Isolation and Identification of Mycobacteria from Soils at an Illegal Dumping Site and Landfills in Japan

Abstract

In order to study the diversity and community of genus Mycobacterium in polluted soils, we tried to isolate mycobacteria from 11 soil samples collected from an illegal dumping site and 3 landfills in Japan. Using culture methods with or without Acanthamoeba culbertsoni, a total of 19 isolates of mycobacteria were obtained from 5 soil samples and 3 of them were isolated only by the co-culture method with the amoeba. Conventional biochemical tests and sequencing of the hsp65, rpoB, and 16S rRNA genes were performed for species identification of 17 of the 19 isolates. Among the 17 isolates, there was one isolate each of Mycobacterium vanbaalenii, Mycobacterium mageritense, Mycobacterium frederiksbergense, M. vanbaalenii or Mycobacterium austroafricanum, and Mycobacterium chubuense or Mycobacterium chlorophenolicum. The remaining 12 isolates could not be precisely identified at the species level. A phylogenic tree based on the hsp65 sequences indicated that 2 of the 12 isolates were novel species. In addition, 4 isolates were phylogenically close to species that degrade polycyclic aromatic hydrocarbons, which induce some cancers in humans. These results demonstrated that there were many hitherto-unreported mycobacteria in the polluted soils, and suggested that some mycobacteria might play roles in the natural attenuation and engineered bioremediation of contaminated sites with other microorganisms.Key words: Mycobacteria, Amoebal co-culture, hsp65, Landfills 513 Microbiol. Immunol., 50(7), [513][514][515][516][517][518][519][520][521][522][523][524] 2006 *Address correspondence to Dr. Hatsumi Taniguchi, Department of Microbiology, School of Medicine, University of Occupational and Environmental Health, Japan, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka 807-8555, Japan. Fax: ϩ81-93-602-4799. E-mail: hatsumi@med.uoeh-u.ac.jp Abbreviations: ARB, amoeba resistant bacteria; CFU, colony forming unit; DSMZ, the German National Resource Centre for Biological Material; hsp65, heat shock protein gene 65-kilodalton; MOI, multiplicity of infection; PAH, polycyclic aromatic hydrocarbons; PBS, phosphate buffered saline; PCDDS, polychlorinated dibenzo-p-dioxins; PCR, polymerase chain reaction; rpoB, RNA polymerase β-subunit gene; SDS, sodium dodecyl sulfate; Z-N, Ziehl-Neelsen staining; 16S rRNA, 16S ribosomal RNA.tion of contaminated sites with other microorganisms (5,10,17,19,22,41,44). Detailed analyses of the interaction between environmental conditions and mycobacteria are needed. However, purposeful isolation of mycobacteria from soil is difficult because of their relatively slow growth rate and susceptibility to overgrowth by faster-growing organisms on solid media. Therefore, an amoebal co-culture method was applied in this study.Generally, amoebae serve as a reservoir to protect internalized bacteria from environmental stress or toxicity (7,15,36). An amoebal co-culture procedure has also been reported to be useful to isolate amoeba resistant bacteria (ARB) and fastidious bacteria in water supplies and...

BacillusSpecies Predominated in an Incineration Ash Layer at a Landfill

“…We have reported the isolation of the genus Mycobacterium in landfills, which suggests the possibility that there were many hitherto unreported mycobacteria in polluted soils. 15) In that study, however, no mycobacterium was detected in the incineration ash. If there is any specific bacterium in the MSWI ash layer, it is potentially useful for industrial purposes, such as alkaline tolerant enzyme production and the adsorption of toxic or valuable metals.…”

“…Recently, there have been several reports on the bacterial diversity of landfills. 15,[25][26][27][28][29] In this study, we focused on a landfill that contained a large amount of municipal solid waste incineration (MSWI) ash. To our The tree was rooted using Escherichia coli as the outgroup, and constructed by the CLUSTAL W algorithm.…”

Mycobacterium Chlorophenolicum Isolation in Sirolimus-Associated Pneumonitis

“…Originally named Rhodococcus chlorophenolicus, M. chlorophenolicum is most closely related to Mycobacterium chubuense and is generally regarded to be nonpathogenic although little is known about its potential for human disease [11,12]. In the report discussed above, our patient was found to meet criteria for both an NTM pulmonary infection with M. chlorophenolicum and sirolimus-associated pneumonitis.…”

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

“…A broad range of microorganisms could be isolated with this technique. The most common bacteria isolated by amoebal coculture are members of the Mycobacterium genus that could be recovered from water treatment plants and from water networks 13,14,24,27,28 . Legionella and α-proteobacteria species could also be isolated from water treatment plants and from hospital water networks 14,24,[28][29][30][31] .…”