ORIGINAL ARTICLE

Trans R Soc Trop Med Hyg 2015; 109: 462–468 doi:10.1093/trstmh/trv040 Advance Access publication 4 June 2015

Direct detection of Burkholderia pseudomallei and biological factors in soil Rasana W. Sermswana,b,*, Phairat Royrosa,b, Nittaya Khakhuma,b, Surasakdi Wongratanacheewinb,c and Apichai Tuanyokd a

Department of Biochemistry, Faculty of Medicine, Khon Kean University, 123 Mitraparp Rd, Khon Kaen 40002, Thailand; bMelioidosis Research Center, Faculty of Medicine, Khon Kean University, Khon Kaen, Thailand; cDepartment of Microbiology, Faculty of Medicine, Khon Kean University, Khon Kaen, Thailand; dDepartment of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida, USA

Received 15 February 2015; revised 21 April 2015; accepted 29 April 2015 Background: Burkholderia pseudomallei, a Gram-negative saprophytic bacillus, is a severe infectious agent that causes melioidosis and soil is the most important reservoir. Methods: One hundred and forty soil samples were tested for pH, moisture content and total C and N measurements and used for DNA extraction and culture for B. pseudomallei. The quantitative real-time PCR (qPCR) targeting wcbG, a putative capsular polysaccharide biosynthesis protein gene of B. pseudomallei, was developed to detect the bacterium, and random amplified polymorphic DNA (RAPD) was used to detect the microbial diversity in soil. Results: The acidic pH was correlated with the presence of the bacterium. Forty-four soil sites (44/140, 31.4%) were positive for B. pseudomallei by qPCR, of which 21 were positive by culture. The limit of detection is 32 fg of DNA (about 4 genomes). The RAPD method could classify the soil samples into low diversity (LD) and high diversity (HD) sites. The trend of LD was found with B. pseudomallei positive soil sites. Conclusions: The acidity of the soil or metabolites from organisms in the sites may contribute to the presence of the bacterium. Further investigation of microbes by a more robust method should elucidate biological factors that promote the presence of B. pseudomallei and may be used for controlling the bacterium in the environment. Keywords: Burkholderia pseudomallei, Diversity, Quantitative real-time PCR, Random amplified polymorphic DNA, Soil

Introduction Burkholderia pseudomallei is a Gram-negative saprophytic motile bacillus that causes melioidosis, a serious disease in humans and animals.1 This organism is mostly found in soil and stagnant water in endemic areas including Southeast Asia and northern Australia.2 More countries such as Sri Lanka, Taiwan, Papua New Guinea and a few countries in South America and Africa have also been reported as definite sites for environmental distribution of the bacteria.3 Transmission occurs by direct contact with contaminated soil and water through skin abrasion or by ingestion or inhalation. The organism was categorized in the category B list of critical agents published by the US Centers for Disease Control and Prevention (CDC) by its potential to be used as a bio-weapon.4 As soil is the most important source of infection, knowledge of physicochemical and biological properties of soils that are correlated with the presence of this bacterium should facilitate in controlling the source of the disease. The presence of B. pseudomallei

is known to be associated with the acidic pH range and suitable ratios of organic materials in soils.5 The information on biological factors, however, is still limited. The evaluation of diversity of organisms in soil by molecular techniques in the presence or absence of B. pseudomallei could predict the richness of microbes in each condition and provide biological information of the soil. To detect the presence of B. pseudomallei in soil, culturing techniques are generally used as the gold standard. To culture bacteria from soil, however, can be problematic due to inherent unculturable forms and difficulty of bacterial dissociation from soil particles.6 The PCR technique has been applied to detect unculturable forms of microorganisms in soil. Several quantitative real-time PCR (qPCR) methods have been reported for detection in pure cultures or clinical samples of B. pseudomallei using various gene targets such as the type III secretion system, ATP-binding transportrelated membrane protein, 16S rDNA, and flagellin genes that provide a wide range of sensitivities.7–10 The information of physicochemical and biological factors in the soil with the

