Melioidosis is an often fatal infectious disease affecting humans and animals in tropical regions and is caused by the saprophytic environmental bacterium Burkholderia pseudomallei. Domestic gardens are not only a common source of exposure to soil and thus to B. pseudomallei, but they also have been found to contain more B. pseudomallei than other environments. In this study we addressed whether anthropogenic manipulations common to gardens such as irrigation or fertilizers change the occurrence of B. pseudomallei. We conducted a soil microcosm experiment with a range of fertilizers and soil types as well as a longitudinal interventional study over three years on an experimental fertilized field site in an area naturally positive for B. pseudomallei. Irrigation was the only consistent treatment to increase B. pseudomallei occurrence over time. The effects of fertilizers upon these bacteria depended on soil texture, physicochemical soil properties and biotic factors. Nitrates and urea increased B. pseudomallei load in sand while phosphates had a positive effect in clay. The high buffering and cation exchange capacities of organic material found in a commercial potting mix led to a marked increase in soil salinity with no survival of B. pseudomallei after four weeks in the potting mix sampled. Imported grasses were also associated with B. pseudomallei occurrence in a multivariate model. With increasing population density in endemic areas these findings inform the identification of areas in the anthropogenic environment with increased risk of exposure to B. pseudomallei.
Southeast Asia and tropical Australia have recently experienced a surge in melioidosis, an often fatal infectious disease caused by the saprophytic environmental bacterium Burkholderia pseudomallei [1,2]. Case numbers in the Top End of Australia have substantially increased in recent years. In the 20 years from 1989 until 2009 there was a median of 27 cases annually [3]. In the last 5 years there has been a median of 64 cases annually and in each of two recent years, 1 in every 2,000 people living in the Top End has had culture confirmed melioidosis [4]. B. pseudomallei are found in soil and water world-wide in the tropical belt with the major endemic region being southeast Asia and tropical Australia [5–10]. B. pseudomallei is an opportunistic pathogen able to infect humans [11] and a large variety of animals [12]. Humans with a compromised immune system such as from diabetes, hazardous alcohol use, chronic renal disease and immunosuppressive therapy are at particular risk of acquiring and dying from melioidosis [13]. Clinical presentations vary widely and include skin and soft tissue abscesses, pneumonia and disseminated infection with septic shock, the latter having mortality rates above 80% [14]. The Darwin area (12° S latitude) in the tropical north of Australia is endemic for melioidosis and gardening is considered to be an important recreational and occupational source of exposure to and ultimately, infection with B. pseudomallei [3]. In the 20-year Darwin prospective melioidosis study, 407 (75%) of 540 consecutive melioidosis patients had documented recreational activities such as gardening or outdoor sporting activities where exposure to B. pseudomallei was considered likely to occur [3]. Domestic gardens are not only a common ground for humans to be exposed to the environment, but B. pseudomallei might also thrive in the garden habitat. While B. pseudomallei and melioidosis predominate in the monsoonal wet season [3], previous work in rural Darwin found that in the dry season B. pseudomallei is more often present in domestic gardens than in farms or environmentally less disturbed areas [15]. This might be attributed to the widespread use of irrigation during the dry season. Being a non-spore forming, gram negative bacterium, B. pseudomallei is often, but not exclusively associated with moist soil close to a water source and with surface water or alluvial areas as well as rice fields [7,15–19]. At environmentally disturbed sites, B. pseudomallei was associated with pens or paddocks for pigs, chickens or horses with an average odds ratio of 3. 8 [15]. This raises the possibility that soil aeration through digging activities or organic material and nitrogen from animal waste support growth of B. pseudomallei [15]. In this study, we addressed the hypothesis that anthropogenic manipulations associated with gardens such as the use of irrigation, fertilizers, commercial potting mix or keeping pets influence the habitat of B. pseudomallei and change its abundance and/or occurrence. We conducted a soil microcosm experiment with a selection of fertilizers as well as a longitudinal study over three years on an experimental fertilized field site in a location naturally endemic for B. pseudomallei.
