LEl lERS TO THE EDITORS

In-vitro acquisition of iron from the iron- desf e rri oxam i ne corn p I ex by Aeromonas hydrophila

To the Editors: Iron is essential for the growth of many micro- organisms. Most bacteria need 0.4 to 4 pM for survival. To establish an infection, pathogenic bacteria must compete with the host to acquire iron. Microorganisms have therefore developed several strategies, amongst which are the production of siderophores and the synthesis of iron-regulated outer membrane proteins, the latter serving as receptors for the former [l]. Desferrioxamine (DFO), produced by Stveptomyces pilorus, is a trihydroxamate siderophore with a high affinity for iron. The methanesulfonate salt of this compound is commercially available as Desferal@ (CIBA-Geigy, Basel, Switzerland). DFO is a t the moment the only iron chelator clinically available for therapy of iron or aluminum overload [2].

A patient on hemodialysis in Taiwan, treated with DFO for aluminum bone disease, died after developing Aevomonas hydrophila bacteremia with myonecrosis and gas gangrene. The purpose of the present in vitro study was to determine whether the iron complex of desferrioxamine (Fe.DF0) promotes the growth of Aevomonas hydrophila under iron-limiting conditions by acting as source of iron for this microorganism.

The patient’s strain, isolated from blood as well as from necrotic tissue, was obtained from Dr Shih-Hua Lin, division of nephrology of the Tri-Service General Hospital of Tapei, Taiwan. Cultures were maintained on Luria agar (DIFCO Laboratories, Detroit, Michigan, USA). The Luria agar was made iron-deficient by adding 1 ,2-dimethyl-3-hydroqy-4-pyridone (‘Ll’ or deferiprone) at a final concentration of 2 mM. The iron chelates tested were: desferrioxamine bound to iron (Fe.DF0) at a concentration of 1, 10, 50 and 100 pM, Fe.(L1)3 and N-N’-bis(2-hydroxybenzyl) ethylenediamine-N,N’-diacetic acid (HBED) bound to iron (Fe.HBED) at a concentration of 50 pM. L1 and HBED were kindly provided by Duchefa, Haarlem, The Netherlands, and by Dr R. W. Grady, New York, USA, respectively. The iron chelates were dissolved in water purified by the Mill-Q System, and sterilized by filtration through non-pyrogenic filters (Minisart NML Sartorius, Gottingen, Germany) prior to use. All glassware was made iron-free by acid washing.

The diffusion method was used in two different ways. Molten iron-deficient Luria agar was inoculated with an overnight culture, adjusted to a density of a 0.5 McFarland (1 to 2 x lo8 CFU/mL), and then paper

disks, impregnated with 20 pL of the iron chelate to be tested, were placed on the solidified iron-deficient Luria agar. Alternatively, a well with a diameter of 4.5 nim was punched in the solidified inoculated iron- deficient Luria agar and 53 pL of the iron chelate to be tested was poured into the well. Plates were incubated at 37 “C and examined at 24 h and 48 h for zones of growth around the disks and the wells. The growth of Aeromonas hydrophila was completely inhibited on the Luria agar containing only Ll . However, when Fe.DFO was added to the Luria agar, Aeromonas hydrophila grew after 24 h of incubation around disks and wells containing Fe.DF0. At the lowest Fe.DFO concentration tested (1 pM), the growth-enhancing effect was already clearly visible. The higher the Fe.DFO concentration, the greater the diameters of the growth around &sks and wells (Figure 1). In contrast, no growth was seen around disks and wells containing the iron complex of two unrelated chelators, L1 (Figure 2) and HBED. The growth-promoting effect of Fe.DF0 on the patient’s AeroMzonas hydrophila strain, when tested in iron-restricted conditions, is therefore chelator-specific. The mechanism of the iron uptake from the iron complex Fe.DFO is not known. An iron- deficient environment stimulates the expression of

Figure 1 hydrophila on an iron-restricted plate. Filter paper disks contained 20 pl of 1 (a), 10 (b), 50 (c) and 100 (d) pM Fe.DF0. The greater the concentration, the greater the diameter of the growth of Aevomonas hydrophila around the disks.

Effect of Fe.DFO on the growth of Aeromonns

2 7 4 C l in ica l M i c r o b i o l o g y and In fec t i on , Vo lume 1 Number 4

the mechanism responsible for the iron uptake from Fe.DFO by Aeromonas hydrophila.

Aoki and Holland [3] and Massad et a1 [4] found that Aeromonas hydrophila, under iron-restrictive conditions, synthesizes new iron-regulated outer membrane proteins, serving for the transportation of siderophore-bound iron. Most isolates of Aeromonus hydrophila were found to produce one or both of the following siderophores: enterobactin, which is the prototypical catecholate siderophore produced by Enterobacteriaceae and amonabactin, which is another catecholate siderophore. A few isolates of Aeromonus hydrophila apparently produce no siderophore [S-71. We did not study the siderophore production pattern of our strain. Whether these endogenously produced sidero- phores (enterobactin and amonabactin) play a role in the uptake of iron from Fe.DFO is not known.

We conclude that the studied clinical Aeromonas hydrophila strain can acquire iron in vitro from Fe.DFO under iron-restricted conditions. Likewise, the 495A2 strain of the same species, isolated from a sick reptile [5], as well as two strains derived from strain 495A2 by mutagenesis (SB22 and SBlOl), and unable to produce amonabactin, were all reported to be capable of Fe.DFO utilization [5,8,9]. It is not known whether all Aeromonas hydrophila strains, of environmental and of clinical origin, share this property and whether or not this is dependent on the type of siderophore produced.

Figure 2 After 48 h of incubation, no growth of Aeromonas hydrophila was visible around the disk containing 50 pM iron complex of 1,2-dimethyl-3-hydroxy-4- pyridone [Fe.(L1)3] (a), as contrasted with the growth- enhancing effect on Aeromonas hydrophila visible around the disk containing 100 pM Fe.DFO (b).

This iron utilization from the exogenous siderophore DFO was probably responsible for the septicemia due to Aeromonas hydrophila, which occurred in this dialysis patient during treatment with DFO. Other micro- organisms have been reported to share the same in vitro property (reviewed in Boelaert et a1 [2]). The microbial-growth-enhancing effect of DFO is known to be of particular concern in cases of infection by Yersinia enterocolitica and the Zygomycetes [2].

E. De Brauwer' H . W I/an Ldnduytl

B. Gordts' 1. R. Boelaer?

'Department of Microbiology Algemeen Ziekenhuis St Jan,

Ruddershore 10, B-8000 Brugge, Belgium;

'Unit of Renal and Infectious Diseases, Algemeen Ziekenhuis St Jan,

B-8000 Brugge, Belgium

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