# The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected].

462

Downloaded from http://trstmh.oxfordjournals.org/ at University of Bath on July 13, 2015

*Corresponding author: Tel: +66 43 348386; Fax: +66 43 348386; E-mail: [email protected]

Transactions of the Royal Society of Tropical Medicine and Hygiene

presence and absence of B. pseudomallei investigated by both culture and molecular methods is quite limited. A qPCR assay targeting wcbG, a putative capsular polysaccharide biosynthesis gene which is specific to B. pseudomallei and the culture method were therefore used together along with the evaluation of some physicochemical factors and diversity of microbes in soil using random amplified polymorphic DNA (RAPD). Burkholderia thailandensis, a non-pathogenic and a closely related species that is also present in soil in the endemic areas such as Thailand, was also detected using qPCR targeting B. thailandensis-specific glycosyltransferase gene (BTH_I1329). This closely related species might be one of the biological factors that influence the presence of B. pseudomallei in soil.

Soil sampling During September to December 2007 (the end of the rainy to the early winter season), 140 soil sites were sampled from an area approximately 20 km2 in Nam Phong and Muang Districts, Khon Kaen Province, northeastern Thailand. The soil sites were randomly selected mostly from unused land located close to rice paddies or cassava plantations. A GPS unit (GPS Pathfinder Basic, Trimble Navigation Ltd, Sunnyvale, CA, USA) was used to record the geographic coordinates of each sampling site. ArcViewGis version 3.1 (ESRI, Redlands, CA, USA) was used to map the bacterial detection sites. At each sampling site, soil samples were collected from three holes one meter apart as a triangle at the depth of 30 cm, and then pooled as a single sample. The soil classification and physicochemical properties of soil (pH, moisture content and total organic carbon) were measured as described previously.5

Culture of Burkholderia pseudomallei and Burkholderia thailandensis from soil Two milliliters of sterile distilled water were vigorously mixed with one gram of soil from each site and the steps were followed as previously described.5 The dilutions of 1021 to 1029 from selective enrichment broth (Threonine-basal salt solution with 20 mg/L of colistin [TBSS-C20])11 were spread on modified Ashdown’s agar and incubated at 378C for 4 days.12 All suspected colonies with a wrinkled, violet-purple appearance among other bacteria on the dilution plates that have the Burkholderia-like morphology, approximately 10 colonies, were identified by biochemical tests, antibiotic susceptibility, arabinose assimilation and latex agglutination to identify B. pseudomallei and B. thailandensis.13 As some other microbes can also grow on Ashdown’s medium, higher density of colonies may cause overgrow of other bacteria and interfere with the selection.

DNA extraction from soil DNA extraction was performed by the bead beating method as described by Yeates and colleagues14 using 2 gm wet weight of soil with 3 mL of extraction buffer and 2 gm of sterile glass beads (BioSpec Products, Bartlesville, OK, USA). The pellet was resuspended in 50 mL TE buffer and humic acids were removed by polyvinylpolypyrrolidone (PVPP) as described previously.15

The wcbG in B. pseudomallei K96243 was amplified by standard PCR using primers: F: 5′ -AACGAGTCGGTCATTTCCCTGA-3′ and R: 5′ -CCGATATTGCCGACTTCCA CTGTGAT-3′ with an annealing temperature at 608C. The 320 bp amplicon was generated and purified from agarose gel by the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). The amplicon was cloned into the pCR 2.1-TOPO plasmid using the TOPO-TA Cloning kit and transformed into TOP10 Competent E. coli (Invitrogen Grand Island, NY, USA). The positive clones were selected on LB agar containing 50mg/mL kanamycin. The recombinant wcbG plasmid named pCR-wcbG was extracted from the positive clones using a QIAGEN QIAprep Spin Miniprep Kit (QIAGEN). To generate the standard curve, the pCR-wcbG was 10 fold diluted to obtain 1.0×103–1.0×106 pg/mL and amplified by real-time PCR for five replicates (LightCycler, Roche, Indianapolis, IN, USA). The reaction mixture of LightCyclerw FastStart DNA MasterPLUS SYBR Green I reaction mix (Roche Applied Science, Madison, WI, USA) was used with 0.30 mM each of wcbG forward and reverse primers and DNA templates. The amplification condition was 508C for 2 min, 958C for 10 min, and 40 cycles of denaturation at 958C for 15 sec, annealing at 608C for 30 sec and extension at 728C for 30 sec followed by one cycle of 15 sec each at 958C, 608C and 958C.

