Comparison of four polymerase chain reaction assays for specificity in the identification of Streptococcus pneumoniae

Messmer, Trudy O.; Sampson, Jacquelyn S.; Stinson, Annie; Wong, Betty; Carlone, George M.; Facklam, Richard R.

doi:10.1016/j.diagmicrobio.2004.04.013

Cited by 58 publications

(50 citation statements)

References 39 publications

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“…PCR detection of genes ubiquitous in pneumococci, particularly lytA (Messmer et al, 1997), psaA (Morrison et al, 2000) and ply (CimaCabal et al, 1999), has been used as an alternative method for species identification. We found that, with the primers and conditions used, lytA detection performed better than psaA, in agreement with previous findings (Messmer et al, 2004). However, since homologues of both genes have been detected in strains of closely related streptococcal species (Dowson, 2004;Jado et al, 2001), we advocate, for each atypical strain, the detection of both genes, since the odds of having a non-pneumococcal isolate carrying lytA and psaA simultaneously will be smaller.…”

Section: Discussionsupporting

confidence: 77%

Identification, prevalence and population structure of non-typable Streptococcus pneumoniae in carriage samples isolated from preschoolers attending day-care centres

et al. 2006

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The authors aimed to get insights into the population structure of non-(sero)typable pneumococci (NTPn), a specific group of natural atypical pneumococci whose identification is often difficult, and which has remained insufficiently studied. A total of 265 presumptive NTPn, isolated between 1997 and 2003 from the nasopharynx of children, were characterized. Strains were confirmed to be pneumococci on the basis of bile solubility, and PCR detection or Southern blotting hybridization of lytA and psaA, genes ubiquitous in this species. Multilocus sequence typing (MLST) was used to exclude two isolates that gave ambiguous results. Non-typability was confirmed by the Quellung reaction using Omniserum. A total of 213 isolates were considered to be true NTPn. The molecular analysis of the true NTPn by PFGE and MLST showed that this population was genetically diverse, although a dominant cluster, accounting for 66 % of the isolates, was identified. Antimicrobial resistance was observed in most genetic backgrounds, and multidrug resistance to penicillin, erythromycin, clindamycin, tetracycline and sulfamethoxazole-trimethoprim was associated with strains belonging to the dominant cluster. Comparison with PFGE fingerprints and sequence types of large collections of serotypable strains showed that the genetic backgrounds of all but two NTPn were different from those found in serotypable strains. In addition, we found that NTPn strains with similar genetic backgrounds to those identified in our study had been isolated from disease sources in other countries. These observations seem to indicate that NTPn have diverse genetic backgrounds and may have evolved as a distinct group of pneumococcal isolates. INTRODUCTIONStreptococcus pneumoniae is a respiratory pathogen that colonizes asymptomatically the nasopharynx of humans. Although the mortality and morbidity associated with pneumococci are high, they are considered to be facultative pathogens: colonization is the rule, and disease the exception (Centers for Disease Control and Prevention, 2000).Most pneumococci are shielded by a polysaccharide capsule, a major virulence factor that hinders phagocytosis. Up to now, at least 90 different capsular types have been described (Henrichsen, 1995). The capsule can be detected by reaction with specific antisera. However, although some isolates are presumptively identified as pneumococci by routine identification tests, a negative result with type-specific antisera for the 90 serotypes is obtained. These atypical isolates are designated non-typable pneumococci (NTPn), and their correct discrimination from commensal viridans streptococci is often difficult (Carvalho et al., 2003;Hanage et al., 2005;Shayegani et al., 1982;Whatmore et al., 2000). The distinction between these closely related species is clinically important, since S. pneumoniae can cause serious disease and viridans streptococci are commensal organisms. Some NTPn have been described as extremely contagious and have been associated with pneumococcal conjunctivitis, causing ...

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Section: Discussionsupporting

confidence: 77%

Identification, prevalence and population structure of non-typable Streptococcus pneumoniae in carriage samples isolated from preschoolers attending day-care centres

et al. 2006

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“…As an alternative, PCR amplification of several genes usually considered specific for S. pneumoniae (namely, lytA, ply, and psaA) has been used to determine which SMG isolate corresponds to a true pneumococcus. In a recent comparative study, Messmer et al reported that PCR amplification of an internal fragment of lytA was the most appropriate approach to the correct identification of S. pneumoniae (26). A similar conclusion had been reached in previous studies (17,25).…”

supporting

confidence: 64%

Characteristic Signatures of the lytA Gene Provide a Basis for Rapid and Reliable Diagnosis of Streptococcus pneumoniae Infections

2006

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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.

