Isolation, identification and characterization of Burkholderia pseudomallei from soil of coastal region of India
https://doi.org/10.1186/2193-1801-3-438
© Prakash et al.; licensee Springer. 2014
Received: 31 July 2013
Accepted: 7 August 2014
Published: 16 August 2014
Abstract
Melioidosis is an emerging infectious disease caused by a free living soil dwelling Gram-negative bacterium Burkholderia pseudomallei. The disease is endemic to most parts of Southeast Asia and northern Australia and the organism has been isolated from moist soil and water. In India clinical cases are recently reported from the states of Tamilnadu, Kerala, Karnataka, Maharashtra, Orissa, Assam, West Bengal, Pondicherry and Tripura. This study is aimed to confirm the prevalence of this important bacterial species in soil samples collected from coastal areas of Tamilnadu. Forty five soil samples from five different sites were collected from Parangipettai, Tamilnadu and screened for the presence of B. pseudomallei. The study confirmed 4 isolates as B. pseudomallei with the help of conventional bacteriological methods and molecular methods that include; 16S rDNA sequencing, B. pseudomallei specific PCR, fli C gene RFLP and MALDI-TOF mass spectrometry based bacterial identification. This study reveals the prevalence and distribution of B. pseudomallei in the soil environment in coastal areas of southern India and further necessitates studies from other parts of the country. It will also be helpful to understand the distribution of B. pseudomallei and to access its epidemiological importance.
Keywords
Introduction
Melioidosis is caused by soil dwelling Gram-negative bacterium Burkholderia pseudomallei and is an emerging infectious disease in India. The disease is mainly endemic in Southeast Asia and northern Australia with highest number of melioidosis cases reported from Thailand. The global distribution boundaries of melioidosis continue to expand well beyond the traditionally recognized endemic regions (Currie et al., 2008). In India, clinical cases have been reported from states of Tamilnadu, Kerala, Karnataka, Maharashtra, Orissa, Assam, West Bengal, Pondicherry and Tripura. Burkholderia pseudomallei has been isolated from clinical samples like blood, sputum, pus, urine, synovial, peritoneal and pericardial fluids mostly from tertiary care hospitals located at Vellore, Tamil Nadu and Mangalore, Karnataka (Raghavan et al., 1991; Kavitha et al., 2008). The true incidence of melioidosis is not known in India and recently larger numbers of cases have been reported from the western coastal areas (Vidyalakshmi et al., 2007). Melioidosis is referred to as “a great imitator” because of its wide spectrum of clinical presentations, ranging from mild subclinical infection to fatal septicaemia that can be chronic, localized or disseminated. The infection occurs through inhalation, or skin abrasions that come in contact with contaminated soil or water. Diabetes is the most common risk factor that is associated with the disease and other risk factors include thalassaemia, alcoholism and renal impairment. Isolation of the organism from soil is required to define the epidemiology and distribution of B. pseudomallei, and the associated risk to humans and livestock (White, 2003; Leelarasamee, 2004). Earlier studies have shown the presence of B. pseudomallei in the environment based on culture of soil and water from different geographic regions, particularly from Southeast Asia and northern Australia (Strauss et al., 1969). The description of B. thailandensis, a non-virulent but closely related species present in the soil, has made the isolation and characterization of B. pseudomallei from soil very challenging (Brett et al., 1998). This species has similar colony morphology characteristics to B. pseudomallei on solid media and biochemical and molecular techniques are needed to distinguish between them. The isolation of B. pseudomallei from different soil depths and during different seasons of the year has been studied, and quantitative culture of B. pseudomallei from soil samples has also been done in many countries previously (Smith et al., 1995; Brook et al., 1997). Recently a review for the global presence and distribution of B. pseudomallei clearly indicates that isolation of this species from soil has not been reported from India, despite its isolation from human cases (Limmathurotsakul et al., 2013). The present study was undertaken to attempt the isolation of B. pseudomallei from the coastal rice cultivating areas of Tamil Nadu, India to confirm the identity of isolates by conventional and molecular methods.