Spiking DNA for sensitivity of detection The sensitivity of amplification by using wcbG and BTH_I1329 primers were investigated by spiking experiments. Ten-fold serial dilutions of B. pseudomallei DNA (K96243) ranging from 320 ng down to 3.2 fg (4×107 to 4 genome copies) and B. thailandensis ranging from 363 ng to 36.3 fg (5×107to 5 genome copies) were used to spike negative soil and the DNA extracted from each dilution was used as templates for amplification.

Real-time PCR for the detection of Burkholderia pseudomallei and Burkholderia thailandensis in soil The specific primers for B. pseudomallei and B. thailandensis were designed by genomic comparisons between B. pseudomallei K96243 and B. thailandensis E243. Burkholderia pseudomallei has a specific capsular polysaccharide biosynthesis gene cluster (also known as CPS-1) which is not present in B. thailandensis. WcbG, one of the CPS-1 biosynthesis genes, was selected as a PCR target to detect B. pseudomallei in soil. BTH_I1329, glycosyltransferase gene, is specific to B. thailandensis that was selected for the PCR target. These primers were tested against B. pseudomallei, B. thailandensis, B. mallei EY2236, B. mallei EY2233, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and closely related bacteria in soil, Ralstonia solanaciarum, and common bacteria in soil Bacillus subtilis and Bacillus amyloliquefaciens. It is noted that B. mallei can also produce CPS-1, but it has not been reported in soil. Two primers were then designed using IDT PrimerQuest. The primer sequences were checked for specificity using BLAST against microbial genomes (www.ncbi.nlm. nih.gov). Quantitative real-time PCR was performed using LightCycler w FastStart DNA MasterPLUS SYBR Green I reaction

463

Downloaded from http://trstmh.oxfordjournals.org/ at University of Bath on July 13, 2015

Materials and methods

Quantification of Burkholderia pseudomallei by real-time PCR

R. W. Sermswan et al.

mix (Roche Applied Science) with 5 mL of soil DNA template with conditions as mentioned earlier. For B. thailandensis detection, 0.3 mM of each primer was used to amplify the glycosyltransferase gene (BTH_I1329 F; 5′ -AAGATCCTGACGCTCAGCACGTAT-3′ and BTH_I1329 R; 5′ -TCAGCAGCGGAT TGTCGATGTACT-3′ ). DNA extracted from overnight cultures of B. pseudomallei K96243 and B. thailandensis UE5 by the proteinase K extraction method were used as positive controls.16

Random amplified polymorphic DNA of DNA extracted from soil

Statistical analysis The analysis of covariance (ANCOVA) for binary response variable was used to analyze the physicochemical properties in B. pseudomallei positive and negative soil. The Mann-Whitney U test (nonparametric) was used for a statistic test of the RAPD.

Results From 140 soil sites, the pH of soil was the only factor that significantly correlated with the presence of B. pseudomallei (p,0.01). The data of each physicochemical factor are shown in Table 1. Colony morphology and biochemical tests of B. pseudomallei and B. thailandensis were similar. Both of them were resistant to colistin but sensitive to Augmentin (amoxicillin/clavulanic acid), which can be used to distinguish them from other soil bacteria such as Burkholderia cepacia. Negative arabinose assimilation

Table 1. The values of physicochemical factors in the positive and negative soil for Burkholderia pseudomallei Physicochemical factors

pH Moisture content (%) Total organic carbon (TOC) NS: not significant.

