Download Isolation of a novel siderophore from Pseudornonas cepacia

J . Med. Microbiol. - Vol. 36 (1992), 184-189
01992 The Pathological Society of Great Britain and Ireland
Isolation of a novel siderophore from Pseudornonas
cepacia
P. A. SOKOL,
C. J. LEWIS and J. J. DENNIS
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta,
Canada TZN 4N 1
Summary. A novel iron-binding compound was identified in ethyl acetate extracts of the
supernates from Pseudornonas cepacia cultures. This compound, named azurechelin, was
produced by 88% of P . cepacia strains isolated from the respiratory tract. Production of
azurechelin was regulated by the iron concentration in the culture medium. Azurechelin
enhanced the growth of P . cepacia in a medium containing transferrin 200 mg/L. Azurechelin
released iron from transferrin in an equilibrium dialysis assay, suggesting that it could
compete with transferrin for iron. Azurechelin could also stimulate iron uptake by P . cepacia.
This siderophore appeared to have a novel structure with neither the typical characteristics
of catechol nor of hydroxamate compounds.
Introduction
Siderophore-mediated iron acquisition has been
correlated with the ability of various bacteria to
The fact that
establish and maintain infection.
many bacterial species produce more than one siderophore is also indicative of the importance of iron in
bacterial proliferation.
Pseudornonas cepacia can cause respiratory infections in cystic fibrosis patients.’ In some cases, these
P . cepacia infections have been correlated with a
deterioration in clinical status. In a previous study, we
reported a correlation between production of the
siderophore pyochelin by P . cepacia isolates from
cystic fibrosis patients and morbidity and mortality in
these patients.6 We have also shown that exogeneously
supplied pyochelin can enhance the virulence of
pyochelin-non-producing P . cepacia strains in a
chronic pulmonary infection model in rats.’ While
performing these studies, we identified another ironbinding compound present in ethyl acetate extracts of
the culture supernates from several strains. In the
present study we describe the production, purification
and preliminary characterisation of this iron-binding
compound, which we have given the trivial name
azurechelin because of its blue fluorescence under
ultraviolet (UV) light.
1v
39
Hospital for Sick Children, Toronto, Ontario, Canada
and J. D. Klinger, Rainbow Babies’ and Children’s
Hospital, Cleveland, Ohio, USA and have been
described
* Strains Pc275c and Pc7lOm
produced both pyochelin and azurechelin. Strains
K30-6 and H1724-1 produced only azurechelin and
strain H1721 did not produce detectable levels of
either siderophore.
Culture conditions
For the detection of azurechelin, cultures were
grown in 50-ml amounts of Casamino Acids Medium
(CAA; Difco) 0.5% which was deferrated as described
previously.6 Assays to assess the effects of azurechelin
and pyochelin on the growth of P . cepacia were
performed in M9 minimal medium supplemented
with glucose 0.5%, 10 mM NaHC03 and transferrin
(Sigma) 200 mg/L (Transferrin Medium).6Production
of azurechelin was also determined in M9 medium
with 20 mM sodium succinate or sodium citrate instead
of glucose as the carbon s o ~ r c e The
. ~ average iron
content of the CAA and M9 media was approximately
3-5 p ~All
. glassware was washed with acid to remove
iron. All reagents were made with water purified by
the Milli-Q System (Millipore, Missisauga, Ontario,
Canada).
Pyochelin pur fication
Materials and methods
Strains
Pyochelin was purified from P . aeruginosa strain
P A 0 as described previously.6**
The P . cepacia isolates from the sputum of cystic
fibrosis patients were a gift from C. L. Prober, the
Azurechelin purification
-
Received 1 1 March 1991; revised version accepted 28 May 1991.
