สรุป: B. amyloliquefaciens N3-8 ในสัดส่วน 1:300 สามารถลดปริมาณ B. pseudomallei p37ในดินได้ชัดเจน การทดสอบเพิ่มในดินธรรมชาติ จะช่วยประเมินศักยภาพการนำไปประยุกต์ใช้ลดปริมาณ B. pseudomalleiในพื้นที่ระบาดได้
Background and Objectives: Burkholderia pseudomallei, a Gram-negative bacterium causes a disease called Melioidosis. It is mostly found in soil and stagnant water in endemic areas. B. amyloliquefaciens N3-8 was isolated from soil and its secondary metabolites can kill B. pseudomallei that may be used to control B. pseudomallei in soil.
Methods: B. pseudomallei p37 and B. amyloliquefaciens N3-8 were separately cultured to obtain 1x 106and 1X109CFU.mL-1. Then mixed B. pseudomallei p37:B. amyloliquefaciens N3-8 with 1:50, 1:100 and 1:300 ratios of cell into 10 g of soil and incubated at room temperature for 4 weeks. The soil has pH=6.98.
Results:The ratio of 1:300 can reduce the number of B. pseudomallei p37 in soil starting from the second week. The pH of soil in the presence of B. amyloliquefaciens N3-8 was significantly increased when compared at the beginning with the 4th week (p< 0.01). At the end, soil with B. pseudomallei p37 had pH = 7.35±0.05, soil with B. amyloliquefaciens N3-8 had pH = 8.26 ±0.06, soil with ratios of 1:50, 1:100 and 1:300 had pH = 8.08 ±0.05, 8.03 ±0.05 and 8.81 ±0.05.
Conclusions: B. amyloliquefaciens N3-8 at 1:300 ratio can clearly reduce the number of B. pseudomallei p37 in soil. Further investigation in natural soil environment could support the potential use of B. amyloliquefaciensN3-8 as a bio-control to reduce B. pseudomallei in endemic areas.
B. pseudomallei can be isolated from soil and water in endemic areas such as Southeast Asia and northern Australia 1. The bacterium causes the disease called melioidosis that occurred in both human and animal2. Routes of B. pseudomallei infection are direct contact of soil and water that contaminated with B. pseudomallei to open wound or by inhalation and ingestion. It is an important cause of septicemia and approximately 20% of community-acquired bacteremia reported in Ubon Ratchathani, northeastern Thailand3. Moreover, melioidosis is the third most common cause of death in the northeast, Thailand from infectious disease inferior to HIV and Tuberculosis 4. Soil is the most important reservoir of the bacterium and also the source of infection.
Biological control is a bio-effector-method for controlling one organism by its antagonistic microorganism. The mechanisms can be competition, parasitism or antibiosis 5. As it does not use chemicals, the method will not cause environmental pollution. Bacillus spp. is a group of Gram-positive spore-forming bacterium commonly found in nature. They were known to produce a variety of secondary metabolites that can compete against other organisms in the same environment6, 7. From previous study in our laboratory, Bacillus amyloliquefaciens isolates N3-8 were isolated from soil in Khon Kaen that found negative for B. pseudomallei. The culture supernatant of B. amyloliquefaciensN3-8 can inhibit several isolates of B. pseudomallei including drug resistant isolates and co-culture of B. amyloliquefaciensN3-8 with B. pseudomallei in liquid medium clearly showed dramatically decrease of B. pseudomallei8. In this study, we therefore investigated if B. amyloliquefaciens N3-8 can be used as a bio-control for controlling B. pseudomallei in soil in laboratory setting condition.
Materials and methods
A batch of 10 kg of soil was taken from a grass field belonging to the Faculty of Agriculture. The soil was grinded and filtered through 2 mm sieve to get rid of roots, pebbles and others. The water holding capacity (WHC) of the soil was culculated and adjusted by sterile distilled water to have 100% WHC9.Then, the whole lot of soil was autoclaved at 121 Co for30 min and check by culture on nutrient agar (NA) to confirm sterility.
Strains and growth conditions
B. amyloliquefaciens strains N3-8 and B. pseudomallei p37 were obtained from the melioidosis research center, Faculty of Medicine, Khon Kaen university, Thailand. B. amyloliquefaciens strains N3-8 was isolated from soil and can produce secondary metabolites that can kill B. pseudomallei. B. pseudomallei p37 was isolated from blood of a patient in Khon Kaen province. It was proved to contain no inducible phages. For propagation, B. amyloliquefaciens N3-8 and B. pseudomallei p37 from the stock were cultured separately on nutrient agar and Ashdowns agar plates, respectively and incubated at 37 °C to obtain pure single colony.
The propagation of B. amyloliquefaciens N3-8 was performed by inoculated a single colony into LB broth and incubated in shaking incubator of 200 rpm at 37 °C for 18 h. One percent inoculum of the culture was used to inoculate into LB broth and culture for 24 h to obtain 1x109 CFU.mL-1 and used in the bio-control experiment. For B. pseudomallei p37, the bacterium was grown in LB broth and culture for 4 h to obtain 1x 106 CFU.mL-1.
