Isolation of Mycobacterium malmoense from the environment in Zaire

Tubercle and Lung Disease (1995) 76, 160-162 © 1995 Pearson Professional Ltd

Tubercleand LungDisease

Isolation of Mycobacterium malmoense from the environment in Zaire F. Portaels*, L. Larsson t, P. A. Jenkins*

*Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium, tDepartment of Medical Microbiology, University of Lund, Lurid, Sweden, tPHLS Mycobacterium Reference Unit, University Hospital of Wales, Cardiff, UK S U M M A R Y. Two strains of mycobacteria isolated f r o m water and soil in Zaire were identified as Mycobacterium malmoense by biochemical tests and lipid analysis. A p a r t f r o m the previously reported fatty acids characteristic of this species, both strains, as well as 5 clinical isolates of M. malmoense, contained 2,4dimethyl-docosanoic acid. One of the environmental strains, with a glycolipid pattern II, additionally contained 2-methyltetradecanoic acid. The results confirm that M. malmoense m a y be subdivided in 2 sub-groups according to its lipid patterns. They also show that M. malmoense can be isolated f r o m the environment which m a y be the source of the infection. R/~ S U M/~. Deux souches de mycobact6ries isol~es h p a r t i r de l'eau et de la terre au Za'ire ont 6tfi identifi6es comme Mycobacterium malmoense p a r tests biochimiques et analyse lipidique. A p a r t les acides gras pr6c~demment not6s, qui sont caract~ristiques de cette esp~ce, les deux souches, ainsi que 5 isolats cliniques de M. malmoense, contenaient de l'acide 2,4-dimethyl-docosanoique. L ' u n e des souches de l'environnement, profil glycolipidique II, contenait 6galement de l'acide 2-methylt6tradecanoique. Les r~sultats confirment que M. malmoense pourrait 6tre sous-divis~ en 2 sous-groupes selon son profil lipidique. Ils m o n t r e n t ~galement que M. malmoense peut 8tre isol~ de l'environnement, lequel p o u r r a i t 8tre la source de l'infection.

R E S U M E N . Dos cepas de mieobacterias aisladas a partir del agua y de la tierra en Zaire fueron identificadas como Mycobacterium malmoense con tests bioquimicos y amilisis lipidico. F u e r a de los tlcidos grasos previamente descritos, caracteristicos de esta especie, a m b a s cepas, asi como 5 aislados clinicos de M. malmoense, contenian ticido 2, 4-dimetil-docosanoico. Una de las cepas ambientales con un perfil glicolipidico I I contenia ademfis ticido 2-metiltetradecanoico. Los resultados confirman que M. malmoense podria estar dividido en 2 subgrupos, segdn el perfil lipidico. Tambi6n demuestran que M. malmoense puede ser aislado a p a r t i r del medio ambiente, el cual podrla constituir una fuente de infecci6n.

sifted as MAI (M. avium-intracellulare) intermediates. 5 A recent reanalysis of these MAI intermediates including lipid analyses allows us to reclassify two of these strains as M. malmoense. This report presents our findings.

INTRODUCTION

Mycobacterium malmoense is a slow-growing pathogen 1 which is isolated with increasing frequency in several countriesY Interestingly, this species has not yet been reported from the environment. However, several observations suggest that M. malmoense, like other atypical mycobacteria which may colonize or cause disease in humans and animals (e.g.M. avium-intracellulare), may be present in the environment too. 3 From 1970-74, as part of a study on environmental mycobacteria in Zaire, 956 mycobacterial strains were isolated. 4 Some of these isolates were provisionally clas-

M A T E R I A L S AND M E T H O D S We studied two mycobacterial strains which had been isolated 20 years ago from soil and water in Zaire. They were isolated from the specimens using the N a 3 P O 4 decontamination procedure of Corper and Stoner 6 modified as described by Portaels et al.7 The isolates were identified as described by Vincent-L6vy-Fr6bault and Portaels. 8 As biochemical tests suggested that both strains belonged to M. malmoense we decided to study

Correspondenceto: F. Portaels, Departmentof Microbiology,Institute of TropicalMedicine, Nationalestraat 155, 2000 Antwerp, Belgium.

