Twelve of 16 M. haemophilum isolates (75%) collected in the New York Metropolitan Area from 1990 to 1991 shared the same pattern, including all six isolates ...
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1994, p. 1083-1084 0095-1137/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Vol. 32, No. 4
DNA Polymorphisms Detected in Mycobacterium haemophilum by Pulsed-Field Gel Electrophoresis WALTER L. STRAUS Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30333 MITCHELL A. YAKRUS*
AND
Received 8 October 1993/Returned for modification 15 November 1993/Accepted 20 December 1993
Nineteen isolates of Mycobacterium haemophilum were analyzed by pulsed-field gel electrophoresis of large restriction fragments generated by digestion of chromosomal DNA with XbaI. Six patterns were observed. Twelve of 16 M. haemophilum isolates (75%) collected in the New York Metropolitan Area from 1990 to 1991 shared the same pattern, including all six isolates submitted from one hospital. Two different patterns were seen among the other four isolates. Individual isolates from Albany, N.Y., Florida, and Texas had unique patterns. Pulsed-field gel electrophoresis is the first method reported with the capability to type strains of M. haemophilum and will hopefully provide insight into the source and transmission of this emerging pathogen.
Mycobacterium haemophilum is being reported with greater frequency since it was first described in 1978 (9). This increase is associated with an increase in the severely immunocompromised population caused by AIDS and organ transplantation procedures. The rise is also associated with improved laboratory identification of the organism resulting from the recognition of its requirement for iron-supplemented media and low temperatures (30 to 32°C) for optimal growth (7). Sixteen isolates from Australia were recently described (8). All were found to be identical by biochemical testing and fatty acid composition analysis. To identify specific strains of M. haemophilum and begin to understand the epidemiology of this organism, we analyzed 19 isolates from several locations by pulsed-field gel electrophoresis (PFGE) of large restriction fragments (LRFs) produced by digestion of chromosomal DNA with the restriction endonuclease XbaI. Organisms. Sixteen isolates of M. haemophilum were originally obtained from six hospitals in the New York metropolitan area from 1990 to 1991 as part of a previously described epidemiologic investigation (3). Individual isolates were from Florida, Texas, and Albany, N.Y., and were originally submitted for identification from state health departments. Two of the New York City isolates were from the same patient but submitted by different hospitals. Strains were confirmed as being M. haemophilum by high-performance liquid chromatography patterns of their mycolic acids (2). DNA extraction. Strains were grown in 20 ml of complete 7H9 medium supplemented with hemin for 3 to 4 weeks at 32°C. Cycloserine (1 mg/ml) was added, and cultures were incubated for 24 h before being harvested by centrifugation. Pellets were suspended in 10 ml of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and the cells were killed by heating in a 75°C water bath for 20 min. The cells were pelleted and frozen overnight. DNA was then extracted by a method described by Zhang et al. (10) with modification. Each pellet was suspended in 1 ml of lysozyme (2 mg/ml) in TE buffer and incubated for 1 h at 37°C. One milliliter of 2% chromosomal-grade agarose (Bio-Rad Laboratories, Melville, N.Y.) in TE buffer was added to the suspension to prepare plugs with a final agarose concentration of 1%. Plugs were incubated in 0.5 M EDTA, *
pH 8.0, containing 1 mg of proteinase K per ml and 1% sodium dodecyl sulfate for 48 h at 50°C. The fluids were removed by aspiration, and the plugs were washed twice with TE buffer containing 1 mM phenylmethylsulfonyl fluoride for 30 min at 50°C and then twice with TE buffer for 2 h at 50°C. The plugs were given a final wash with TE buffer overnight at 50°C and stored in TE buffer at 4°C until needed. Digestion and PFGE of DNA. Five-millimeter slices of each plug were transferred to Eppendorf tubes containing 200 pul of restriction buffer, which was supplied by the manufacturer (Promega, Madison, Wis.) and allowed to equilibrate for 2 h on ice. Twenty units of XbaI was then added, and digestion was allowed to occur overnight at 37°C. Plug slices were inserted into 1% agarose gels prepared with Tris-borate-EDTA buffer (25 mM Tris base, 25 mM boric acid, 0.5 mM EDTA). Separation of LRFs by PFGE was achieved with a CHEF-DR II system (Bio-Rad Laboratories) using Tris-borate-EDTA buffer at 14°C. Pulse time was ramped from 0.1 to 8 s for 18 h at 200 V. Gel bands were visualized by staining with ethidium bromide, followed by UV illumination. Bacteriophage lambda DNA concatemers (Bio-Rad) were included as size standards with each run. LRF patterns. When M. haemophilum genomic DNA was digested with XbaI, four to seven sharp fragment bands were produced in a range from just below 97 kb to 194 kb when compared to lambda DNA concatemers (Fig. 1). Fragments smaller than 245 kb were also visible but could not be interpreted easily for typing strains. Among the 19 strains analyzed, six different banding patterns were observed. Three patterns were seen among 16 isolates from six hospitals in the New York metropolitan area (Table 1). Within that same area, 12 isolates from five different hospitals had identical LRF patterns (LRF pattern 1). These included all six isolates from hospital A, two from hospital B, two from hospital C, and one each from hospitals D and E. Two of these isolates, one from hospital A and another from hospital C, were from the same patient. Three other isolates, one each from hospitals B, C, and F, were shown to have a second banding pattern (LRF pattern 2). A third pattern (LRF pattern 3) was found with an isolate from hospital B. Individual patterns were observed from isolates received from Albany, N.Y. (LRF pattern 4), Florida (LRF pattern 5), and Texas (LRF pattern 6). This study represents the first successful attempt to type strains of M. haemophilum. Analysis by PFGE indicated that
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FIG. 1. LRF patterns of DNA from M. haemophilum strains digested with XbaI. Lanes: 2 and 3, pattern 1, isolates CDC91-859 and CDC91-719; 4 and 5, pattern 2, isolates CDC91-3709 and CDC922283; 6, pattern 3, isolate CDC91-818; 7, pattern 4, isolate CDC911016; 8, pattern 5, isolate CDC90-989; 9, pattern 6, CDC91-816; 1 and 10, lambda DNA concatemers. Sizes (in kilobases) are indicated to the left and right of the gel.