Agar well diffusion
To confirm the production of metabolites fromB. amyloliquefaciens strains N3-8 that can inhibitB. pseudomallei, agar well diffusion method was used to determine the antimicrobial activity10. In brief, B. pseudomallei were grown in Luria Bertani (LB) broth for 16-18 h and 10% inoculum were used to inoculate into 3 ml LB and incubated for 4 h until log phase. A hundred microliters of the culturewere spread on Mueller Hinton agar (MHA) plates, dried in a biohazard laminar flow cabinet (Esco Technologies, Horsham, PA) and then punched 5-6 wells/plate using a sterile micropipette tip. A hundred microliters of supernatant from B. amyloliquefaciens strains N3-8 were then added into each well and incubated at 37 °C for 24 hours. Ceftazidime, the drug of choice for B. pseudomallei was used as a positive control and the production medium was used as a negative control.
Bio-control in soil conditions
All experiments were operated in triplicate in sterile 50 ml self-standing conical plastic tubes. B. amyloliquefaciens N3-8 and B. pseudomalleip37 weremixed into 10 g, 100% WHC soil to obtain 3 conditions 1.) Control B. amyloliquefaciensN3-8,1x108 CFU.g-1soil, 2.) Control B.pseudomalleip37, 1x105CFU.g-1soil and 3.) Co-culture between B. pseudomalleip37 and B. amyloliquefaciens N3-8 with the CFU ratios of 1:50, 1:100 and 1:300. Each condition had 4 tubes to be sampled each week. The tubes with loosely closed cap were incubated at room temperature for 4 weeks.
On the 2nd week, 5x107 CFU.g-1 (1:50 ratio), 1x108 CFU.g-1 (1:100) and 3x108 CFU.g-1 (1:300) of B. amyloliquefaciens N3-8 were added into the co-culture soil, mixed well and incubate further for another 2 weeks. To determine the number of B. pseudomallei p37 in the control and co-culture at 0, 1, 2, 3 and 4 weeks, 10 ml of sterile DW was added, shake vigorously and stand for 30 minutes to let soil particles settle. The supernatant was collected to measure pH and also 10-fold serially diluted and then drop 10 l on Ashdowns agar for B. pseudomalleip37 colony count. For B. amyloliquefaciens N3-8 colony count, the process was similar and performed only in B. amyloliquefaciens N3-8 control and Phenylethyl alcohol agar (PAA) was used for B. amyloliquefaciens N3-8 colony count (Figure 1).
Figure 1 The schematic diagram of steps in the bio-control experiment.
Mann-Whitney U Test was used to compare mean-values of pH of soil in bio-control experiment and the statistic considered to be significance whenp<0.01.
B. amyloliquefaciens N3-8 and its antimicrobial activity
The morphologies of B. amyloliquefaciens N3-8 are being white and large with wavy, lobed margins were observed on nutrient agar plates (Figure 2A) that confirmed by Grams stain to be Gram-positive rods (Figure 2B) and confirmed for the production of antimicrobial activity as seen by clear zone on B. pseudomallei lawn (Figure 2C).
Figure 2 Morphology and antimicrobial activity of B. amyloliquefaciens N3-8. A) colonies of B. amyloliquefaciens N3-8 on nutrient agar plate, B) Grams stain and C) clear zone from action of antimicrobial activity on B. pseudomallei p37 lawn. Ceftazidime (50 g.mL-1) was served as a positive control and LB as a negative control.
Bio-control in soil
The soil pH before the experiments was 6.89 and changed to be 7.26 when adjusted to have 100% WHC. The colony count of the control B. pseudomallei p37 in 10 g soil was 1x 105 CFU.g-1 at the beginning and increased to 1x 108 CFU.g-1 on the 1st week before slightly decreased on the second week and increased again until the 4th week (Figure 3). The number of B. amyloliquefaciens N3-8 in the control soil was 1x 108 CFU.g-1 at the beginning and slightly increased until the 4th week.
For the bio-control experiments, the pattern of CFU.g-1 of B. pseudomallei p37 in the 1:50 and 1:100 ratios were similar to what observed in the B. pseudomallei p37 control soil but the number of B. pseudomallei p37 was decreased about 0.5-1 log10 on the 2nd week onward when compared to the control. For the 1:300 ratio, the number of B. pseudomallei p37 was abruptly decreased from the 1st week and about 4 log10 on the 3rd week. The inoculation of B. amyloliquefaciens N3-8 into the bio-control experiments on the 2nd week could not decrease the pathogen in 1:50 and 1:100 ratios but can drop it down when1:300 ratio was used.
Figure 3 The colony count of B. amyloliquefaciens N3-8 and B. pseudomallei p37 in soil. The Y axis showed CFU.g-1soil and the X axis showed time in week. The black line showed the CFU.g-1 soil of B. pseudomallei p37 control, dark brown line was B. amyloliquefaciens N3-8 control and green dashed, black dot and black dashed line were CFU.g-1soil of B. pseudomallei p37 when co-culture with B. amyloliquefaciens N3-8 at the ratios of 1:50, 1:100 and 1:300. The arrow indicated the time when B. amyloliquefaciens N3-8 was added.