In August 2008 an experimental site was established on a private property in rural Darwin in an area that previously tested positive for B. pseudomallei. The soil at this site was a hydrosol [20] and the soil texture of the topsoil was clay with a subsoil consisting of grey clays and siltstone. The site consisted of two plots, 0. 75 metres apart and each plot had six 1x1 metre quadrants (Fig. 1), which included a control quadrant and five quadrants with different treatments which represent common garden practices in the Darwin region (Table 1). Treatments were applied every two weeks with water application every 2nd day for three years. Timing and dose reflected local garden practices. The water used was unchlorinated water from the property’s bore with a pH of 7. 5 containing 50 mg/L calcium carbonate and which repeatedly tested negative for B. pseudomallei by culture. There were 14 rounds of soil sampling and in each round 2 random soil samples were collected from each of the 12 quadrants to give a total of 336 soil samples. The first sampling round was before the start of the experiment in August 2008 followed by sampling every two months in year-1, every three months in year-2 and every four months in year-3 of the experiment, with the last round in August 2011. Soil from a depth of 20–30 cm was collected into sterile 50 mL specimen containers and auger and spade were cleaned with 70% ethanol between soil collections [21]. Soil moisture was determined as described previously using the Australian Soil and Land Survey Field Handbook [21,22]. Soil pH was measured using a soil pH field test kit (Inoculo, Australia). In the last 6 months, soil electrical conductivity (EC) was measured using the Field Scout EC Meter (Spectrum Technologies, USA). Grasses covering the experimental field site were identified by the Northern Territory Government Herbarium and were either Sorghum spp. (spear grass) or Pennisetum pedicellatum (annual “mission grass”). At the time of sampling, the presence or absence of live specimens of these grasses at the sampling hole was noted. Of 120 250-mL clean and autoclaved plastic containers, 30 were each filled with either 130 g of commercial “garden soil”, sandy clay loam, clay or sand (Table 2). The non-commercial soil was collected in rural Darwin and tested negative for B. pseudomallei by culture. Nine different treatments plus controls (no change) were applied in triplicate to the containers (Table 3). Treatments included the addition of distilled water or distilled water in combination with eight fertilizers which are commonly used in residential gardens in the Darwin region. After two weeks of soil conditioning at 32 degrees Celsius in the dark, all soils were inoculated with 5x10e4 CFU of an environmental strain of B. pseudomallei (MSHR2817) which has the commonly found multi-locus sequence type (ST) 144 [23] and incubated at 32 degrees Celsius for four weeks in the dark. Soil DNA was extracted and B. pseudomallei DNA detected as described below. Soil DNA extraction was done as previously described [15,21]. Briefly, 20 g of soil were incubated with 20 mL of Ashdown’s broth for 39 hours shaking at 37°C, the soil supernatant was centrifuged twice and the pellet processed using the PowerSoil Kit (MoBio Laboratories, USA). Modifications included the addition of 0. 8 mg of aurintricarboxylic acid (ATA) and 20 μL of proteinase K (20 mg / mL). B. pseudomallei DNA was targeted by the well validated B. pseudomallei specific Type Three Secretion System-1 TTS1 real-time PCR [24,25]. For the microcosm experiment, DNA was extracted from 20 g of soil using the previously described semi-quantification method with an internal extraction and amplification plasmid control [23]. TTS1 copy numbers were normalized by dividing them by the copy number of the internal pt7 plasmid control which was added to the soil samples prior to extraction, in order to account for differences in DNA extraction and amplification efficiency as a result of varying amounts of inhibitors present in soil samples [23]. Statistical analysis was carried out using Stata (Intercooled Stata, version 12. 1, USA). For bivariate analyses, Fisher’s exact test and Mann-Whitney U test were used. All tests were 2-tailed and considered significant if P values were less than 0. 05. Graphs were generated in Stata and GraphPad Prism 6. For the experimental field site, a conditional logistic regression model was used to model the odds of B. pseudomallei occurrence once the experiment had started, with fixed effects for treatments and dates of sampling. Fractional treatment effects were assumed for the first 12 months (e. g. 50% of full treatment effect after 6 months) to allow the application of fertilizers to have a gradual effect on the soil environment and Burkholderia community [26]. Heat maps for the experimental field site were generated using the thin-plate-spline interpolation method in ArcGIS 10. 1 (ESRI 2012).