Materials and methods
Study site and collection of samples
Soil sampling and five study sites of Parangipettai, Tamilnadu, India indicated by arrow as shown in map.
Soil processing and isolation
Schematic presentation of isolation and identification procedure used in study.
Biochemical and phenotypic confirmation
Initial screening of isolates was performed according to the standard protocols followed for the identification of B. pseudomallei (Sentinel Laboratory guidelines, 2003). Two standard strains, NCTC 1688 and NCTC 10274, were used as reference type strains along with the isolates. The commercially available API 20NE (Biomerieux) was also used for the generation of biochemical profiles of all soil isolates along with standard strain (Dance et al., 1989; Amornchai et al., 2007). Results were recorded after incubation of 24 hr to 48 hr at 28°C and interpreted referring to the data interpretation table from the API 20NE manual. In vitro antibiotic susceptibility of isolates for polymyxin B (100 units/disc) and colistin (25 mcg/disc) were also tested on Mueller–Hinton agar by the Kirby Bauer disk diffusion method (CLSI, 2007). Assimilation of L-arabinose was also tested conventionally as because it plays a key role in the discrimination of virulent B. pseudomallei from the nonvirulent species B. thailandensis (Wuthiekanum et al., 1996). The suspected isolates were further screened for yellow haze production on Francis agar for the discrimination of B. pseudomallei from B. cepacia (Francis et al., 2006).
Molecular confirmation
Identification by Specific PCR
Primers used in molecular confirmation study
Primer name | Primer sequences | Annealing temperature | Amplification size | Purpose/Reference |
---|---|---|---|---|
VMP 23-1-F | 5′ CTTTTGGGTCATCCTRGA 3′ | 48°C | 1,051 bp | Identification (Bauernfeind et al., 1998) |
MP 23-2-R | 5′ TCCTACCATGCGAGACT 3′ | |||
BPTTS1-F | 5′CGTCTCTATACTGTCGAGCAATCG 3′ | 58°C | 548 bp | Identification (Novak et al., 2006) |
BPTTS1-R | 5′ CGTGCACACCGGTCAGTATC 3′ | |||
F8 | 5′ AGTTTGATCCTGGCTCAG 3′ | 50°C | 1,488 bp | 16S sequencing (Gee et al., 2003) |
R1492 | 5′ ACCTTGTTACGACTT 3′ | |||
fli C-F | 5′ CTCGGATCCAACAGCAAC 3′ | 52°C | 1,167 bp | PCR-RFLP (Primer designed) |
fli C-R | R- 5′ TATTGCAGGTACCTTCAG 3′ |
16S rDNA based phylogenic analysis
16S rDNA sequencing was used to confirm PCR identified isolates and 16S rDNA sequence of each isolate was BLAST analysed (Gee et al., 2003). The PCR reaction mixture for the amplification of the 16S rDNA gene consisted of 200 mM of each dNTP, 1.5 mM MgCl2, 1 × PCR buffer, 10 pmol of each primer, 1 U of Taq DNA polymerase (Fermentas) and 10 ng DNA. The reaction was made up to 25 μl with sterile distilled water and the cycle consisted of initial denaturation at 95°C for 6 min and 30 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 2 min with the final extension at 72°C for 10 min. PCR products were electrophoresed on 1.0% agarose gel and visualised under UV in a gel documentation system as above. Amplified 16S rDNA PCR products were sequenced by the dideoxy chain termination method using the Big dye Terminator v 3.1 cycle sequencing kit and Big dye X Terminator Purification kit in an ABI 10 sequencer (Applied Biosystems). The derived sequences were aligned using DNASTAR lasergene 9 Core Suit and BLAST analysis was performed for comparison with other bacterial species, sequences available in the NCBI database. A dendogram based on 16S rDNA sequence was also constructed by the neighbor joining method using Molecular Evolutionary Genetics Analysis version 5.0 (MEGA 5) analytical software (Tamura et al., 2011).