Azurechelin was purified by essentially the same
method as was used for pyochelin. Cultures (500 ml)
184
Downloaded from www.microbiologyresearch.org
by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:48:43
SIDEROPHORE FROM P . CEPACZA
were grown in CAA medium for 24 h at 30°C. The
cells were removed by centrifugation at 10 000 g. The
supernates were collected, adjusted to pH 2.0 with
HCl, and extracted with ethyl acetate (2 vol:5 vol
supernate). The extracts were concentrated to dryness
by rotary evaporation then dissolved in 1-ml amounts
of methanol and chromatographed on a thin layer of
)
Scientific) with chloSilica Gel G (250 p ~ (Mandel
roform :acetic acid :ethanol 90 : 5 : 2.5 as the mobile
phase. The fluorescent blue band visible under
UV light was scraped from the plate and eluted
into methanol. The methanolic extracts were rechromatographed and developed with chloroform :acetic :ethanol 90 :5 : 5. The azurechelin band
(R,=O.SO) was detected by its fluorescence, eluted into
methanol, and stored at - 20°C. In some purifications,
azurechelin was subjected to gel filtration chromatography on a Sephadex G-10 column (1-5x 30 cm) with
aqueous methanol 10%v/v as the mobile phase instead
of to a second thin layer chromatography step. For
most experiments, azurechelin was used within 2 days
of purification.
To quantify azurechelin, methanol extracts were
filtered through 0 . 2 2 - p ~filters (Millipore) and dried
under vacuum into pre-weighed acid-washed vials.
Azurechelin was also quantified in culture supernates
by measuring the absorbance at 310 nm (the absorbance maximum for azurechelin) and determining the
concentration from a standard curve for purified
azurechelin.
Assay for azurechelinproduction
Strains were grown in 50 ml of CAA medium for
24 h. The supernates were processed according to the
purification procedure above, through to the first thinlayer chromatogram. Azurechelin was identified by its
relative mobility (R,= 0-8), its blue fluorescence under
UV light, and its red-brown reaction when sprayed
with 0.1 M FeCl, in 0.1 M HCl. Purified azurechelin
was chromatographed as a control.
Chemical characterisation
Absorption spectra for purified azurechelin and
pyochelin were obtained with a Model 80 spectrophotometer (Pye-Unicam, Cambridge, Cambs.). Siderophore activity was determined by the Chrome Azurol
S assay." This assay is based on the ability of a
siderophore to remove iron from a dye-containing
complex. Purified azurechelin was analysed for
catechol structures by the assays of Arnow12 and
Rioux et aZ.13 Bound and free hydroxylamine compounds were analysed by the method of Csaky.l4
Growthpromotion assays
185
chelin or pyochelin, dissolved in ethanol, was added
to the medium to a final concentration of 10 mg/L.
Equivalent volumes of ethanol were added to the
control cultures. Growth was monitored by measuring
the increase in A600with time.
Equilibrium dialysis assay
Release of iron from transferrin was investigated
by the method described by Sriyosachati and Cox"
with the following modifications. Apotransferrin was
prepared by dialysing transferrin (20 g/L ; Sigma)
against 1 L of 0 . 2 ~
sodium citrate-sodium acetate
buffer, pH 4.5. Portions of the apotransferrin were
brought to 100% saturation by the procedure of
Simonson et aZ.16 Excess 59Fe was removed by
extensive dialysis against 40 mM Tris, 20 mM sodium
bicarbonate buffer pH 7.4 until the amount of 59Fein
the dialysate remained at a minimal constant level.
Reactions were performed in 20 mM morpholine
propane sulphonic acid (MOPS), 20 mM pyrophosphate. The pH was adjusted to 5.0,6.0 or 7-4with 1 M
HC1. 59Fe-transferrin was added to this buffer to a
concentration of 12 mg/L. Azurechelin was added to
some reactions at a final concentration of 25 mg/L.
Apotransferrin was included at 100 mg/L. This mixture was placed in dialysis tubing (6.2 mm diameter,
mol. wt 10 000 exclusion limit) and dialysed against
2ml of the same buffer containing apotransferrin
(100 mg/L). Portions of the dialysate were removed at
intervals and the amount of 59Fe released into the
dialysate was determined by measuring radioactivity
in a gamma counter (Compugamma LKB Instruments, Inc. Rockville, MD, USA). The percentage of
the available iron released was calculated relative to
the initial counts in the reaction mixture.
