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Journal of Clinical Microbiology, March 2002, p. 1103-1105, Vol. 40, No. 3
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.3.1103-1105.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Epidemiology of Mycobacterium malmoense Infections in Scotland

C. Doig,¹ L. Muckersie,² B. Watt,¹ and K. J. Forbes^2*

Scottish Mycobacteria Reference Laboratory, The City Hospital, Edinburgh EH10 5SB,¹ Medical Microbiology, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom²

Received 10 September 2001/ Returned for modification 31 October 2001/ Accepted 16 December 2001

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Clinical isolates of Mycobacterium malmoense collected over 5 years from patients across Scotland with a variety of diseases have been characterized by pulsed-field gel electrophoresis (PFGE), ribotyping, and 16S ribosomal DNA gene sequencing. Results indicate that this species harbors little genetic diversity and that the different strain types that were identified by PFGE showed no correlation with geographical origin or date of isolation.

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Mycobacterium malmoense is an important respiratory pathogen (7), especially in northern Europe and in Scotland in particular. It is a slow-growing organism that was first described as a respiratory tract pathogen in 1977 (19) and since recognized as a species distinct from the other slow-growing mycobacteria (5, 6, 9, 12, 17). It can colonize the respiratory tract of patients with preexisting lung disease, and it may cause disease indistinguishable from tuberculosis in such patients and in those with an otherwise normal respiratory tract; it has also been isolated from nonrespiratory sites (20). It is, however, unclear to what extent the presence of this organism is indicative of infection or colonization. Although normally classed as an atypical or environmental mycobacterial species, little is known about its environmental occurrence. There have been reports of isolation from small numbers of samples in Zaire (16) and Japan (18) and from 4% of water samples in Finland (10). It is assumed that M. malmoense is not infectious among humans and that person-to-person spread does not occur, although anecdotal evidence of the clustering of cases in Edinburgh, Scotland (3), has suggested that this is not wholly correct.

A number of molecular techniques have been used to identify isolates to species level (5, 6, 9, 12, 17). The differentiation of strains has been noted using glycolipid profiling (11), random amplified polymorphic DNA PCR (13), and ribotyping (14); however, there was little correlation of the groupings of these isolates by these different tests (13), possibly due to the poor discrimination that these methods achieved.

In this study, M. malmoense clinical isolates were collected between 1997 and 2000 from 79 patients with respiratory disease across Scotland. In all cases, M. malmoense was isolated from the respiratory tract. The species of all isolates was determined by using phenotypic characteristics (15). 16S ribosomal DNA (rDNA) gene sequencing was additionally carried out with 18 of these isolates, which were dispersed throughout the diversity of pulsed-field gel electrophoresis (PFGE) types shown in Fig. 1, with a PCR product generated from the center of the 16S rDNA (2). A 550-nucleotide double-stranded DNA sequence from the PCR product was found to be identical in all of the isolates and to the published sequence of M. malmoense (EMBL accession no. AF152560), implying that all of these isolates were indeed members of the same species, M. malmoense.

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FIG. 1. PFGE of DraI digests of genomic DNA of M. malmoense. The dendrogram was constructed with the BioNumerics program (Applied Maths) using unweighted pair group method using arithmetic average and Dice percent coefficients of similarity (calculated with a band tolerance ranging from 1% at the top of the gel to 2% at the bottom). Isolate code numbers are indicated.

Ribotyping was not found to be a useful strain-typing method. With a probe (4) based on the 16S region amplified by PCR described above, the restriction enzymes BamHI and EcoRV were found to be the more-useful restriction enzymes of those tested (including AluI, ApaI, EcoRI, PstI, SalI, and TaqI). Previous studies indicated that M. malmoense, like the other slow-growing mycobacteria, probably possesses only a single rDNA operon (1, 5,14, 17) and therefore restriction fragment length polymorphism (RFLP) results associated with the rDNA operon are likely to be much reduced in this species. This was indeed observed here with BamHI (33 isolates tested) and EcoRV (55 isolates tested), each revealing a predominant pattern (73 and 82% of tested isolates) and only a few, rare RFLP variants which were dispersed throughout the dendrogram shown in Fig. 1. Certainly, there was insufficient polymorphism with which to establish a typing scheme. This absence of polymorphism is likely to be a contributory factor in the poor discrimination previously noted for ribotyping (14) and is indicative of the highly restricted genetic diversity of this species.

PFGE was carried out by the method of Hughes et al. (8) with restriction enzyme DraI, which was found to give an adequate number of fragments in the size range of ca. 50 to 500 kb and reproducible patterns (Fig. 1). Isolates from repeat specimens from 10 patients were all found to have indistinguishable PFGE patterns, indicating the reproducibility of the method. This is the first report of the PFGE analysis of M. malmoense, and a number of features are noteworthy.

Estimates of the total length of the fragments resolved in a selection of the patterns indicated that the genome of M. malmoense is at least 3 Mb in size. Given that many fragments smaller than 50 kb were not resolved, it is likely that M. malmoense will have a genome length comparable to the 4.70 Mb of the phylogenetically close Mycobacterium avium-M. intracellulare complex (http://www.tigr.org/).

Perhaps most apparent in Fig. 1 is the extensive similarity of the patterns to each other. There are fragments of all sizes that are present in a large proportion, if not all, of the isolates. Given the large size of these fragments, this would suggest that large sections of the genome of M. malmoense are not genetically heterogeneous and that there is little genetic difference between isolates. This is in agreement with our ribotyping findings, which also indicated little genetic diversity, and the random amplified polymorphic DNA findings of Kauppinen et al. (13).

Sufficient PFGE RFLP variation was detectable, over the full size range of the fragments, to identify different strain types. Many RFLP patterns were represented by more than one isolate. At a Dice coefficient of similarity of >90%, at which the patterns were visually very similar to each other (Fig. 1), there were 10 multi-isolate clusters (strains) with 2 to 17 members in each, the largest comprising nearly one quarter of the isolates. Three of the repeat isolates were from specimens collected 13 to 18 months after the first, and this suggests that these patients had sustained infection by a single, invariant, strain over that period. From the perspective of the epidemiology of disease caused by these M. malmoense isolates, there was no correlation of strain type with any of the patient or infection characteristics (date of birth, sex, location of current residence, referring hospital, or date of specimen collection). There was no evidence of transmission between patients, even in those cases of geographical proximity. Conversely, and perhaps more intriguingly, the strains identified were dispersed both geographically and temporally across Scotland.

This is the first molecular epidemiological study of M. malmoense to be carried out in such depth. If patients do not acquire M. malmoense from others who harbor the organism, then from where is it acquired? Given that the results here do not support person-to-person spread, it may be that these patients acquired their isolates from environmental sources, where other investigators have indeed isolated the organism (10, 16, 18). It will now be possible to test this hypothesis by typing such environmental isolates and comparing them to those found in humans from the same area.

 
This study was financially supported by Chest, Heart and Stroke Scotland.

 
* Corresponding author. Mailing address: Medical Microbiology, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. Phone: 44 1224 554953. Fax: 44 1224 685604. E-mail: k.forbes{at}abdn.ac.uk.

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Journal of Clinical Microbiology, March 2002, p. 1103-1105, Vol. 40, No. 3
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.3.1103-1105.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Doig, C. Articles by Forbes, K. J. Search for Related Content PubMed PubMed Citation Articles by Doig, C. Articles by Forbes, K. J.