B. pseudomallei occurrence was monitored over three years on a field site with five different treatments applied in an area in rural Darwin naturally positive for B. pseudomallei (Fig. 1) The microcosm experiment was used to determine whether commercial fertilizers commonly used in gardens in the Darwin region increased B. pseudomallei load in soil. Eight different treatments of garden fertilizers were applied to each of four different soil types in triplicate. There were also triplicate water controls with only distilled water added and triplicate controls with nothing added. Four weeks after inoculation, no B. pseudomallei were detected in commercial garden soil for any of the treatments. Sand and clay contained on average 733 times more B. pseudomallei than sandy clay loam which only contained minimal B. pseudomallei cells (Fig. 5). After four weeks, no B. pseudomallei (10/12) or only minimal B. pseudomallei cells (2/12) were detected in the 12 control samples. The effect of fertilizers upon B. pseudomallei differed between soil type (Fig. 5). In sand, the addition of a fertilizer rich in urea showed the highest B. pseudomallei load compared to controls but the same fertilizer only had a moderate effect in clay and no effect in sandy clay loam. The pH and EC of urea in sand were lower with a mean of 4. 6 and 56 μS/cm in comparison to sandy clay loam (pH 5. 8 and EC 216 μS/cm) and clay (8. 4 and 172 μS/cm). The addition of an organic and a phosphorus rich fertilizer resulted in the two highest mean B. pseudomallei counts in clay but no such effect was seen for the other soils. The addition of water alone caused a similarly high increase in B. pseudomallei load in sand and clay which had a low VSW of 0–2% before addition of water but there was no load increase in sandy clay loam with a higher initial VSW of 5%.
In summary there was clear evidence for irrigation increasing B. pseudomallei occurrence. The effect of fertilizer application upon B. pseudomallei was more complex and was dependant on soil type and physicochemical properties as well as on vegetation, with nutrients also causing an increase in plant root development beneficial to B. pseudomallei. The use of fertilizers is causing drastic changes to the global nutrient cycle with a significant rise in supply of otherwise limiting nutrients. These changes have a major impact upon the soil and water microbial community structure and likely also upon host pathogen interactions [50], including those involving B. pseudomallei.
Melioidosis cases are on the rise in endemic areas of northern Australia and Thailand. This potentially severe infectious disease affecting humans and animals in the tropical belt is caused by the gram negative bacterium Burkholderia pseudomallei. Domestic gardens are a common point of exposure to these environmental bacteria and B. pseudomallei are more prevalent in the dry season in gardens when compared to other areas. This is why we analysed whether common gardening practices such as regular watering (irrigation) or soil fertilizing change the occurrence of B. pseudomallei. We conducted a soil microcosm experiment with a range of fertilizers and soil types as well as a longitudinal interventional study over three years on an experimental fertilized field site in an area naturally positive for B. pseudomallei. Irrigation was the only consistent treatment to increase B. pseudomallei occurrence over time. The effects of fertilizers upon these bacteria depended on soil texture, physicochemical properties such as pH or salinity and vegetation. B. pseudomallei occurrence was also associated with imported grasses. With increasing populations in endemic areas, these findings inform the identification of areas in the anthropogenic environment with increased risk of exposure to B. pseudomallei.
What Drives the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Domestic Gardens?