1887

Journal of Medical Microbiology logo

Volume 63, Issue 4

Development of a 4-valent genotyping assay for direct identification of the most frequent serotypes from respiratory specimens of pneumonia patients Free

Abstract

is a common cause of nosocomial infections and is associated with high rates of mortality. In order to facilitate rapid and sensitive identification of the most prevalent serotypes of , we have developed a 4-valent real-time PCR-based assay using oligonucleotides specific for open-reading frames constituting the O-antigen-specific lipopolysaccharide loci of . The assay simultaneously detects and differentiates between each of the four serotypes IATS-O1, -O6, -O11 and serogroup 2 (IATS-O2, -O5, and -O16) with high sensitivity and specificity in a single-tube reaction. No cross-reactivity was observed with other serotypes of or other microbial species, and reproducibility was demonstrated regardless of template, i.e. purified DNA, bacterial culture and clinical specimens (broncho-alveolar lavage). The limit of detection of the assay was approximately 100 copies per reaction for IATS-O1, -O2 and -O11, and 50 copies per reaction for IATS-O6. Comparison of the assay specificity with a commercially available slide agglutination kit showed consistent results; however, the number of non-typable isolates was reduced by 15 % using the genotyping assay. Use of the 4-valent genotyping assay in the context of a clinical trial resulted in identification of pneumonia patients positive for the IATS-O11 serotype, and hence eligible for therapy with panobacumab (an investigational monoclonal antibody against the O-polysaccharide of serotype IATS-O11).

  • Received:
  • Accepted:
  • Published Online:
© 2014 SGM
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.066043-0
2014-04-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jmm/63/4/508.html?itemId=/content/journal/jmm/10.1099/jmm.0.066043-0&mimeType=html&fmt=ahah

