Effectiveness of a Simplified Method for Isolation of Burkholderia pseudomallei from Soil Direk Limmathurotsakul,a,b Vanaporn Wuthiekanun,b Premjit Amornchai,b Gumphol Wongsuwan,b Nicholas P. J. Day,b,c and Sharon J. Peacockb,d,e Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailanda; Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailandb; Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, United Kingdomc; Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailandd; and Department of Medicine, Cambridge University, Addenbrooke’s Hospital, Cambridge, United Kingdome
Detection of environmental Burkholderia pseudomallei indicates a risk for melioidosis and is important for the development of a global risk map. We describe a simple method for detecting B. pseudomallei using direct culture of soil in enrichment broth. This gives a rate of positivity comparable to that obtained with a standard method but is cheaper and labor saving.
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elioidosis, a fatal infectious disease caused by the Gramnegative bacterium Burkholderia pseudomallei, is highly endemic in Thailand, where it is the third most common cause of death due to infectious diseases, after HIV/AIDS and tuberculosis (8), and in northern Australia, where it is the most common cause of fatal community-acquired septicemic pneumonia (5). The natural habitat of B. pseudomallei is soil and surface water, from which humans acquire melioidosis following bacterial inoculation, inhalation, or ingestion. Indigenous melioidosis is being increasingly reported in many countries, including China, India, Kenya, Madagascar, Costa Rica, El Salvador, Brazil, and Venezuela (4, 7), suggesting that the global distribution of environmental B. pseudomallei is wider than appreciated. Environmental sampling to detect the presence of B. pseudomallei is fundamental to defining geographical areas where humans and animals are at risk of melioidosis. The current standard for detection of B. pseudomallei in soil is culture, a method that has the advantage over other approaches such as PCR in that it has 100% specificity, requires only basic equipment, and provides live organisms for further use. We use a technique (method 1) (Table 1) in which 100 g of soil and 100 ml of distilled water are placed in a plastic bag and shaken vigorously for about 1 min, the bag is left to stand overnight, and then the upper layer of water is collected for culture (9, 11, 13). Aliquots are spread plated onto Ashdown agar (1), incubated at 40°C in air, and visually inspected daily for 4 days for colonies of B. pseudomallei. An additional 1 ml of sample is added to 9 ml of a selective enrichment broth, incubated at 40°C in air for 48 h, and plated as before onto Ashdown agar. This is labor intensive and limits the number of samples that can be tested at any one time. This becomes a problem when sampling is being done in an area where there is uncertainty about the likelihood of positivity or where the proportion of positive samples may be very low. Under such circumstances, the number of samples required to demonstrate positivity or provide a robust result for a true negative is high (9, 11). The objective of this study was to determine the B. pseudomallei yield from soil using our current method versus a range of alternative methods that are labor saving. In March 2011 (pre-rainy season), 200 soil samples were taken from two rice fields (100 samples per field) in Ubon Ratchathani, Thailand. The soil type was sandy loam, and the soil was moist at the time of sampling. Each field was divided into a grid system, in
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which 100 sampling points (10 by 10) were plotted 2.5 m apart. At each sampling point, around 200 g of soil was removed from the base of a 30-cm hole, placed in a plastic bag, and transferred on the same day at ambient temperature to the laboratory. Four different methods of soil culture were performed in parallel for the 200 samples (Table 1). The B. pseudomallei yield of our current method (method 1) was compared that of with methods that introduced reduction in the volume of soil cultured (methods 2, 3, and 4). Method 3 also shortened the sedimentation time after the soil was mixed with distilled water, while method 4 omitted the sedimentation step by adding the soil directly to culture broth. Of 200 soil specimens, 94 were culture positive for B. pseudomallei by at least one method (Table 2). The sensitivity of each method was defined by comparing yield against the cumulative yield for all four methods. The standard method (method 1) yielded 70 positive samples out of 94 (74.5%), which was comparable to the results of method 4 (direct culture), which yielded 79 positive samples (84.0%; P ⫽ 0.15, McNemar’s exact test). This indicates that our standard method could be replaced with this labor-saving alternative. Other methods gave a significantly lower yield (Table 2). Method 2, which used a smaller volume of soil sample for culture but no other significant changes, gave a poor yield, probably reflecting a lower number of bacteria in 10-g versus 100-g soil samples, with either an absence of B. pseudomallei in the sample or more likely a fall below the limit of detection imposed by the method used for plating or subculturing in selective broth. The further reduction in yield in method 3, which combined reduced soil sample volume and reduced sedimentation time, may be due to a reduction in the proportion of B. pseudomallei organisms that transit from soil to the liquid fraction over a more limited time frame or to the fact that B. pseudomallei organisms undergo replication during overnight sedimentation.