MALDI-TOF analysis for bacterial identification
Biochemical profile of standard strains and confirmed soil isolates
Substrate | Standard strains | Soil isolates | ||||
---|---|---|---|---|---|---|
Burkholderia pseudomallei NTCC10274 | Burkholderia pseudomallei NTCC1688 | DRDEBPS 1001 | DRDEBPS 1002 | DRDEBPS 1003 | DRDEBPS 1004 | |
Nitrate | + | + | + | + | + | + |
Tryptophan | + | + | + | + | + | + |
Glucose (acidification) | + | + | + | + | + | + |
Arginine | + | + | + | + | + | + |
Urea* | - | - | - | - | - | - |
Esculin* | - | - | + | + | + | + |
Gelatin | + | + | + | + | + | + |
PNPG | - | - | - | - | - | - |
Assimilation of | ||||||
Glucose | + | + | + | + | + | + |
Arabinose | - | - | - | - | - | - |
Mannose | + | + | + | + | + | + |
Mannitol | + | + | + | + | + | + |
N-Acetyl glucosamine | + | + | + | + | + | + |
Maltose | - | - | - | - | - | - |
Gluconate | + | + | + | + | + | + |
Caprate | + | + | + | + | + | + |
Adipate | + | + | + | + | + | + |
Malate | + | + | + | + | + | + |
Citrate | + | + | + | + | + | + |
Phenyl acetate | + | + | + | + | + | + |
Flagellin C (fli C) gene based restriction fragment length analysis
Restriction fragment length analysis was performed on the specific PCR, 16S rDNA sequencing and MALDI-TOF/MS confirmed isolates to observe that whether these isolates belongs to similar strains (clone of a single strain) or belongs to different strains. The primers fli C forward 5′-CTC GGA TCCAAC AGC AAC-3′ and fli C reverse 5′-TAT TGC AGG TAC CTT CAG-3′ were used for the amplification of the 1167 bp fli C gene. The PCR amplification was carried out in a 50 μl reaction volume with the following PCR conditions: initial denaturation at 95°C for 6 min and 30 cycles of denaturation at 95°C for 1 min, annealing at 52°C for 1 min and extension at 72°C for 2 min with the final extension at 72°C for 10 min. PCR products were purified using a gel extraction purification kit (Qiagen) and concentration was determined spectrophotometrically. Restriction digestion was performed with three restriction enzymes Dde I, Msp I and Pst I (Fermentas, fast digest). Restriction digestion reaction mixture contained 7 μl of purified fli C PCR products at a conc. of 100 ng/μl and 1U of restriction enzymes. Restriction digestion reaction was carried out in 0.5 ml tubes incubated at 37°C for 2 hrs and then inactivated by keeping tubes in a dry bath at 65°C for 10 minutes. Then digested products were electrophoresed on 1.8% agarose gel and visualized under UV in an Alpha Innotech Gel Imager.