464

Positive soil

Negative soil

p-value

Range

Mean

Range

Mean

4.77–7.66+0.6 1.37–36.86+9.2 2.56–31.86+6.4

5.85 13.08 15.25

5.35–7.7+0.6 2.79–31.07+8.1 2.56–27.16+6.06

6.35 13.90 17.69

,0.01 NS NS

Downloaded from http://trstmh.oxfordjournals.org/ at University of Bath on July 13, 2015

Random amplified polymorphic DNA was used to study the diversity of organisms in the soil using the method as previously described.17 Briefly, each 50 ml RAPD reaction was composed of 1.25 units of Taq DNA polymerase, 100 ng of DNA samples, 0.2 mM of dNTP, 4 mM MgCl2, 1x Taq polymerase buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl) and 1 mM of RSP primer; 5′ -GGAAACAGCTATGACCATGA-3′ . The amplification was started with two cycles of low-stringency PCR (denaturation at 948C for 5 min, annealing at 408C for 5 min and extension at 728C for 5 min) and then 40 cycles of high-stringency PCR (948C for 1 min, 608C for 1 min and 728C for 1 min). The amplified product was analyzed on 1.5% agarose gel electrophoresis. The RAPD of each soil sample was performed at least twice to get consistency in the number of bands.

and positive latex agglutination distinguished B. pseudomallei from B. thailandensis. Out of 140 soil sites 15.0% (21) and 8.6% (12) were culture positive for B. pseudomallei and B. thailandensis. Only three sites were positive by culture for both of them. From 150 colonies identified from Ashdown’s agar, 70.0% (105/150) were B. pseudomallei and the rest were B. thailandensis. The ratio of B. pseudomallei to B. thailandensis colonies was 2.3 (105/45). The ranges of colony-forming units (CFU) of B. pseudomallei and B. thailandensis in the soil were 1.52×104–7.8×1010 CFU/g and 5×104–4×1010 CFU/g. For pCR-wcbG, the amplification plot demonstrating an observable difference in threshold cycle (Ct) values between different dilutions was shown to be approximately three cycles and the end-point dilution factor was 1×1028 ng (Figure 1). The end point of detection was confirmed by visualization on agarose gel electrophoresis (Figure 2). For B. pseudomallei quantification, the standard curve by the wcbG gene (R2 value¼0.99), was as little as two copies of pCR-wcbG that were detectable by this qPCR assay (4.61×1026 pg/copy). The amplification plot demonstrates agreement between the five replicates that were run with qPCR (data not shown). The amplification results showed that wcbG and BTH_I1329 primers can detect B. pseudomallei and B. thailandensis DNA templates at concentrations as low as 32 fg (about four genomes) and 363 fg (about 50 genomes). The PCR products for B. pseudomallei and B. thailandensis assays were 323 bp and 211 bp. The qPCR showed 44 positive soil sites (44/140, 31.4%) for B. pseudomallei, of which only 15.0% (21/140,) were detected by bacterial culture. For B. thailandensis, 12.1% (17/140,) were positive by qPCR but only 12 sites were positive by culture (12 sites by qPCR, seven by culture and five by both methods, 24 positive sites). There were 11 sites detected as positive for B. pseudomallei and B. thailandensis by both methods and three sites by culture (Bp: Bt¼5:1, 8:1 and 9:2). The ratio of B. pseudomallei/ B. thailandensis positive soil sites was about 1.8 (44/24). ArcViewGIS was used to localize the positive soil sites of B. pseudomallei on the map of Phong river area (Figure 3) that has the river and its branches running through it as indicated with nearby places covered by rice paddies and cassava plantations. The soil classification indicated that the majority of soil in this area is clayed sand. The RAPD technique that used the RSP primer to amplify DNA extracted from soil generated amplicons ranging in size from

Transactions of the Royal Society of Tropical Medicine and Hygiene

Downloaded from http://trstmh.oxfordjournals.org/ at University of Bath on July 13, 2015

Figure 1. The quantitative real-time PCR (qPCR) amplification plot demonstrating an observable difference in threshold cycle values with different dilution factors of pCR-wcbG. The end-point dilution factor is 1×1028 ng. This figure is available in black and white in print and in color at Transactions online.

