Comparison of Five Genotypic Techniques for Identification of Optochin-Resistant Pneumococcus-Like Isolates

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 ...

Characterization of Nontypeable and Atypical Streptococcus pneumoniae Pediatric Isolates from 1994 to 2010

“…Furthermore, pneumococci can often be distinguished from nonpneumococci by amplification of the genes encoding pneumolysin (ply) and pneumococcal surface antigen A (psaA). The presence of psaA in particular has been found to correspond with encapsulation in strains and has been suggested to be the best indicator for genuine pneumococci (19).…”

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

“…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.…”

Meningitis neonatal por Streptococcus pneumoniae atípico: Reporte de un caso y revisión

“…Además, las cepas acapsuladas no pueden ser detectadas por pruebas serológicas utilizadas para diagnóstico y/o tipificación. Este hecho se ha reportado en 2% de las cepas provenientes de líquidos estériles y hasta en 20% de los aislados de muestras de secreción conjuntival 16 . Debido a lo anterior, se ha propuesto el uso de técnicas moleculares que identifiquen genes específi-cos de S. pneumoniae para un diagnóstico certero.…”

“…Algunos de los métodos descritos incluyen RPC para los genes de virulencia de S. pneumoniae: autolisina (gen lytA) y pneumolisina (gen ply) y para el antígeno de superficie neumocóccico A (psaA). Sin embargo, se ha demostrado la presencia de estos genes en otros miembros del grupo viridans, lo que los inhabilita como herramientas únicas para el diagnóstico de certeza 3,16,17 . Otra técnica genética propuesta es la hibridación de una sonda de ADN con secuencias 16S rARN especí-ficas para S. pneumoniae (AccuProbe; GenProbe, San Diego, California), la que, aunque se ha usado como estándar de oro en algunos trabajos, no permite una adecuada discriminación entre especies en todos los casos 3 .…”

“…Otra técnica genética propuesta es la hibridación de una sonda de ADN con secuencias 16S rARN especí-ficas para S. pneumoniae (AccuProbe; GenProbe, San Diego, California), la que, aunque se ha usado como estándar de oro en algunos trabajos, no permite una adecuada discriminación entre especies en todos los casos 3 . Si bien los métodos genotípicos entregan nuevas posibilidades para la identificación de esta bacteria, la presencia de genes de virulencia de S. pneumoniae en cepas no neumocóccicas de S. mitis, probablemente debido a su estrecha relación génica y la transferencia horizontal de genes, dificultan su interpretacion 16 . Es un hecho bien conocido que los miembros del grupo mitis muestran valores de similitud ADN-ADN de 40 a 60% y algunas especies comparten más de 99% de homología en la secuencia 16S rARN 15 .…”