotypes for this species, delineated CSP6.1 (10/14, 79%), CSP6.3 (3/14, 21%), and SK674 (1/14, 7%). Pseudopneumococcal ComC sequences formed a discrete cluster within those of other oral streptococci. This suggests that the comC sequence could be used to identify S. pseudopneumoniae, thus simplifying the study of the pathogenic potential of this organism. To avoid confusion between pneumococcal and pseudopneumococcal pherotypes, we have renamed the competence pherotype CSP6.1, formerly reported as an "atypical" pneumococcus, CSPps1 to reflect its occurrence in S. pseudopneumoniae.     

Accuracy of phenotypic and genotypic testing for identification of Streptococcus pneumoniae and description of Streptococcus pseudopneumoniae sp. nov

We have identified an unusual group of viridans group streptococci that resemble Streptococcus pneumoniae. DNA-DNA homology studies suggested that a subset of these isolates represent a novel species that may be included in the S. oralis-S. mitis group of viridans group streptococci. We suggest that this novel species be termed Streptococcus pseudopneumoniae. A combination of phenotypic and genetic reactions allows its identification. S. pseudopneumoniae strains do not have pneumococcal capsules, are resistant to optochin (inhibition zones, less than 14 mm) when they are incubated under an atmosphere of increased CO2 but are susceptible to optochin (inhibition zones, >14 mm) when they are incubated in ambient atmospheres, are not soluble in bile, and are positive by the GenProbe AccuProbe Pneumococcus test. The bile solubility test is more specific than the optochin test for identification of S. pneumoniae. Genetic tests for pneumolysin (ply) and manganese-dependent superoxide dismutase (sodA) and identification tests with a commercial probe, AccuProbe Pneumococcus, do not discriminate between the new species and S. pneumoniae.     

Protein serotyping of Streptococcus pneumoniae based on reactivity to six monoclonal antibodies

Six monoclonal antibodies to proteins of Streptococcus pneumoniae were tested in a dot blot assay for reactivity with 499 clinical isolates of pneumococci. Forty-four percent of the isolates reacted with at least one of the antibodies. Nineteen patterns of reactivity were identified and each designated as a provisional protein serotype. Protein serotyping identified pneumococcal strains independently of their capsular type and made it possible to differentiate strains within most capsular types. A protein serotyping system provides a new dimension to the phenotypic identification of S. pneumoniae and may eventually provide a basis for assessing the population structure of these organisms.