Results & Discussion
Biochemical profiles of Ashdown grown suspected soil isolates confirmed by API20NE
Species name | NO3/N2 | TRP | GLU | ADH | URE | ESC | GEL | PNG | GLU | ARA | MNE | MAN | NAG | MAL | GNT | CAP | ADI | MLT | CIT | PAC |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Ochrobactrum anthropi (n = 3) | +/+ | - | - | - | + | - | - | - | + | + | + | - | + | + | + | + | - | + | - | - |
Achromobacter xylosoxidans (n = 5) | +/+ | - | - | - | - | - | - | - | - | - | - | - | + | - | + | + | + | + | + | + |
Chromobacterium violaceum (n = 9) | +/+ | - | + | + | - | - | + | - | + | - | + | + | + | - | + | + | + | + | + | + |
Stenotrophomonas maltophilia (n = 10) | +/+ | - | - | - | - | + | + | + | + | - | + | - | + | + | - | - | - | + | + | - |
Rizobium radiobacter (n = 1) | +/+ | - | - | - | + | + | - | + | + | + | + | + | + | + | + | - | - | + | + | - |
Burkholderia cepacia (n = 4) | +/+ | - | + | - | - | + | + | + | + | + | + | + | + | - | + | + | - | + | + | + |
Delftia acidovorans (n = 5) | +/− | - | - | - | - | - | - | - | - | - | - | + | - | - | + | + | + | + | + | + |
Ralstonia pickettii (n = 3) | - | - | - | - | - | - | - | - | + | - | - | - | - | - | + | + | + | + | + | - |
Enterococcus faecalis (n = 2) | +/+ | - | + | - | - | + | - | + | - | - | - | - | - | - | - | + | - | - | + | - |
Brevundimonas vesicularis (n = 1) | - | - | - | - | - | + | - | - | + | - | - | - | - | + | - | - | - | - | - | - |
Pseudomonas luteola (n = 2) | +/+ | - | - | + | - | + | - | + | + | + | + | + | - | + | + | + | - | + | + | - |
Comamonas testosterone (n = 1) | +/+ | - | - | - | - | - | - | - | - | - | - | - | - | - | - | + | - | + | - | - |
Pseudomonas aeruginosa (n = 5) | +/+ | - | - | + | + | - | + | - | + | - | - | + | + | - | + | + | + | + | + | - |
Acinetobacter lwoffii (n = 1) | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | + | - | + | - | + |
Acinetobacter dentrificans (n = 2) | +/+ | - | - | - | - | - | - | - | - | - | - | - | - | - | + | - | + | + | + | + |
Pseudomonas mendocina (n = 1) | +/+ | - | - | + | - | - | - | - | + | - | - | - | - | - | + | + | - | + | + | - |
Chryseobacterium indologenens(n = 4) | +/+ | + | - | - | + | + | + | - | + | - | - | - | - | + | - | - | - | - | - | - |
VMP PCR for the confirmation of isolates- Lane1: 100 bp plus Marker, Lane 2: B. pseudomallei NTCC 10274, Lane 3: B. pseudomallei NTCC 1688, Lane 4: Soil isolate DRDEBPS1001, Lane 5: DRDEBPS1002, Lane 6: DRDEBPS1003, Lane 7: DRDEBPS1004, Lane 8: Negative control.
TTSS PCR for the confirmation of isolates- Lane1: 100 bp plus Marker, Lane 2: B. pseudomallei NTCC 10274, Lane 3: Soil isolate DRDEBPS1001, Lane 4: DRDEBPS1002, Lane 5: DRDEBPS1003, Lane 6: DRDEBPS1004, Lane 7: 100 bp plus Marker.
Dendogram based on 16S rDNA sequencing constructed by neighbor joining method (Bar represents 0.005 substitution per site). Soil isolates DRDEBPS1001, DRDEBPS1002, DRDEBPS1003 and DRDEBPS1004 showing proximity to B. pseudomallei standard strains.
fli C gene Restriction patterns of isolates Lane: M 100 bp plus Marker, Lane 1, 7, 13: B. pseudomallei NTCC 10274, Lane 2, 8, 14: B. pseudomallei NTCC 1688, Lane 3, 9, 15: Soil isolate DRDEBPS1001, Lane 4, 10, 16: DRDEBPS1002, Lane 5, 11, 17: Soil isolate DRDEBPS1003, Lane 6,12,18: Soil isolate DRDEBPS1004.
Recently MALDI-TOF mass spectrum analysis has been considered an easy and discriminatory tool for identification of bacterial species (Lista et al., 2011). The results of MALDI-TOF mass spectrum analysis of the eight suspected isolates matched with that of the 16S rDNA sequence analysis. The four isolates DRDEBPS1001, DRDEBPS1002, DRDEBPS1003 and DRDEBPS1004 were confirmed as B. pseudomallei on the basis of score values 2.601, 2.099, 2.362 and 2.047. The other four isolates had been biochemically suspected but not supported by both the PCRs were also identified by MALDI-TOF spectrum analysis as Cupriavidus necator and Enterobacter cloacae with score value of 2.112, 2.122 and 2.341, 2.241 respectively, confirming the results of the 16S analysis.