300 to 900 base pairs. DNA patterns composed of 1–5 bands were categorized into high diversity (HD) consisting of 3–5 bands and low diversity (LD) of 1–2 bands (data not shown). The statistical analysis indicated the trend of increasing number of band with B. pseudomallei negative soil but does not significantly differ as the 3 bands profile appeared almost equally in B. pseudomallei positive and negative soil (B.p. positive median¼2.5) (B.p. negative median¼3.0) (U¼24, p¼0.4187). Furthermore, soil physicochemical properties that were analyzed were not different between HD and LD groups but the amount of total genomic DNA in the HD group was higher than the LD group (data not shown).

Discussion The most important reservoir of B. pseudomallei is the soil; 50.1% of the soil sampling sites in the northeast of Thailand were reported to be positive for B. pseudomallei by the culture technique.18 The number of melioidosis patients found in this area was also higher than other parts of the country.19 Therefore, information of the physicochemical and biological factors that contribute to the occurrence of B. pseudomallei in soils is important and may be used to control the bacteria in soil in some areas such as zoos and recreation areas.

465

R. W. Sermswan et al.

Figure 3. ArcViewGis version 3.1 (ESRI, Redlands, CA, USA) of positive and negative soil sites for Burkholderia pseudomallei detected by culture and quantitative real-time PCR (qPCR). The Phong River area of approximately 20 km2 is demonstrated with positive (triangle) and negative (circle) sites for B. pseudomallei. The river is illustrated as a dense line. The distance bar indicates a 6 km scale. This figure is available in black and white in print and in color at Transactions online.

Although bacterial culture is well known as the gold standard for bacterial detection and identification, some limitations such as the efficiency of the extraction, methods to dissociate the bacteria from soil components, a low number of bacteria compared to other microbes in the soil and the presence of unculturable forms of the bacteria could reduce the possibility of detection.20 The qPCR targeting the type III secretion system (TTS1) has been developed to specifically detect B. pseudomallei in soil samples21 and the

466

comparisons were made between direct culture and qPCR that can detect B. pseudomallei at two genome equivalents.20 The method, however, requires a probe and the process is impaired by interference with humic substances from soil. The qPCR in this study was targeted to wcbG that specifically detected B. pseudomallei and did not react to B. thailandensis, B. mallei or B. cepacia (data not shown). The bioinformatics analysis also showed no homology with other saprophytic bacteria including other Burkholderia spp. The sensitivity to detect B. pseudomallei is at four genomes that is not much different from detection using TTS1 but, as mentioned earlier, does not require a probe for detection. As wcbG has only one copy per B. pseudomallei genome, the actual cell count contained within a soil sample should be closely parallel to the standardized dilution curve developed in this study. All culture positive soil sites were positive by qPCR for B. pseudomallei but not for B. thailandensis. The disagreement of detection may be due to the poor distribution of B. thailandensis in soil. There were 11 sites that were positive by qPCR for both B. pseudomallei and B. thailandensis. This may indicate that they could be found together and this study has shown that B. pseudomallei is more abundant than B. thailandensis in soils if they are found together. This finding is in agreement with a recent report by Ngamdee et al.22 that B. pseudomallei can inhibit both growth and motility of B. thailandensis. A previous study identified some physicochemical properties of soil including pH, percent of moisture content (%MC) and chemical oxygen demand (COD) that was correlated with the presence of B. pseudomallei in soil samples.15 The modification of soil physicochemical factors in the microcosm under laboratory conditions also demonstrated the effects of pH, salinity, C/N ratio and Fe to the bacterial survival by culture.23 In this study, however, only the pH was significantly correlated with the presence of B. pseudomallei in soils (p,0.01). Because all soil samples were

Downloaded from http://trstmh.oxfordjournals.org/ at University of Bath on July 13, 2015

Figure 2. The end-point dilution for pCR-wcbG amplification. Agarose gel electrophoresis showed five replicates of pCR-wcbG dilutions detected by quantitative real-time PCR (qPCR). The end point dilution for detection from five replicates as indicated by arrows is the consensus at 1×1028 ng. NTC: negative control.