Iron uptake assays
Cultures were grown in M9-glucose medium to a
density of lo8 cfu/ml, centrifuged, washed once, and
resuspended in fresh medium. Uptake reactions were
initiated by the addition of 100 pg (1 pCi) of 59FeC1,
(Amersham Corpn., Arlington Heights, IL, USA) and
either 1.0 pg of pyochelin or 4 pg of azurechelin in a
total volume of 1OOpl. One-ml samples of these
reaction mixtures were removed at 10-min intervals
and filtered through cellulose acetate 0.2 pm pore
filters (Sartorius GmbH, Germany). The amount of
"Fe accumulated was determined as previously
described6 in a gamma counter (LKB).
Results
Production of azurechelin
Growth assays were performed as described previously.6 The cultures were inoculated at an initial
density of lo5 cfu/ml in Transferrin Medium. Azure-
When ethyl acetate extracts of the supernates from
cultures of P.cepacia were chromatographed on Silica
Gel G plates, two fluorescent bands were visible under
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:48:43
I86
P. A. SOKOL, C. J. LEWIS AND J. J. DENNIS
UV light, both of which turned red-brown when
sprayed with 0.1 M FeC1,. A yellow-green fluorescent
band at an Rfof 0.4 has previously been reported to
be pyocheh6 A blue fluorescent band, which turned
brown with FeCl,, was also visible and had an Rf of
0.8. This latter spot also turned brown when sprayed
with 0.1 M ferric ammonium sulphate, ferric citrate,
and ferric ammonium citrate in 0.1 M HC1. Forty-six
(88%) out of 52 respiratory isolates of P . cepacia from
patients with cystic fibrosis produced this compound
at concentrations detectable by thin layer chromatography. Eight of the 46 positive strains did not produce
detectable pyochelin, so this compound could not be a
degradation product of pyochelin. We have given this
compound the trivial name azurechelin because of its
fluorescent blue colour and to prevent confusion with
cepabactin, a siderophore produced by some strains
of P . cepacia and described by Meyer et a!.l 7
To determine the optimal medium for production
of azurechelin, the yields from strains Pc275c and
K30-6 were compared in four different media (table
I). Yields were highest in CAA medium, which,
therefore, was used for subsequent studies. The effect
of iron on production of azurechelin was examined for
three strains of P . cepacia grown in CAA medium
(table 11). Production of azurechelin in strains Pc275c
and H1724-1 was highly iron-regulated. In strain
Pc7 10, iron concentrationsof 50-1 00 C(Mwere required
to repress azurechelin expression. Thus, although
expression of this compound was iron-regulated,there
was some inter-strain variation in the effect of iron on
its yield.
Table I. Comparison of azurechelin production by P .
cepacia strains in different culture media
Azurechelin (mg/L) from
strain
Medium
Pc275c
K30-6
5.9
1.1
1.9
0.3
4.6
1.0
1.4
0
CAA
M9 +glucose 0.5%
M9 + 20 mM succinate
M9 + 20 mM citrate
Table 11. Effect of iron concentration on production of
azurechelin by P . cepacia
~
~~
~
Azurechelin (mg/L)/ ODeooUnit
at FeCI, concentrations*
Strain no.
1
Pc275c
H1724-1
Pc7 10m
10.8
6.8
3.8
3.4
3.4
3.6
1.2
3.2
3.2
1.1
0.7
2.4
1.0
0-2
0.7
*Final concentration of FeC1, added to the CAA medium.
iron-free azurechelin dissolved in methanol. The
absorption spectrum for iron-free pyochelin was
obtained for comparative purposes. Azurechelin had
absorbance maxima at 240 and 310 nm (fig. 1). By
contrast, pyochelin gave peaks at 210,250 and 310 nm
(fig. 1 p 9
The Chrome Azurol S (CAS) assay is a universal
assay for siderophores, exploiting their ability to
release iron from the Chrome Azurol S/iron/hexadeyltrimethyl-ammonium bromide complex. * Purified
azurechelin removed iron from the dye complex in the
CAS assay and changed the colour of the solution
Characterisation of azurechelin
Azurechelin was purified by preparative thin-layer
chromatography on Silica Gel G or by gel filtration on
Sephadex G-10. UV-visible spectra were obtained for
3-51
3.02.52.0 1-5-
1.00.5y
ih
I
I
. L
..
