iron chelator for investigating mycobactin production in mycobacteria. The optimal conditions of EDDA concentration and time of exposure were established and ...
CURRENT MICROBIOLOGY Vol. 26 (1993), pp. 353-358
Current Microbiology 9 Springer-Verlag New York Inc. 1993
A Simple and Reliable EDDA Method for Mycobactin Production in Mycobacteria: Optimal Conditions and Use in Mycobacterial Speciation Suzana Bosne, Fernand Papa, Sabine Clavel-S6r6s, and Nalin Rastogi Tuberculosis and Mycobacteriology Unit, Institut Pasteur, Paris, France
Abstract. Ethylenediamino-di-(0-hydroxyphenylacetic acid) (EDDA) was found to be a suitable
iron chelator for investigating mycobactin production in mycobacteria. The optimal conditions of EDDA concentration and time of exposure were established and were used to develop a twostep method for mycobactin production. Applied to representative strains of selected species, the method was found to yield reliable results useful for identification and speciation of mycobacteria.
Mycobacteria, like most other aerobic and facultative anaerobic organisms, have the capacity of synthesizing high-affinity iron chelators, or siderophores, that enable them to capture this essential metal in low iron environments [10]. Since iron exists in its insoluble ferric form in aerobiosis and at neutral pH, it is virtually unavailable under these conditions for microbial metabolism unless captured by a ferric ligand which in itself is regulated by the presence of the metal [10]. In mycobacteria one of the siderophore systems developed is lipid-soluble siderophores known as the mycobactins [16], which appear located within the mycobacterial cell wall [12]. The mycobactins are of considerable interest, not only because of their lipoidal nature, unique among siderophores, but also because they are growth factors of Mycobacterium paratuberculosis [15-17]. Mycobactins have also been reported to be potentially useful taxonomic markers [1-3, 6-8, 17]. The practical use of mycobactins in clinical diagnosis and the development of other technologies has been hindered by the difficulty of obtaining significant yields of the natural compounds in a reasonable period of time and by the complexity of the purification of the crude extract [12]. Mycobactin is produced under conditions of iron starvation [5, 16]. In previously proposed methods, a minimal synthetic medium with low iron content was used [7, 9, 15-17]. Mycobacteria were grown as pellicles on the surface or as submerged growth with stirring in liquid synthetic medium [16,
18] or on the surface of a solid synthetic medium [7]. Not only is growth under these conditions considerably slowed down, but, when using liquid medium, media and glassware have to be carefully treated to remove all traces of iron, since a concentration as low as 1 /xg/ml is sufficient to repress mycobactin synthesis [6]. Attempts at using iron chelators to simplify the removal of excess iron from the medium failed [13]. In a previous paper [3] a simple and practical method for obtaining iron starvation conditions and mycobactin synthesis in rich medium with the iron chelator EDDA gave sufficient mycobactin for TLC analysis of Mycobacterium fortuitum and Mycobacterium chelonae species with heavy inoculum directly in EDDA medium (the EDDA being present in excess, the eventual carryover of iron was not a significant problem). However, further experimentation with slowly growing strains was not so successful, and further studies were required. It is the purpose of this paper to propose a two-step method for mycobactin production. Once established, the use of mycobactin analysis for the identification and speciation of mycobacteria was further investigated. Materials and Methods Bacteria and growth. M y c o b a c t e r i u m a u r u m was selected as the reference strain to examine production conditions for mycobactins. M . a u r u m presents the advantage of being a rapidly growing, nonpathogenic strain that synthesizes only one mycobactin based on TLC analysis of the product [17]. Once established, these
Address reprint requests to: Dr. S. Bosne, Unit6 de la Tuberculose & des Mycobact6ries, Institut Pasteur, 75724-Paris Cedex 15, France.
354 conditions were tested on strains of M. avium complex (one strain from each serotype, except serotypes 20, 23, and 24), M. scrofulaceum (one strain), M. simiae (three strains), M. gastri (six strains), M. triviale (one strain), M. terrae (three strains), M. kansasii (six strains), and M. marinum (six strains). Liquid medium containing, per liter, 5 g dehydrated Bacto Nutrient Broth (Difco, Detroit, Michigan, USA), 5.2 g Middlebrook 7H9 broth (Difco), 10 ml glycerol, was used as previously described (this medium will be referred to as NB-7Hgglycerol medium in the text [3]). EDDA [16, 17] was added in varying concentrations, 0-5000/~g/ml. Cultures were incubated at 30~ or 37~ for varying lengths of time according to experiment and type of strain (slow or rapid growers).
CURRENT MICROBIOLOGY Vol. 26 (1993)
| 80
EDDA-O
E ,0
EDDA-IO0
' ~
60
0
o
40
X g~
ZO L
OC--'~-~,~ , O 10
Chelator preparation. EDDA was obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). It was freed from contaminating iron by the method of Rogers [14]. Briefly, a solution containing 10 g of EDDA in 190 ml of 1 N HCI was boiled and stirred for 1 h. After cooling and subsequent filtration through a 0.45-btm pore-size filter, the solution was diluted with 1500 ml of pure acetone, and the pH was raised to 6.0 by adding 1 N NaOH. After standing overnight at 4~ the precipitate was collected by filtration and washed with cold acetone. Dilution in acetone and filtration were repeated twice. The precipitate was dried overnight at 40~ and then collected into a plastic container for storage. The deferrated EDDA was incorporated into the growth medium by the procedure of Ong et al. [11]. To prepare a stock solution, 1 g of deferrated EDDA was dissolved in 15 ml of 1 N NaOH. The pH was adjusted to 9 with concentrated hydrochloric acid, and the total volume was adjusted to 20 ml with distilled water to give a final concentration of 5% (w/v).
EDDA"~O
~
~-"-~, 20 30
~
i
~
40
,~ 50
60
DAYS
,.,
40
| ~
O
~
DAY-15
30
0
o
20
X {a
10
