Genotypic identification of presumptive Streptococcus pneumoniae by PCR using four genes highly specific for S. pneumoniae

Suzuki, Nao; Yuyama, Mayumi; Maeda, Sinsaku; Ogawa, Haruhiko; Mashiko, Kazuyuki; Kiyoura, Yusuke

doi:10.1099/jmm.0.46296-0

Cited by 51 publications

(41 citation statements)

References 22 publications

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“…This demonstrates the ability of the assay to detect S. pneumoniae DNA. It has been reported in the literature that strains of closely related oral streptococci, in particular the SMG strains, may also harbor the lytA gene in addition to other S. pneumoniae-specific virulence factors such as pneumolysin (20,21). These atypical isolates often display unusual phenotypic characteristics, such as optochin sensitivity, that make them appear more like S. pneumoniae isolates.…”

Section: Discussionmentioning

confidence: 99%

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Duplex Real-Time PCR Assay for Detection of Streptococcus pneumoniae in Clinical Samples and Determination of Penicillin Susceptibility

et al. 2008

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We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of >1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of <0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.

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Section: Discussionmentioning

confidence: 99%

“…Such SMG strains appear to be associated with severe disease. Recent studies have found that lytA PCR was more specific than ply PCR for the identification of S. pneumoniae (11,20,21).…”

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confidence: 99%

Duplex Real-Time PCR Assay for Detection of Streptococcus pneumoniae in Clinical Samples and Determination of Penicillin Susceptibility

et al. 2008

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“…Indeed, ply appears to be less specific than lytA [8,14]. Other studies have also targeted genes which may be more specific than lytA for pneumococcus such as, spy, spn9802 and spn9828 [15].…”

Section: Discussionmentioning

confidence: 99%

Invasive Pneumococcal Disease in Children: The Contribution of Real-Time PCR on Blood

Chantreuil

Cantagrel

Maakaroun-Vermesse

et al. 2015

JMEN

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Objectives: This study aimed to determine the benefits of a real-time PCR test specific for S. pneumoniae performed directly on blood samples in children with suspected invasive pneumococcal disease (IPD). Methods:We performed real-time PCR of the ply and lytA genes on blood samples from children with a suspicion of IPD, taken early after admission. Results:We prospectively enrolled 76 children suspected of IPD over a 6-month period. Out of the 76 patients, 5 IPD were confirmed with two positive blood cultures and three positive pleural cultures for S. pneumoniae. Using specific realtime PCR on blood samples, eight pneumococcal infections were positive allowing the identification of five more cases of IPD than standard methods alone, including three of the four cases of pneumonia with pleurisy. The overall sensitivity of realtime PCR tests on blood samples was 80 % and the specificity was 98%. Conclusion:Our study demonstrates the value of real-time PCR especially in case of pleurisy.

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“…The presence of two virulence genes of S. pneumoniae (pneumolysin; ply, and autolysin; lytA) was detected by specific primers [14]. Erythromycin resistance genes were detected with primers specific for ermA, ermB and mefA/E genes [15,16].…”

Section: Pcr Detection For S Pneumoniae and Erythromycin/tetracyclinmentioning

confidence: 99%

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

et al. 2016

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Transmission of antibiotic resistance genes among Streptococcus pneumoniae and beta-hemolytic streptococcus (BHS) was generally associated with transmissible genetic elements. The objectives of this study were to investigate carriage rate, antibiotic resistance and related mobile genetic elements of pneumococci and BHS from school-children. The pneumococci and BHS were recovered from 220 Thai school-children, and then tested for antibiotic susceptibility pattern by disc diffusion. Antibiotic resistance genes and related genetic elements were detected by PCR with specific primers. A total of 77 pneumococcal isolates were resistant to erythromycin (42 %), tetracycline (44 %), clindamycin (8 %), or penicillin (3 %). Fifty-four BHS isolates were resistant to erythromycin (28 %), tetracycline (52 %), or clindamycin (13 %). All isolates tested were 100 % sensitive to penicillin and levofloxacin. Among erythromycin-resistant streptococcal isolates showed different phenotypes of clindamycin resistance. It was found that isolated pneumococci showed constitutive clindamycin resistance (19 %), and inducible clindamycin resistance (12 %). The BHS isolates exhibited constitutive clindamycin resistance (40 %), and inducible resistance (20 %) phenotypes. The predominant erythromycin resistance genes in pneumococci and BHS were mefE and ermB, while the most common tetracycline resistance gene in this population was tetM. Furthermore, almost all erythromycin-and tetracycline-resistant streptococci (97 %) mainly contained various genetic elements, including mega elements and six different transposon types (Tn2009, Tn2017, Tn917, Tn3872, Tn6002 and Tn916). Therefore, carriages of pneumococci and BHS with multidrug resistance in children might be important reservoirs of antibiotic-resistance genes carried by transposons. Tn916-like elements could lead to dissemination of the antibiotic resistance genes among genus streptococcus in human oral cavity and nasopharynx.

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Genotypic identification of presumptive Streptococcus pneumoniae by PCR using four genes highly specific for S. pneumoniae

Cited by 51 publications

References 22 publications

Duplex Real-Time PCR Assay for Detection of Streptococcus pneumoniae in Clinical Samples and Determination of Penicillin Susceptibility

Duplex Real-Time PCR Assay for Detection of Streptococcus pneumoniae in Clinical Samples and Determination of Penicillin Susceptibility

Invasive Pneumococcal Disease in Children: The Contribution of Real-Time PCR on Blood

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

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