Transactions of the Royal Society of Tropical Medicine and Hygiene

Authors’ contributions: PR, RWS carried out the experiments and NK confirmed the qPCR work and analysis. PR, NK and RWS drafted the manuscript. AT designed and analyzed the bioinformatics part. AT, SW and RWS conceived the idea and contributed in experimental designs and laboratory expenses and RW is the guarantor of the paper. All authors have read and approved the manuscript. RWS is guarantor of the paper. Acknowledgements: We would like to thanks Prof James A. Will for editing the manuscript via Publication Clinic, Khon Kaen University, Thailand. The statistical analysis was suggested and help given by Dr Thotsapol Chaianunporn, Faculty of Science, Khon Kaen University. Funding: This study was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of

the Higher Education Commission, through the heath cluster, project ‘Specific Health Problems in the Greater Mekong Sub-region (SHeP-GMS)’ of Khon Kaen University, Faculty of Medicine, Khon Kaen University and Melioidosis Research Center, Khon Kaen University, Thailand. Competing interests: None declared. Ethical approval: Not required.

References 1 Cheng AC, Currie BJ. Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev 2005;18:383–416. 2 White N. Melioidosis. Lancet 2003;361:1715–22. 3 Limmathurotsakul D, Dance D, Wuthiekanun V et al. Systematic review and consensus guidelines for environmental sampling of Burkholderia pseudomallei. PLoS Negl Trop Dis 2013;7:e2105. 4 Aldhous P. Tropical medicine: melioidosis? Never heard of it. Nature 2005;434:692–3. 5 Palasatien S, Lertsirivorakul R, Royros P et al. Soil physicochemical properties related to the presence of Burkholderia pseudomallei. Trans R Soc Trop Med Hyg 2008;102 (Suppl):S5–9. 6 Inglis TJ, Sagripanti JL. Environmental factors that affect the survival and persistence of Burkholderia pseudomallei. Appl Environ Microbiol 2006;72:6865–75. 7 Andresen K, Dargis R, Kemp M, Christeinsen K. Detection of Burkholderia pseudomallei by SYBR green real time PCR. Open Pathol J 2009;3:30–2. 8 Ma G, Zheng D, Cai Q, Yuan Z. Prevalence of Burkholderia pseudomallei in Guangxi, China. Epidemiol infect 2010;138:37–9. 9 Supaprom C, Wang D, Leelayuwat C. Development of real-time PCR assays and evaluation of their potential use for rapid detection of Burkholderia pseudomallei in clinical blood specimens. J Clin Microbiol 2007;45:2894–901. 10 Thibault FM, Valade E, Vidal DR et al. Development of real-time PCR assays and evaluation of their potential use for rapid detection of Burkholderia pseudomallei in clinical blood specimens. J Clin Microbiol 2004;3:5871–4. 11 Wuthiekanun V, Dance DA, Wattanagoon Y et al. The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice. J Med Microbiol 1990;33:121–6. 12 Ashdown LR, Clarke SG. Evaluation of culture techniques for isolation of Pseudomonas pseudomallei from soil. Appl Environl Microbiol 1992;58: 4011–5. 13 Anuntagool N, Naigowit P, Petkanchanapong V et al. Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. J Med Microbiol 2000;49:1075–8. 14 Yeates C, Gillings MR, Davison AD et al. Methods for microbial DNA extraction from soil for PCR amplification. Biol Proced Online 1998;1:40–7. 15 Trochimchuk T, Fotheringham J, Topp E et al. A comparison of DNA extraction and purification methods to detect Escherichia coli O157:H7 in cattle manure. J Microbiol Met 2003;54:165–75. 16 Sambrook JF, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbour Laboratory Press; 2001. 17 Perolat P, Merien F, Ellis WA, Baranton G. Characterization of Leptospira isolates from serovar hardjo by ribotyping, arbitrarily primed PCR, and mapped restriction site polymorphisms. J Clin Microbiol 1994;32: 1949–57.

