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Applied and Environmental Microbiology, August 2000, p. 3206-3213, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of Mycobacterium ulcerans in the
Environment from Regions in Southeast Australia in Which It Is
Endemic with Sequence Capture-PCR
Timothy
Stinear,1,*
John K.
Davies,1
Grant A.
Jenkin,1
John A.
Hayman,2
Frances
Oppedisano,3 and
Paul
D. R.
Johnson1,3,4
Department of Microbiology, Monash
University, Clayton,1 Pathology
Department, Box Hill Hospital,2
Microbiology Research Unit, Royal Children's
Hospital,3 and Department of Infectious
Diseases and Clinical Epidemiology, Monash Medical
Centre,4 Victoria, Australia
Received 27 March 2000/Accepted 19 May 2000
We recently described the use of PCR to identify the environmental
source of Mycobacterium ulcerans during an outbreak of ulcerative disease that occurred in a localized region of southeast Australia. The PCR used was based on amplification of the M. ulcerans-specific insertion sequence, IS2404. In this
study we developed a new test that is a substantial improvement over
the original PCR method in terms of sensitivity, reliability, and ease
of use. In the new method magnetic bead sequence capture-PCR is used to
detect two M. ulcerans sequences (IS2404 and
IS2606) and total mycobacterial 16S ribosomal DNA. We used
sequence capture-PCR to test water and plant material collected over a
12-month period during 1998 and 1999 from sites near the centers of two
distinct foci of M. ulcerans infections. A golf course
irrigation system in one area and a small shallow lake in another area
repeatedly were PCR positive for M. ulcerans. Nearby sites
and sites unrelated to the endemic areas were negative. Based on the
PCR data, a most-probable-number method was used to estimate the
concentration of M. ulcerans cells in positive samples from
both regions. This procedure resulted in average concentrations of 0.5 cell per 100 ml of water and 40 cells per 100 g of detritus. Loss
of the PCR signal coincided with a decrease in ulcerative disease in
each area. These results provide further evidence that M. ulcerans may be transmitted from a point environmental source and
demonstrate the utility of magnetic bead sequence capture-PCR for
identification of nonculturable microbial pathogens in the environment.
*
Corresponding author. Mailing address: Department of
Microbiology, Monash University, Wellington Rd., Clayton 3168, Australia. Phone: 61 3 9905 4809. Fax: 61 3 9905 4811. E-mail:
tim.stinear{at}med.monash.edu.au.
Applied and Environmental Microbiology, August 2000, p. 3206-3213, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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