Burkholderia paludis sp. nov., an Antibiotic-Siderophore Producing Novel Burkholderia cepacia Complex Species, Isolated from Malaysian Tropical Peat Swamp Soil

Abstract

A novel Gram negative rod-shaped bacterium, designated strain MSh1^T, was isolated from Southeast Pahang tropical peat swamp forest soil in Malaysia and characterized using a polyphasic taxonomy approach. The predominant cellular fatty acids (>10.0%) were C_16:0 (31.7%), C_17:0 cyclo (26.6%), and C_19:0 cyclo ω8c (16.1%). The polar lipids detected were phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. The predominant ubiquinone was Q-8. This revealed that strain MSh1^T belongs to the genus Burkholderia. The type strain MSh1^T can be differentiated from other Burkholderia cepacia complex (Bcc) species by phylogenetic analysis of 16S rRNA gene sequence, multilocus sequence analysis (MLSA), average nucleotide identity (ANI) and biochemical tests. DNA-DNA relatedness values between strain MSh1^T and closely related type strains were below the 70% threshold value. Based on this polyphasic study of MSh1^T, it can be concluded that this strain represents a novel species within the Bcc, for which the name Burkholderia paludis sp. nov. is proposed. The type strain is MSh1^T (= DSM 100703^T = MCCC 1K01245^T). The dichloromethane extract of MSh1^T exhibited antimicrobial activity against four Gram positive bacteria (Enterococcus faecalis ATCC 29212, E. faecalis ATCC 700802, Staphylococcus aureus ATCC 29213, S. aureus ATCC 700699) and a Gram negative bacteria (Escherichia coli ATCC 25922). Further purification work has led to the isolation of Compound 1, pyochelin. Pyochelin demonstrated antimicrobial activity against four S. aureus strains and three E. faecalis strains with MIC-values of 3.13 μg/ml and 6.26 μg/ml, respectively. SEM analysis showed that the cellular morphology of E. faecalis ATCC 700802 was not affected by pyochelin; suggesting that it might target the intracellular components. Pyochelin, a siderophore with antimicrobial activity might be useful in treating bacterial infections caused by S. aureus and E. faecalis, however further work has to be done.

Keywords: Burkholderia cepacia complex; antimicrobial; multilocus sequence analysis (MLSA); polyphasic taxonomy; tropical peat swamp forest.

Figure 1

Neighbor-joining tree based on 16S rRNA sequences showing relationship between strain MSh1^T and representatives of some other related taxa. Bootstrap values (>50%) based on 1000 resampled datasets are shown at branch nodes. Bar, 5 substitutions per 1000 nucleotide positions.

Figure 2

Phylogenetic tree based on the concatenated sequences of seven housekeeping gene fragments of established Bcc species and strain MSh1^T. Bootstrap values (>50%) based on 1000 resampled datasets are shown at branch nodes. Bar, 2 substitutions per 100 nucleotide positions.

Figure 3

SEM images of Burkholderia paludis MSh1^T. Images were taken under 25,000 × magnification at 1 kV. The bacterial cells sizes are approximately 0.6–0.8 × 1.6–2.1 μm.

Figure 4

Thin layer chromatogram polar lipid detected in Burkholderia paludis MSh1^T. AL, aminolipid; PN, aminophospholipid; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; DPG, diphosphatidylglycerol.

Figure 5

FE-SEM images of E. faecalis ATCC 700802 showing (A) negative control, (B) treated with MIC of Compound 1 and (C) treated with 100 μg/ml of chloramphenicol (positive control). Images were taken under 5000x magnification at 1 kV.