the organism exists as a heterogeneous population. The fact that the same LRF patterns were observed from isolates from more than one hospital in the New York metropolitan area suggests that infections are being acquired from an environmental source shared by all locations. TABLE 1. LRF patterns and origin of isolates of M. haemophilum pattern
Isolate no.
Origin'
1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 3 4 5 6
CDC91-722 CDC91-723 CDC91-724 CDC91-725 CDC91-726 CDC91-727 CDC91-817 CDC91-819 CDC91-967 CDC91-968 CDC91-719 CDC91-859 CDC92-2283 CDC91-966 CDC91-3709 CDC91-818 CDC91-1016 CDC90-989 CDC91-816
NYMA hospital A NYMA hospital Ab NYMA hospital A NYMA hospital A NYMA hospital A NYMA hospital A NYMA hospital B NYMA hospital B NYMA hospital Cb NYMA hospital C NYMA hospital D NYMA hospital E NYMA hospital B NYMA hospital C NYMA hospital F NYMA hospital B Albany, N.Y. Florida Texas
aNYMA, New York metropolitan area, which includes isolates from New York City, New Jersey, and Long Island. " Isolated from the same patient.
It has been suggested that water is associated with infection by M. haemophilum since most cases have been reported in persons who reside near large bodies of water (1). Standard methods for treating drinking water probably have little, if any, effect upon the viability of mycobacteria (4). Obviously, to demonstrate that water is a potential source of infection, strains of M. haemophilum need to be isolated from natural bodies of water, reservoirs, or hospital water supplies and typed by PFGE to determine whether their LRF patterns match those from patient isolates. Unfortunately, initial attempts to isolate the organism from water have been unsuccessful (5, 6). Individual isolates of M. haemophilum from Albany, N.Y., Florida, and Texas were examined by PFGE. Their LRF patterns were found to be distinct when compared. with those from the New York metropolitan area. This might indicate that some wild types of M. haemophilum are geographically clustered. Strains isolated from other areas within and outside the United States need to be examined to evaluate the genetic diversity of this emerging pathogen. REFERENCES 1. Becherer, P., and R. L. Hopfer. 1992. Infection with Mycobacterium haemophilum. Clin. Infect. Dis. 14:793. 2. Butler, W. R., K. C. Jost, Jr., and J. 0. Kilburn. 1991. Identification of mycobacteria by high-performance liquid chromatography. J. Clin. Microbiol. 29:2468-2472. 3. Centers for Disease Control. 1991. Mycobacterium haemophilum infections: NYC metro area, 1990-1991. Morbid. Mortal. Weekly Rep. 40:636-637, 643. 4. Collins, C. H., J. M. Grange, and M. D. Yates. 1984. A review: mycobacteria in water. J. Appl. Bacteriol. 57:193-211. 5. Davis, B. R., J. Brumbach, W. J. Sanders, and E. Wolinsky. 1982. Skin lesions caused by Mycobacterium haemophilum. Ann. Intern. Med. 97:723-724. 6. Gouby, A., B. Branger, R. Oules, and M. Ramuz. 1988. Two cases of Mycobacterium haemophilum infection in a renal-dialysis unit. J. Med. Microbiol. 25:299-300. 7. Kristjansson, M., V. M. Bieluch, and P. D. Byeff. 1991. Mycobacterium haemophilum infection in immunocompromised patients: case report and review of the literature. Rev. Infect. Dis. 13:906910. 8. Portaels, F., D. J. Dawson, L. Larsson, and L. Rigouts. 1993. Biochemical properties and fatty acid composition of Mycobacterium haemophilum: study of 16 isolates from Australian patients. J. Clin. Microbiol. 31:26-30. 9. Sompolinsky, D., A. Lagziel, D. Naveh, and T. Yankilevitz. 1978. Mycobacterium haemophilum sp. nov., a new pathogen of humans. Int. J. Syst. Bacteriol. 28:67-75. 10. Zhang, Y., G. H. Mazurek, M. D. Cave, K. D. Eisenach, Y. Pang, D. T. Murphy, and R. J. Wallace, Jr. 1992. DNA polymorphisms in strains of Mycobacterium tuberculosis analyzed by pulsed-field gel electrophoresis: a tool for epidemiology. J. Clin. Microbiol. 30:1551-1556.
