Rapid and sensitive method for the detection of Mycobacterium chlorophenolicum PCP-1 in soil based on 16S rRNA gene-targeted PCR (original) (raw)
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Detection of Mycobacterium leprae DNA from soil samples by PCR targeting RLEP sequences
The Journal of communicable diseases, 2006
Despite near elimination of leprosy as a public health problem, several problems in leprosy still remain. These include early detection, determining efficacy of the treatment and differentiating relapses from re-infection. These aspects have important impact on the patients undergoing treatment and also have a bearing on understanding transmission dynamics in the community. While early diagnosis and management do not need major technological inputs, various reports have suggested that M. leprae is found in the environment and may have a role in continued transmission of disease. In earlier studies from other parts of world the presence of M. leprae DNA in the environment has been investigated both by microbiological and molecular studies. In the present study, an attempt was made to extract M. leprae DNA from soil samples, which were collected from eighteen different locations including 3 from our Institute area and 15 from different villages of Ghatampur area. We optimized a protoc...
FEMS Microbiology Ecology, 2000
Fast-growing mycobacteria are considered essential members of the polycyclic aromatic hydrocarbons (PAH) degrading bacterial community in PAH-contaminated soils. To study the natural role and diversity of the Mycobacterium community in contaminated soils, a culture-independent fingerprinting method based on PCR combined with denaturing gradient gel electrophoresis (DGGE) was developed. New PCR primers were selected which specifically targeted the 16S rRNA genes of fast-growing mycobacteria, and single-band DGGE profiles of amplicons were obtained for most Mycobacterium strains tested. Strains belonging to the same species revealed identical DGGE fingerprints, and in most cases, but not all, these fingerprints were typical for one species, allowing partial differentiation between species in a Mycobacterium community. Mycobacterium strains inoculated in soil were detected with a detection limit of 10 6 CFU g À1 of soil using the new primer set as such, or approximately 10 2 CFU g À1 in a nested PCR approach combining eubacterial and the Mycobacterium specific primers. Using the PCR-DGGE method, different species could be individually recognized in a mixed Mycobacterium community. This approach was used to rapidly assess the Mycobacterium community structure of several PAH-contaminated soils of diverse origin with different overall contamination profiles, pollution concentrations and chemical-physical soil characteristics. In the non-contaminated soil, most of the recovered 16S rRNA gene sequence did not match with previous described PAH-degrading Mycobacterium strains. In most PAH-contaminated soils, mycobacteria were detected which were closely related to fast-growing species such as Mycobacterium frederiksbergense and Mycobacterium austroafricanum, species that are known to include strains with PAH-degrading capacities. Interestingly, 16S rRNA genes related to M. tusciae sequences, a Mycobacterium species so far not reported in relation to biodegradation of PAHs, were detected in all contaminated soils.
Molecular Detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in Soil
Applied and Environmental Microbiology, 2005
PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 ؋ 10 3 to 3.6 ؋ 10 3 gene copies g of soil ؊1 , depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37°C with moist soil (؊20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.
Applied Microbiology and Biotechnology, 2006
An environmental Mycobacterium able to degrade phenanthrene, pyrene and fluoranthene was transformed with an IS1096-based transposon marker system. Electroporation and subsequent delivery of the transposon enabled formation of constitutive lacZ transformants, with similar growth rates on pyrene and R2A media to the parental strain. A semi-selective medium was developed to recover and detect colonies of the transformed strain after inoculation into polycyclic aromatic hydrocarbon-contaminated soil. Microcosm experiments involving inoculation of the tagged Mycobacterium strain into a historically PAH-contaminated soil indicated survival when an appropriate carbon source was available. The results reported show that transposon systems developed for clinical mycobacterial isolates are also applicable for use in environ-mental isolates. The results also show that inoculated Mycobacterium strains could survive for at least 100 days at 10 6 -10 7 cfu g −1 in the PAH-contaminated soil tested here.
Applied and environmental microbiology, 2004
For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.
The Indian journal of medical research, 2009
Several environmental mycobacteria have been shown to be important human pathogens linked to immunomodulation especially in relation to effect on vaccination. Hence identification of mycobacteria to the species level is not only relevant to patient management but also to understand epidemiology of mycobacterial diseases and effect on vaccination. We undertook this study to assess the usefulness of various conventional and molecular methods in identification of environmental mycobacterial species from Agra, north India. One hundred nineteen isolates of environmental mycobacteria were grown from 291 (116 soil and 175 water) samples. These isolates were identified by standard biochemical tests, and a simple, rapid and cost-effective in-house developed gene amplification restriction analysis targeting 16S-23S rRNA spacer and flanking region and 16S rRNA sequencing. Biochemical tests could clearly identify only 68.1 per cent (81/119) of isolates to species level. An in-house developed ge...