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“…All of the atypical Opt r streptococci presented colony morphology, alpha-hemolysis, and bile solubility characteristics that were typical of pneumococci. To confirm pneumococcal identification, a battery of genetic tests was applied, such as PCR amplification of the cpsAB (15), ply (31), lytA, and psaA genes (19) and sodA partial sequence analysis (13,30). All Opt r strains showed positive PCR results for the genes analyzed (Table 1), and the sodA sequences confirmed that the isolates were pneumococci.…”

Section: Characterization Of the Optmentioning

confidence: 99%

Characterization of In Vitro-Generated and Clinical Optochin-Resistant Strains of Streptococcus pneumoniae Isolated from Argentina

et al. 2008

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Optochin susceptibility is a key test used for pneumococcal diagnosis, but optochin-resistant (Opt r ) pneumococci have been reported in the last 2 decades. In this work, we characterized eight Opt r clinical strains which presented a new mutation, G47V, a predominant A49S mutation (recently reported in Brazil) and A49T. These mutations were found in the c subunit of the F 0 F 1 ATPase encoded by the atpC gene, and W206C was found in the a subunit encoded by the atpA gene. The Opt r clinical isolates were analyzed by BOX PCR, multilocus sequence typing, and serotype and antimicrobial resistance profiles, and they showed no epidemiological relationship. To characterize the Opt r mutations that could emerge among clinical strains, we studied a pool of spontaneous Opt r colonies obtained in vitro from the virulent D39 strain. We compared the atpAC mutations of these Opt r pneumococci (with or without passage through C57BL/6 mice) with those described in the clinical isolates. This analysis revealed three new mutations, G47V and L26M in the c subunit and L184S in the a subunit. Most of the mutations identified in the laboratory-generated Opt r strains were also found in clinical strains, with the exception of the L26M and L184S mutations, and we suppose that both mutations could emerge among invasive strains in the future. Considering that atpAC are essential genes, we propose that all spontaneous mutations that confer in vitro optochin resistance would not present severe physiological alterations in S. pneumoniae and may be carried by circulating pneumococcal strains.Streptococcus pneumoniae is one of the most important pathogens in children and in elderly populations, being the most common cause of invasive bacterial infections such as pneumonia, bacteremia, and meningitis. The laboratory characterization of S. pneumoniae is based on phenotypic tests such as optochin susceptibility, bile solubility, the Quellung reaction, and genotypic tests performed in specialized centers. The optochin susceptibility test is critical in the identification of S. pneumoniae, and it has been used for decades in bacteriological laboratories. However, optochin-resistant (Opt r ) strains have been reported since 1987 in the United States, Spain, and Israel and more recently in Portugal and Brazil (1,2,7,9,16,17,[23][24][25]28), thus complicating the pneumococcal diagnosis. When additional tests are not applied, these Opt r strains are probably misidentified and overlooked, resulting in inappropriate antimicrobial therapies for patients. Despite several clinical reports describing Opt r isolates, there are only a few mutants characterized at the molecular level. It was reported that point mutations in the atpAC genes, which encode subunits of F 0 F 1 ATPase, conferred optochin resistance on S. pneumoniae (5,10,29).In this work, our objectives were to characterize the Opt r strains isolated in our country, to compare the atpAC mutations with those identified in Opt r strains isolated in other geographical regions, and to investigate a p...

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Comparison of four polymerase chain reaction assays for specificity in the identification of Streptococcus pneumoniae

Cited by 58 publications

References 39 publications

Identification, prevalence and population structure of non-typable Streptococcus pneumoniae in carriage samples isolated from preschoolers attending day-care centres

Identification, prevalence and population structure of non-typable Streptococcus pneumoniae in carriage samples isolated from preschoolers attending day-care centres

Characteristic Signatures of the lytA Gene Provide a Basis for Rapid and Reliable Diagnosis of Streptococcus pneumoniae Infections

Characterization of In Vitro-Generated and Clinical Optochin-Resistant Strains of Streptococcus pneumoniae Isolated from Argentina

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