467

Downloaded from http://trstmh.oxfordjournals.org/ at University of Bath on July 13, 2015

collected within only a 2 month period in the same season and in a small area, the moisture content and the total organic carbon (TOC) in soil samples at 30 cm depth were not significantly different. From grain size analyses to classify the soil, it was mostly found to be clayed sand (data not shown). The culture proven of B. pseudomallei in a natural habitat together with the help of molecular detection could demonstrate more precise factors affecting the presence of the organism. Microbial communities in which the B. pseudomallei are found may have a direct effect on the presence of B. pseudomallei. Most of these microbes, however, are unculturable under normal laboratory conditions. The RAPD technique was used to investigate soil microbial communities or their diversity in terms of richness and modified richness of soil microbial diversity that was affected by agricultural chemicals.24 Furthermore, RAPD method with 10-mer primers was successfully used to identify and assess genetic diversity between isolates of Streptomyces isolated from soil by comparing with reference strains.25 The current experiment therefore used the RAPD technique with RSP primer17 to investigate soil microbial diversity. The statistical analysis showed trend of higher number of band in the B. pseudomallei negative soil. The low number of DNA fragments indicated a low diversity of microbial communities.24 The lower pH found in positive sites might selectively preserve types of organisms including B. pseudomallei or some compounds produced by these organisms might affect the diversity in that location. Identification of positive and negative soils with the presence and absence of B. pseudomallei with more robust techniques would be helpful to indicate the affecting biological factors and could lead to the control of B. pseudomallei in soil. As only one soil site has been investigated and a small number of positive and negative sites for B. pseudomallei were analyzed by RAPD, more of the soil samples from other sites may help confirm these finding. In conclusion, the qPCR to detect wcbG of B. pseudomallei is specific and sensitive to detect the presence of B. pseudomallei in soil. The low pH of the soil was confirmed to have a strong affect on the presence of B. pseudomallei. It is also the first time that B. thailandensis was reported to be present in the same soil sites as B. pseudomallei. The RAPD revealed a trend of lower diversity of microbes in soil with the presence of B. pseudomallei. Further investigations to indicate significant organisms present in B. pseudomallei positive and negative soil may pave the way to control the bacteria in the most important reservoirs.

R. W. Sermswan et al.

18 Vuddhakul V, Tharavichitkul P, Na-Ngam N et al. Epidemiology of Burkholderia pseudomallei in Thailand. Am J Trop Med Hyg 1999;60: 458–61. 19 Suputtamongkol Y, Chaowagul W, Chetchotisakd P et al. Risk factors for melioidosis and bacteremic melioidosis. Clin Infect Dis 1999;29: 408–13. 20 Trung TT, Hetzer A, Go¨hler A et al. Highly sensitive direct detection and quantification of Burkholderia pseudomallei bacteria in environmental soil samples by using real-time PCR. Appl Environ Microbiol 2011;77:6486–94. 21 Kaestli M, Mayo M, Harrington G et al. Sensitive and specific molecular detection of Burkholderia pseudomallei, the causative agent of

melioidosis, in the soil of tropical northern Australia. Appl Environ Microbiol 2007;73:6891–7. 22 Ngamdee W, Tandhavanant S, Wikraiphat C et al. Competition between Burkholderia pseudomallei and B. thailandensis. BMC Microbiol 2015;15:56. 23 Wang-Ngarm S, Chareonsudjai S, Chareonsudjai P. Physicochemical factors affecting the growth of Burkholderia pseudomallei in soil microcosm. Am J Trop Med Hyg 2014;90: 480–5. 24 Yang S. Melioidosis research in China. Acta Trop 2000;77:157–65. 25 Malkawi HI, Saadoun I, Moumani FA, Meqdam MM. Use of RAPD-PCR fingerprinting to detect genetic diversity of soil Streptomyces isolates. New Microbiol 1999;22: 53–8.

Downloaded from http://trstmh.oxfordjournals.org/ at University of Bath on July 13, 2015

468

Direct detection of Burkholderia pseudomallei and biological factors in soil.

Burkholderia pseudomallei, a Gram-negative saprophytic bacillus, is a severe infectious agent that causes melioidosis and soil is the most important r...
577KB Sizes 2 Downloads 8 Views