Paperreceived7 February1994. Finalversionaccepted27 June 1994. 160

Isolation o f Mycobacterium malmoense f r o m the e n v i r o n m e n t in Zaire T a b l e 1.

161

Characteristics o f M. malmoense isolated f r o m the e n v i r o n m e n t and f r o m clinical specimens

G r o w t h at 3 7 ° C G r o w t h in presence o f Isoniazid (10 g g / m l ) H y d r o x y l a m i n e (250 g g / m l ) p-Nitrobenzoate (500 g g / m l ) E n z y m a t i c properties Urease T w e e n h y d r o l y s i s (10 days) Acid phosphatase M y c o l a t e types c~-mycolates c(-mycolates methoxymycolates ketomycolates c0-carboxymycolates

E n v i r o n m e n t a l isolates no. 6033 no. 6 0 3 5

Clinical isolates* 5 isolates M. malmoense*

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*+ = > 85% o f strains positive; M = 5 0 - 8 5 % o f strains positive; = < 15% strains positive. +Data f r o m V i n c e n t L6vy-Fr6bault a n d P o r t a e l s ?

their cellular composition regarding fatty acids, glycolipids, and mycolic acids. It has previously been shown that lipid analysis is very useful for species identification of M. malmoense. 9 For comparison, 5 clinical isolates of M. malmoense were included (Tables 1 and 2). Fatty acid analysis was made after heating the freezedried mycobacteria in acid methanol. The methyl esters were identified by gas chromatography-mass spectrometry analysis in electron impact mode. 1° Mycolic acids were studied using one-dimensional thin-layer chromatography after heating the bacteria in alkaline methanolic solution and then esterifying the liberated acids with iodomethane? I,~2 Glycolipids were studied by onedimensional thin-layer chromatography of a simple ether/ethanol water extract of the dried bacilli. 9

RESULTS The two mycobacterial strains isolated from the environment were identified as M. malmoense according to their cultural, physiological and biochemical characteristics as indicated in Table 1. This Table also contains data on the 5 clinical isolates which were identical and such as our two environmental isolates had the same properties as those described for M. malmoense, s

T a b l e 2. B r a n c h e d - c h a i n fatty acids and glycolipids detected in 2 e n v i r o n m e n t a l m y c o b a c t e f i a i isolates and in 5 clinical isolates of

M. malmoense

Strain

Source

6033 6035

Soil Water Patients

M. malmoense

B r a n c h e d - c h a i n fatty acids Retention time (min)* 6.20 10.11 12.71 16.75 (A) (B) (C) (D)

Glycolipid pattern*

* -

T y p e II Type I Type I

* * *

* * *

* * *

The 7 tested strains all formed mycolic acid patterns characteristic .of M. malmoense VIZ c~-mycolates, ~'mycolates and ketomycolates2 ° All strains analyzed produced methyl hexacosanoate as the main pyrolysis product of the methyl mycolates. Four branched-chain fatty acids (apart from tuberculostearic acid) were detected (Table 2); all of them produced mass spectra with dominating peaks at m/z 88 and m/z 101 indicating methyl-branching in position 2 of fatty acid methyl esters. The compound called (A) 6.20 (retention time, see Table 2) produced a molecular ion of m/z 256 and was identified as 2-methyletetradecanoate (2-CH 3 14:0). (B) 10.11 was identified as 2-methyl-eicosanoate (2-CH 3 20:0). (C) 12.71 exhibited a molecular ion peak at rrdz 382 and characteristic fragments of m/z 339 (M-43), m/z 311 (M-71), and m/z 129 (cleavage of bonds at position 4), and was identified as 2,4-dimethyldocosanoate (2,4C H 3 22:0). (D) 16.75 (molecular ion m/z 424) was identified as 2,4,6-trimethyltetracosanoate (2,4,6-CH 3 24:0). 13 Of the environmental strains analyzed, one (6035) showed the fatty acid pattern characteristics of M. malmoense (including 2-CH 3 20:0 and 2,4,6,-CH3 24:0) whereas the other (6033) additionally contained 2-CH3 14:0. In addition, 2,4-CH3 22:0 was detected in all strains. The glycolipid analyses revealed features typical of M. malmoense: one (6035) was of glycolipid type I and the other (6033) showed the less commonly found glycolipid type II9 (Table 2).

