title: Multitarget Quantitative PCR Improves Detection and Predicts Cultivability of the Pathogen Burkholderia pseudomallei.
abstract: Burkholderia pseudomalleiis present in the environment in many parts of the world and causes the often-fatal disease melioidosis. The sensitive detection and quantification ofB. pseudomalleiin the environment are a prerequisite for assessing the risk of infection. We recently reported the direct detection ofB. pseudomalleiin soil samples using a quantitative PCR (qPCR) targeting a single type three secretion system 1 (TTSS1) gene. Here, we extend the qPCR-based analysis ofB. pseudomalleiin soil by validating novel qPCR gene targets selected from a comparative genomic analysis. Two hundred soil samples from two rice paddies in northeast Thailand were evaluated, of which 47% (94/200) wereB. pseudomalleiculture positive. The TTSS1 qPCR and two novel qPCR assays that targeted open reading frames (ORFs) BPSS0087 and BPSS0745 exhibited detection rates of 76.5% (153/200), 34.5% (69/200), and 74.5% (150/200), respectively. The combination of TTSS1 and BPSS0745 qPCR increased the detection rate to 90% (180/200). Combining the results of the three qPCR assays and the BPSS1187 nested PCR previously published, all 200 samples were positive by at least one PCR assay. Samples positive by either TTSS1 (n= 153) or BPSS0745 (n= 150) qPCR were more likely to be direct-culture positive, with odds ratios of 4.0 (95% confidence interval [CI], 1.7 to 9.5;P< 0.001) and 9.0 (95% CI, 3.1 to 26.4;P< 0.001), respectively. HighB. pseudomalleigenome equivalents correlated with high CFU counts by culture. In conclusion, multitarget qPCR improved theB. pseudomalleidetection rate in soil samples and predicted culture positivity. This approach has the potential for use as a sensitive environmental screening method forB. pseudomalleiIMPORTANCEThe worldwide environmental distribution of the soil bacteriumBurkholderia pseudomalleiremains to be determined. So far, most environmental studies have relied on culture-based approaches to detect this pathogen. Since current culture methods are laborious, are time consuming, and have limited sensitivity, culture-independent and more sensitive methods are needed. In this study, we show that aB. pseudomallei-specific qPCR approach can detect significantly higher numbers ofB. pseudomallei-positive soil samples from areas where it is endemic compared with that from culture. The use of multiple independentB. pseudomallei-specific qPCR targets further increased the detection rate ofB. pseudomalleicompared with that from single targets. Samples with a high molecularB. pseudomalleiload were more likely to be culture positive. We conclude that our quantitative multitarget approach might be useful in defining areas where there is a risk ofB. pseudomalleiinfections in different parts of the world.
authors.affiliation: Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany.|Institute of Hygiene, Microbiology and Environmental Medicine, Medical University Graz, Graz, Austria.
authors.forename: Andre
authors.initials: A
authors.lastname: Göhler
authors.affiliation: Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany.|Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, Vietnam.
authors.forename: Trinh Thanh
authors.initials: TT
authors.lastname: Trung
authors.affiliation: Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Hopf
authors.affiliation: Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Kohler
authors.affiliation: Institute for Geography and Geology, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Hartleib
authors.affiliation: Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
authors.forename: Vanaporn
authors.initials: V
authors.lastname: Wuthiekanun
authors.affiliation: Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.|London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Peacock
authors.affiliation: Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.|Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
authors.forename: Direk
authors.initials: D
authors.lastname: Limmathurotsakul
authors.affiliation: Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA.
authors.forename: Apichai
authors.initials: A
authors.lastname: Tuanyok
authors.affiliation: Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany ivo.steinmetz@medunigraz.at.|Institute of Hygiene, Microbiology and Environmental Medicine, Medical University Graz, Graz, Austria.
authors.forename: Ivo
authors.initials: I
authors.lastname: Steinmetz
mesh_terms: D001431:Bacteriological Techniques; D016957:Burkholderia pseudomallei / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D004777:Environment; D006801:Humans; D008554:Melioidosis / Q000382:microbiology; D016366:Open Reading Frames; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology; D013785:Thailand; D000069296:Type III Secretion Systems / Q000235:genetics
chemical_list: D000069296:Type III Secretion Systems
keywords: Burkholderia pseudomallei; Thailand; melioidosis; qPCR; rice field; soil