Journal of Clinical Microbiology, 2001
Identification of mycobacteria to the species level by growth-based methodologies is a process that has been fraught with difficulties due to the long generation times of mycobacteria. There is an increasing incidence of unusual nontuberculous mycobacterial infections, especially in patients with concomitant immunocompromised states, which has led to the discovery of new mycobacterial species and the recognition of the pathogenicity of organisms that were once considered nonpathogens. Therefore, there is a need for rapid and sensitive techniques that can accurately identify all mycobacterial species. Multiple-fluorescence-based PCR and subsequent single-strand conformation polymorphism (SSCP) analysis (MF-PCR-SSCP) of four variable regions of the 16S rRNA gene were used to identify species-specific patterns for 30 of the most common mycobacterial human pathogens and environmental isolates. The species-specific SSCP patterns generated were then entered into a database by using BioNumerics, version 1.5, software with a pattern-recognition capability, among its multiple uses. Patient specimens previously identified by 16S rRNA gene sequencing were subsequently tested by this method and were identified by comparing their patterns with those in the reference database. Fourteen species whose SSCP patterns were included in the database were correctly identified. Five other test organisms were correctly identified as unique species or were identified by their closest relative, as they were not in the database. We propose that MF-PCR-SSCP offers a rapid, specific, and relatively inexpensive identification tool for the differentiation of mycobacterial species.
Using Identification of Several Mycobacterium Species by
2011
In order to diagnose MOTT species M. smegmatis and M. kansasii along with M. tuberculosis, species specific primers were designed using PRIMER-BLAST which amplified particular 16S rRNA genes having variable amplicons sizes. All primer sets were found to be specific for their target 16S regions of mycobacterium species. Easy and fast DNA isolation protocol developed that could be implemented directly for PCR reaction of the samples. By involving standardized PCR conditions with defined primers and genome combinations, we have successfully reported the 16S rRNA gene based PCR detection of several mycobacterium species which has highlighted the MOTT detection capabilities.
Detection of Mycobacterium leprae DNA in soil: multiple needles in the haystack
Scientific Reports
Leprosy is an infectious disease caused by Mycobacterium leprae affecting the skin and nerves. Despite decades of availability of adequate treatment, transmission is unabated and transmission routes are not completely understood. Despite the general assumption that untreated M. leprae infected humans represent the major source of transmission, scarce reports indicate that environmental sources could also play a role as a reservoir. We investigated whether M. leprae DNA is present in soil of regions where leprosy is endemic or areas with possible animal reservoirs (armadillos and red squirrels). soil samples (n = 73) were collected in Bangladesh, Suriname and the British Isles. Presence of M. leprae DNA was determined by RLEP PCR and genotypes were further identified by Sanger sequencing. M. leprae DNA was identified in 16.0% of soil from houses of leprosy patients (Bangladesh), in 10.7% from armadillos' holes (Suriname) and in 5% from the habitat of lepromatous red squirrels (British Isles). Genotype 1 was found in Bangladesh whilst in Suriname the genotype was 1 or 2. M. leprae DNA can be detected in soil near human and animal sources, suggesting that environmental sources represent (temporary) reservoirs for M. leprae. Leprosy is a debilitating infectious disease caused by Mycobacterium leprae and Mycobacterium lepromatosis that is still considered a major threat in developing countries by WHO, remaining persistently endemic in regions in Africa, South America and Asia. Every year more than 200,000 new patients are still diagnosed and this new case detection rate has been virtually stable over the last decade 1. These facts indicate that multidrug therapy (MDT), although effective to treat leprosy, is insufficient to prevent transmission 2. Granting M. leprae transmission is not completely understood, risk factors for development of leprosy have been identified including close contact with untreated, multibacillary patients 3 , human susceptibility genes 4,5 , infection with soil transmitted helminths 6 , as well as food shortage 7. The mechanism by which bacteria are transmitted from one organism to another has not been unequivocally demonstrated 8. However, based on existing evidence, skin-to-skin contact, aerosols as well as shedding of bacteria into the environment subsequently followed by infection of other individuals remain the most obvious options for human leprosy 8,9. Still these routes provide no explanation for the occurrence of leprosy in individuals without known contact to leprosy patients or in areas without any reported new cases 9,10. Through PCR amplification of M. leprae DNA, its presence has been detected in environmental samples such as soil 11-16 and water 17,18 in areas inhabited by leprosy patients in Brazil and India. The viability of M. leprae was assessed by its multiplication in footpads of wild type mice and showed that M. leprae can remain alive in wet soil for 46 days 19. Moreover, viability of M. leprae bacilli in soil from India has been studied by 16S ribosomal RNA gene analysis 20. This study showed that 25% of the soil samples collected from patients' areas contained M. leprae 16S ribosomal RNA, suggesting the presence of viable M. leprae in the soil. Additionally, if environment-free