DISCUSSION

(5 strains) *Identity o f peaks: 6.20, 2 - C H 3 14:0; 10.11, 2 - C H 3 20:0; 12.71, 2,4C H 3 22:0; 16.75, 2 , 4 , 6 - C H 3 24:0. See text for explanation o f abbreviations. tSee reference 9.

When M. malmoense was first described in 19771 it was a comparatively rare pathogen and only occasionally isolated as a casual contaminant from clinical specimens. During the early 1980s the incidence of genuine cases of pulmonary disease in England and Wales rose to an average of 30 per year. There was a similar rise

162 Tubercle and Lung Disease in the n u m b e r o f single isolates f r o m sputum w h i c h w e r e o f doubtful significance. In addition, cases o f c e r v i c a l a d e n o p a t h y b e g a n to appear in children. TM This increase in i n c i d e n c e could be attributed to m o r e effective m e t h o d s o f isolation and/or i m p r o v e d m e t h o d s o f identification. H o w e v e r , there has b e e n little if any change in these m e t h o d s in the last 20 years, and the m o r e likely explanation is that M. malmoense has found an e c o l o g i c a l n i c h e w h i c h suits its survival. L i p i d analysis was used to verify the species identify o f the m y c o b a c t e r i a . Interestingly all M. malmoense strains c o n t a i n e d 2, 4-CH3 22:0, an acid w h i c h to date has not b e e n reported for this species. T h e g l y c o l i p i d II type o f M. malmoense strain also contained 2 - C H 3 14:0 - a fatty acid present also in M. gordonae. 15 The simultaneous p r e s e n c e o f 2 - C H 3 20:0, 2,4-CH3 22:0, and 2,4,6CH3 24:0 appears to be u n i q u e for M. malmoense. T h e isolation o f M. malmoense f r o m the e n v i r o n m e n t is o f interest. H o w e v e r these two isolates w e r e obtained 20 years ago in Zaire, w h e r e a s the clinical isolates h a v e b e e n largely c o n f i n e d to N o r t h e r n E u r o p e during the last 10 years. M. malmoense has b e e n isolated f r o m faeces o f H I V p o s i t i v e patients in Zaire 16 w h i c h confirms its o c c u r r e n c e there. It was also isolated f r o m w i l d armadillos captured in L o u i s i a n a ~7 and f r o m stools o f healthy individuals in B e l g i u m . TM R e c e n t l y e n v i r o n m e n t a l isolates h a v e b e e n reported in F i n l a n d (including detection o f 2 , 4 - C H 3 22:0). 19 All these observations indicate that M. malmoense, like m o s t o f the atypical m y c o b a c t e r i a , is p r o b a b l y present in m a n y different g e o g r a p h i c areas. It is thus likely that M. malmoense is an e n v i r o n m e n t a l o r g a n i s m that is ubiquitous and m a y c o l o n i s e or cause infections in h u m a n s and animals. W i t h i m p r o v e d isolation and diagnostic techniques it will p r o b a b l y be isolated m o r e c o m m o n l y f r o m clinical s p e c i m e n s and f r o m the environment.

Acknowledgements

This study was supported by the Swedish Association against Heart and Lung Diseases and by the Science and Technology for Development Programme of the European Community, contract number TS2-0080UK.

References

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