The isolation of B. pseudomallei from soil is very complex as the presence of large numbers of closely related soil microflora interferes with its recovery although use of Ashdown broth and agar for the isolation of B. pseudomallei from soil samples provides some selectivity. This selectivity comes from crystal violet, neutral red and gentamicin present in the Ashdown medium. The vast majority of B. pseudomallei isolates are resistant to aminoglycosides due to expression of a multidrug efflux pump, thus allowing use of gentamicin for selection. This study confirms that, although this medium is useful for initial screening, many other species also grow on this medium thus interfering the recovery of B. pseudomallei. Francis agar is a medium that can clearly differentiate between B. pseudomallei and other closely related species including B. cepacia (Francis et al., 2006). We also analyzed all 8 suspected isolates of B. pseudomallei on Francis agar to observe the yellow haze around colonies 24 hr. Although Francis Agar was not developed for primary soil isolation of this species it can be used to confirm suspected colonies isolated from Ashdown agar. In our study the rate of isolation was found to be 5.97% as 4 isolates were confirmed as B. pseudomallei out of 67 suspected isolates from Ashdown agar and 2 isolates each were obtained from site 1 and site 2 (Figure 1). In similar soil isolation studies conducted in Malaysia and Thailand, high isolation rates were found in wet rice fields and other cleared and cultivated areas (Brett et al., 1998; Smith et al., 1995). This study reports the first isolation of B. pseudomallei from soil collected from paddy fields in the coastal region of India. The study site selected in this study fulfilled with all criteria of soil sampling which were considered as important factors in the isolation of B. pseudomallei. The presence of B. pseudomallei depends on various environmental factors that support the survival of B. pseudomallei in soil. The presence of B. pseudomallei in soil is high during when ploughing and planting of seedlings takes place (Palasatien et al., 2008; Currie et al., 2004). The maintenance of viable bacteria in soil samples during collection, transport and storage before processing in laboratory is also an important factor for the isolation. The presence of B. pseudomallei needs to be initially determined by culture based methods in microbiological laboratories that require only basic equipment and provides live organisms for further confirmation by DNA based molecular methods. The isolates recovered from soil need to be compared with those recovered from clinical cases as it provides vital information on the pathogenicity and virulence of soil isolates (Currie et al., 2004). The presence of identical genetic patterns among clinical and environmental isolates suggests a link between the bacteria present in contaminated soil and the emergence of indigenous melioidosis (Chen et al., 2010). Future studies are also recommended with large number of samples collected from different geographical regions of India to study the pattern of distribution of this important bacterial species and also to correlate the epidemiological relevance of soil isolation to the occurrence of melioidosis.
Conclusion
We report the first isolation and molecular confirmation of B. pseudomallei from soil of paddy fields in the coastal region of India. These isolates were initially identified by conventional biochemical methods and further confirmed by advanced molecular based methods of 16S rDNA sequencing, B. pseudomallei specific PCR, PCR-RFLP at fli C locus and MALDI-TOF based protein profiling. This study confirms the presence of B. pseudomallei in soil in the coastal region of India. The isolation of this important bacterial species from this part of India should initiate further studies on the extent of environmental and clinical impact of melioidosis in India.
Declarations
Acknowledgement
Authors are thankful to Dr M. P. Kaushik, Director, DRDE Gwalior and Dr. Urmil Tuteja Head of Microbiology Division, DRDE Gwalior for their kind supports and providing facilities for this work. The financial support to Archana Prakash by UGC, Govt. of India is also acknowledged.
Authors’ Affiliations
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