The pH of soil in all tubes were significantly increased when incubate for 4 weeks. The average pH of soil in the presence of B. amyloliquefaciens N3-8 control (1x108CFU.g-1) and all co-culture tubes on the 4th week were much higher than the B. pseudomallei p37 control and pH of soil before the experiments.(Figure 4).
Figure 4 The pH of soil in each experiment. The Y axis is pH and the X axis indicated each experiment. The dark gray bars were pH at the beginning and light gray showed pH of soil on the 4th week. The asterisk (**) indicated significant between week 0 and week 4 (p<0.01).
Soil is considered to be a complex environment and is a major reservoir of living organisms either in competitive or symbiotic microbial communities11. Bacillus species can produce a large number of metabolites such as peptides and non-peptides with biological activities, some of which can be used as bio-controls, mostly for plant diseases12. Bacillus amyloliquefaciens has been recognized as a good plant growth promoter and root colonizer13. Several strains of B. amyloliquefaciens were investigated as a bio-control agent against plant pathogens such as B. amyloliquefaciens SQR9 that controls cucumber and water melon wilt disease14. B. amyloliquefaciens FZB42 was also reported as a bio-fertilizer and bio-control agent in agriculture 15. To our knowledge, none of the bio-control concept have been applied to control human pathogen is soil. The difficulties are mostly come from the complexity of soil that compost of a huge number of living organisms and the large area of soil to handle. However, when soil is the most important reservoir of a pathogen, controlling them in some certain areas such as in the zoo where melioidosis is endemic is therefore can be a clear benefit to protect centennial exotic animals that susceptible to B. pseudomallei infection.
B. pseudomallei is unevenly present in soil and the number of the bacterium varies for example from a few to 104 CFU.g-1of soil as reported from Laos16 or 103-106 CFU.g-1in Khon Kaen (unpublished data). The most common route of infection is the exposure of skin abrasion to contaminated soil and water17. Therefore, the reduction of the bacterial number in soil might decrease the risk of infection. The amount of B. pseudomallei p37 used to spike into the soil was 1x105 CFU.g-1 that relatively high comparing to what generally found. The 1:300 ratio of B. pseudomallei p37: B. amyloliquefaciens N3-8 can decrease the amount of B. pseudomallei p37 down to 106within 2 weeks and lower the bacterial count further totally by 4 log10 at the 4th week after B. amyloliquefaciens N3-8 was added. Without adding the bacterium for the second time, the bacterium did not show a sharp decrease (data not shown). After the battle between these organisms was over and soil condition was suitable, B. amyloliquefaciens N3-8 can form spore and persist in soil waiting to germinate again similar to nature of other Bacillus spp. in soil18. The longer observation may help prove and predict if this situation will occur and could prolong controlling of B. pseudomallei.
Soil in the northeast of Thailand showed a few abiotic factors that were significantly different when compared between soil that positive and negative for B. pseudomallei19, 20. Acidic pH range was found to be related to the present of B. pseudomallei. The soil in this experiment was obtained from the area where B. pseudomallei were found, however, the pH was not in the acidic condition. When B. pseudomallei p37 was inoculated into the soil and incubated for 4 weeks, the bacterium showed ability to grow in this soil condition. The soil in B. amyloliquefaciens N3-8 control tube and co-culture in all ratios showed the increased in pH value in alkali range and more than soil with B. pseudomallei alone. This range of pH may not suitable forB. pseudomallei, however, the alkaline pH alone was not enough to decrease a large number of B. pseudomallei as seen in 1:50 and 1:100 ratios. The changes of pH if apply in the soil that found to be acidic may help improve the quality of soil. Nevertheless, more experiments have to be set-up in order to prove this hypothesis. The secondary metabolites from B. amyloliquefaciens N3-8 were reported to inhibit a range of pathogen but not non-pathogenic bacteria in soil even a closely related B. thailandensis 8. Moreover, the bacterium was isolated from soil in the northeast of Thailand that unlikely to do harm to the ecosystem. However, investigation of bacterial diversity in soil after treatment with B. amyloliquefaciens N3-8 should be perform to confirm.
The 1:300 ratio of B. pseudomallei: B. amyloliquefaciensN3-8 bio-control can clearly decrease B. pseudomallei in laboratory soil condition. The pH changed may come from the secondary metabolites of B. amyloliquefaciens N3-8. Further investigation of the bio-control in a field experiment should provide more information if B. amyloliquefaciens N3-8 can be used as a bio-control of B. pseudomallei in nature.
This study was supported by the Melioidosis Research Center (MRC), Faculty of Medicine, Khon Kaen university, Khon Kaen, Thailand and the Royal Golden Jubilee Ph.D. Program (RGJ:4.O.KK/51/I.1.A. XX).