Evaluation of Several Biochemical and Molecular Techniques for Identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and Their Detection in Respiratory Samples

Abstract

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. S treptococcus pneumoniae is a major cause of community-acquired pneumonia (CAP). There is no true gold standard for S. pneumoniae identification, but the bile solubility test has been shown to have a high level of accuracy and is frequently used for the identification of S. pneumoniae (18). Since this test is relatively time-consuming and sometimes difficult to interpret, several other conventional biochemical and phenotypic tests, like Gram stain morphology, optochin susceptibility, colony morphology, and alpha-hemolysis on sheep blood agar, are currently applied as part of routine procedures in the clinical microbiology laboratory. S. pneumoniae belongs to the mitis group of streptococci, and several molecular assays have been applied in an attempt to discriminate S. pneumoniae from other mitis group streptococci. A variety of genes has been used as target for a (real-time) PCR assay to detect S. pneumoniae, including genes encoding the virulence factors autolysin (lytA) and pneumolysin (ply) and the DNA fragment Spn9802 (1,4,7,16,22,25,32). However, most of these PCR assays are not able to discriminate S. pneumoniae from the recently discovered Streptococcus pseudopneumoniae and other mitis group streptococci. As an alternative approach, sequence analysis of several genes has been used for the species-level identifica...

A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for Streptococcus

“…However, it involves more inputs and selection of other genes out of a few thousand genes within the genome is not an easy task. In spite of the fact that around 23 genes have been used frequently in many studies carried out for identifying Streptococcus, no consensus gene has been identified [3,4,11,[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. It has also not been realized that except recA, the rest of the 22 genes are not present in all the species of Streptococcus.…”

“…A few genes which are frequently used for the identification of Streptococcus include genes: cpsA, gdh, groESL, lytA, ply, psaA, pspA, recA, recN, rpoA, rpoB, sodA, tuf, wzg, 16S-23S ribosomal DNA spacer region, and the DNA fragment Spn9802 [3,4,11,[22][23][24][25][26][27][28][29][30][31]. Optochin susceptibility testing (CO 2 atmosphere) in combination with amplification of Spn9802 fragment and the autolysin genes: lytA, rpoB, and tuf, have proved effective in identifying S. pneumoniae [32]. It must be realized that the most idyllic rrs gene is offering no escape as human beings are struggling to reach a consensus, while pathogenic bacteria are threatening our long-term survival.…”

“…Although, multilocus sequence analysis (MLSA) has been relatively quite successful in the identification of a wide range of bacterial species [33], however, using sequences of 7 HKGs: guaA, map, pfl, ppaC, pyk, rpoB, and sodA, also did not prove suitable for rapid identification of Streptococcus species [21]. MLSA using gdh, ddl along with guaA, map, pfl, ppaC, pyk, rnpB, rpoB, sodA, and tuf have been useful in identifying Streptococcus species [32,34,35].…”

Antibiotic Resistance Patterns and Related Mobile Genetic Elements of Pneumococci and β-Hemolytic Streptococci in Thai Healthy Children

“…Betahemolytic colonies were tested with bacitracin susceptibility and a latex agglutination test (Oxioid, USA) for Lancefield classification. Alpha-hemolytic colonies were pursued with the optochin disc test (Oxioid, USA) and bile solubility test [12]. All strains were kept as 15 % glycerol stocks in a -80°C freezer.…”

Streptococcus pseudopneumoniae as an emerging pathogen from patients with respiratory diseases

“…Clearing of the test suspension within 3 h was considered to be positive test (Wessels et al, 2012). All S. pseudopneumoniae isolates included in this study were phonotypically suspected as being capsule free, sensitive to optochin in ambient air but intermediate or resistant by incubation in 5% CO2 and bile insoluble.…”