References

  1. American Thoracic Society 2005; Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 171:388–416 [View Article][PubMed]
     [Google Scholar]
  2. Berthelot P., Attree I., Plésiat P., Chabert J., de Bentzmann S., Pozzetto B., Grattard F.Groupe d’Études des Septicémies à Pseudomonas aeruginosa 2003; Genotypic and phenotypic analysis of type III secretion system in a cohort of Pseudomonas aeruginosa bacteremia isolates: evidence for a possible association between O serotypes and exo genes. J Infect Dis 188:512–518 [View Article][PubMed]
     [Google Scholar]
  3. Chastre J., Combes A., Luyt C. E. 2005; The invasive (quantitative) diagnosis of ventilator-associated pneumonia. Respir Care 50:797–807 discussion 807–812[PubMed]
     [Google Scholar]
  4. Chenna R., Sugawara H., Koike T., Lopez R., Gibson T. J., Higgins D. G., Thompson J. D. 2003; Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497–3500 [View Article][PubMed]
     [Google Scholar]
  5. European Centre for Disease Prevention and Control 2010; Antimicrobial resistance surveillance in Europe 2009. Annual Report of the European Antimicrobial Resistance Surveillance Network. (EARS-Net). http://www.ecdc.europa.eu/en/publications/Publications/1011_SUR_annual_EARS_Net_2009.pdf
  6. Fagon J. Y., Chastre J., Hance A. J., Montravers P., Novara A., Gibert C. 1993; Nosocomial pneumonia in ventilated patients: a cohort study evaluating attributable mortality and hospital stay. Am J Med 94:281–288 [View Article][PubMed]
     [Google Scholar]
  7. Faure K., Shimabukuro D., Ajayi T., Allmond L. R., Sawa T., Wiener-Kronish J. P. 2003; O-antigen serotypes and type III secretory toxins in clinical isolates of Pseudomonas aeruginosa. J Clin Microbiol 41:2158–2160 [View Article][PubMed]
     [Google Scholar]
  8. Finney D. J. 1952 Probit Analysis: a Statistical Treatment of the Sigmoid Response Curve, 2nd edn. vol. xiv p. 318 New York, NY: Cambridge University Press;
     [Google Scholar]
  9. Horn M. P., Zuercher A. W., Imboden M. A., Rudolf M. P., Lazar H., Wu H., Hoiby N., Fas S. C., Lang A. B. 2010; Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11. Antimicrob Agents Chemother 54:2338–2344 [View Article][PubMed]
     [Google Scholar]
  10. Jones R. N. 2010; Microbial etiologies of hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia. Clin Infect Dis 51:Suppl. 1S81–S87 [View Article][PubMed]
     [Google Scholar]
  11. Knaus W. A., Draper E. A., Wagner D. P., Zimmerman J. E. 1985; APACHE II: a severity of disease classification system. Crit Care Med 13:818–829 [View Article][PubMed]
     [Google Scholar]
  12. Kollef K. E., Schramm G. E., Wills A. R., Reichley R. M., Micek S. T., Kollef M. H. 2008; Predictors of 30-day mortality and hospital costs in patients with ventilator-associated pneumonia attributed to potentially antibiotic-resistant Gram-negative bacteria. Chest 134:281–287 [View Article][PubMed]
     [Google Scholar]
  13. Lam J. S., Handelsman M. Y., Chivers T. R., MacDonald L. A. 1992; Monoclonal antibodies as probes to examine serotype-specific and cross-reactive epitopes of lipopolysaccharides from serotypes O2, O5, and O16 of Pseudomonas aeruginosa. J Bacteriol 174:2178–2184[PubMed]
     [Google Scholar]
  14. Le Berre R., Nguyen S., Nowak E., Kipnis E., Pierre M., Quenee L., Ader F., Lancel S., Courcol R.& other authors ( 2011; Relative contribution of three main virulence factors in Pseudomonas aeruginosa pneumonia. Crit Care Med 39:2113–2120 [View Article][PubMed]
     [Google Scholar]
  15. Liu P. V. 1969; Changes in somatic antigens of Pseudomonas aeruginosa induced by bacteriophages. J Infect Dis 119:237–246 [View Article][PubMed]
     [Google Scholar]
  16. Liu P. V., Wang S. 1990; Three new major somatic antigens of Pseudomonas aeruginosa. J Clin Microbiol 28:922–925[PubMed]
     [Google Scholar]
  17. Liu P. V., Matsumoto H., Kusama H., Bergan T. 1983; Survey of heat-stable major somatic antigens of Pseudomonas aeruginosa. Int J Syst Bacteriol 33:256–264 [View Article]
     [Google Scholar]
  18. Lu Q., Rouby J. J., Laterre P. F., Eggimann P., Dugard A., Giamarellos-Bourboulis E. J., Mercier E., Garbino J., Luyt C. E.& other authors ( 2011; Pharmacokinetics and safety of panobacumab: specific adjunctive immunotherapy in critical patients with nosocomial Pseudomonas aeruginosa O11 pneumonia. J Antimicrob Chemother 66:1110–1116 [View Article][PubMed]
     [Google Scholar]
  19. Masuda N., Sakagawa E., Ohya S. 1995; Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 39:645–649 [View Article][PubMed]
     [Google Scholar]
  20. Millar B. C., Xu J., Moore J. E. 2007; Molecular diagnostics of medically important bacterial infections. Curr Issues Mol Biol 9:21–39[PubMed]
     [Google Scholar]
  21. Mittal R., Sharma S., Chhibber S., Aggarwal S., Gupta V., Harjai K. 2010; Correlation between serogroup, in vitro biofilm formation and elaboration of virulence factors by uropathogenic Pseudomonas aeruginosa. FEMS Immunol Med Microbiol 58:237–243 [View Article][PubMed]
     [Google Scholar]
  22. Motoshima M., Yanagihara K., Fukushima K., Matsuda J., Sugahara K., Hirakata Y., Yamada Y., Kohno S., Kamihira S. 2007; Rapid and accurate detection of Pseudomonas aeruginosa by real-time polymerase chain reaction with melting curve analysis targeting gyrB gene. Diagn Microbiol Infect Dis 58:53–58 [View Article][PubMed]
     [Google Scholar]
  23. Motoshima M., Yanagihara K., Yamamoto K., Morinaga Y., Matsuda J., Sugahara K., Hirakata Y., Yamada Y., Kohno S., Kamihira S. 2008; Quantitative detection of metallo-β-lactamase of blaIMP-cluster-producing Pseudomonas aeruginosa by real-time polymerase chain reaction with melting curve analysis for rapid diagnosis and treatment of nosocomial infection. Diagn Microbiol Infect Dis 61:222–226 [View Article][PubMed]
     [Google Scholar]
  24. Secher T., Fauconnier L., Szade A., Rutschi O., Fas S. C., Ryffel B., Rudolf M. P. 2011; Anti-Pseudomonas aeruginosa serotype O11 LPS immunoglobulin M monoclonal antibody panobacumab (KBPA101) confers protection in a murine model of acute lung infection. J Antimicrob Chemother 66:1100–1109 [View Article][PubMed]
     [Google Scholar]
  25. Sekiguchi J., Asagi T., Miyoshi-Akiyama T., Kasai A., Mizuguchi Y., Araake M., Fujino T., Kikuchi H., Sasaki S.& other authors ( 2007; Outbreaks of multidrug-resistant Pseudomonas aeruginosa in community hospitals in Japan. J Clin Microbiol 45:979–989 [View Article][PubMed]
     [Google Scholar]
  26. Vincent J. L., Rello J., Marshall J., Silva E., Anzueto A., Martin C. D., Moreno R., Lipman J., Gomersall C.& other authors ( 2009; International study of the prevalence and outcomes of infection in intensive care units. JAMA J Am Med Assoc) 302:2323–2329 [View Article][PubMed]
     [Google Scholar]
  27. Winstanley C., Kaye S. B., Neal T. J., Chilton H. J., Miksch S., Hart C. A.Microbiology Ophthalmic Group 2005; Genotypic and phenotypic characteristics of Pseudomonas aeruginosa isolates associated with ulcerative keratitis. J Med Microbiol 54:519–526 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.066043-0
Loading
Development of a 4-valent genotyping assay for direct identification of the most frequent Pseudomonas aeruginosa serotypes from respiratory specimens of pneumonia patients
J. Med. Microbiol. 63, 508 (2014); https://doi.org/10.1099/jmm.0.066043-0
/content/journal/jmm/10.1099/jmm.0.066043-0
/content/journal/jmm/10.1099/jmm.0.066043-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error