Received 28 September 2011 Accepted 3 November 2011 Published ahead of print 18 November 2011 Address correspondence to Direk Limmathurotsakul,
[email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.07039-11 The authors have paid a fee to allow immediate free access to this article.
0099-2240/12/$12.00
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Simplified Method for Isolating B. pseudomallei from Soil
TABLE 1 Details of culture methods evaluated Characteristic
Method 1a
Method 2
Method 3
Method 4
Sample size (n) Soil sample (grams) Container Liquid added to make soil suspension Mixing of soil and liquid Postsedimentation step(s)
200 100 Plastic bag Distilled water (100 ml)
200 10 Universal tube Distilled water (10 ml)
200 10 Universal tube Distilled water (10 ml)
Manual shaking, 1 min After overnight sedimentation, (i) direct subculture of 10 l, 10 l, 100 l, and 100 l onto four Ashdown agarc plates and (ii) subculture of 1 ml into TBSS-C50 (9 ml) Broth cultures were incubated at 40°C in air for 48 h, and 10 l was subcultured onto Ashdown agar plates; agar plate cultures were incubated at 40°C in air and inspected visually for 4 days
Vortex mixer, 30 s After overnight sedimentation, subculture of 1 ml into TBSS-C50 (9 ml)
Vortex mixer, 30 s After 1 h sedimentation, subculture of 1 ml into TBSS-C50 (9 ml)
200 10 Universal tube Direct broth culture of soil using TBSS-C50b (10 ml) Vortex mixer, 30 s No sedimentation step required
Same as for method 1
Same as for method 1
Same as for method 1
Culture conditions
a
Method 1 is our standard method. TBSS-C50 broth consists of threonine-basal salt solution (TBSS) plus colistin 50 mg/liter (13). c Ashdown agar consists of Trypticase soy agar with 4% glycerol, crystal violet 5 mg/liter, neutral red 50 mg/liter and gentamicin 4 mg/liter (1). b
This is the first study to compare the effect of different soil sample volumes on the culture detection of B. pseudomallei. In the published literature, the weight of soil samples cultured has ranged from 2 to 1,000 g, and the extraction solution used has varied between distilled water and enrichment media (2, 6, 10). Our study shows that 10 g of soil can be used in place of 100 g provided that a direct culture technique is used. Using 10 g also has the advantage that a greater number of samples can be taken at negligible extra cost. TBSS-C50 (threonine-basal salt solution plus colistin at 50 mg/liter) was selected because it is commonly used as an enrichment broth for soil culture and is inexpensive (13). It is possible that other enrichment broths described in the literature (3, 6, 10, 12), such as modified Ashdown broth or PEG-DOC (polyethylene glycol and sodium deoxycholate) solution, would yield comparable results. In summary, we have found that direct culture of soil in enrichment broth, omitting steps involving the creation of a soilliquid suspension, sedimentation, and culture of the surface liquid, gives a number of positive results comparable to that obtained with a more laborious method currently in use in our laboratory. We intend to adopt the direct culture method and recommend it for its simplicity and effectiveness.
TABLE 2 Sensitivities of variable culture methods for 94 soil samples that were positive for B. pseudomallei by at least one method
Method
No. of positive samples
Sensitivity (%) (95% confidence interval [%])
P valuea
Method 1 Method 2 Method 3 Method 4
70 39 26 79
74.5 (64.4 to 82.9) 41.5 (31.4 to 52.1) 27.7 (18.9 to 37.8) 84.0 (75.0 to 90.8)
⬍0.001 ⬍0.001 0.15
a Calculated by McNemar’s exact test, comparing the sensitivity of methods 2, 3, and 4 to that of method 1, which is the standard method used in our laboratory. The sensitivities of methods 2 and 3 were significantly lower than that of method 1, while the sensitivity of method 4 (direct culture) was not significantly different from that of method 1.
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ACKNOWLEDGMENTS We are grateful to staff at the Mahidol-Oxford Tropical Medicine Research Unit for their support. We thank Sayan Langla, Areeya Faosap, and Sujittra Ngamwilai for technical assistance. This study was funded by The Wellcome Trust.
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