A-
q
0
2i 00 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0
Y
1
I
I
I
I
I
I
Wavelength (nm)
a)
Fig. 1. Comparison of absorption spectra for purified azurechelin
and pyochelin ( 0 ) 50
; pg of azurechelin or pyochelin were dissolved
in methanol and scanned from 190 to 800 nm at 10-nm intervals. Absorbance between 190 and 400 nrn is shown; the values remained at a
background level between 400 and 800 nm.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:48:43
SIDEROPHORE FROM P. CEPACIA
from blue to orange. Culture supernates quickly
changed the colour of the CAS solution, but purified
azurechelin required the addition of 5-sulphosalicylic
acid, which acts as a shuttle between the dye complex
and the siderophore. * When TLC plates were sprayed
with Chrome Azurol S solution, the azurechelincontaining spots turned pink, as has been reported for
other siderophores, including aerobactin, enterochelin
and rhizobactin. *
Most siderophores are either of the catechol or
hydroxamate class. However, azurechelin gave negative results in assays for the detection of catecholcontaining siderophores. l4 It did not react, either,
with a “phenolate detection” spray (50 mM FeC13,
50 mM potassium ferricyanide,l o which normally turns
blue when sprayed on a TLC plate containing phenolic
compounds. Although not expected to be a hydroxamate siderophore, since it was extractable with ethyl
acetate, azurechelin was tested for hydroxylamine by
the Csaky assay,I5 with negative results.
Azurechelin appeared unstable. It lost iron-binding
activity after a few days when stored in a dried form.
When stored in ethanol at - 20°C, the iron-binding
activity decreased by approximately 25% within 7
days.
1 3 9
Eflects of azurechelin on growth and iron transport
Previously, we have shown that pyochelin can
enhance the growth rate of P . cepacia strains in a
medium containing transferrin.6 To determine if
azurechelin also could increase the growth rate of P .
cepacia, the effects of azurechelin and pyochelin on
the growth of three strains of P . cepacia were
determined. Strain Pc275c produced both pyochelin
lo[
t
187
and azurechelin, whereas strains K45- 1 and H 1721
did not produce detectable levels of either siderophore.
Cultures were grown for 24 h in M9 medium with
200 PM ethylene diamine-N-N’-diacetic acid to starve
the bacteria for iron, then diluted into Transferrin
Medium at an initial density of 104-105 cfu/ml. The
cultures were divided into three flasks and pyochelin
(10 mg/L) or azurechelin (10 mg/L) was added to two
of the cultures. Growth was monitored by measuring
A600 at intervals for up to 4 days (fig. 2). Both pyochelin
and azurechelin dramatically increased the growth
rates of the siderophore-negative strains, K45-I and
H1721, in the Transferrin Medium. Although the
effect on strain Pc275c was not as dramatic, both
pyochelin and azurechelin shortened the lag phase for
this organism. Pyochelin increased the growth rates
of strains Pc275 and H1721 to a greater extent than
did azurechelin (fig. 2B, C). These data suggest that
azurechelin may be effective in removing iron from
transferrin. To investigate this aspect further, the
ability of azurechelin to remove iron from transferrin
was examined in an equilibrium dialysis assay (table
111). Removal of iron from transferrin was determined
at pH 7.4, 6.0 and 5.0. Azurechelin was effective in
releasing iron at both pH 6.0 and 5.0 but not at pH
7.4. At pH 6.0, equilibrium was reached within 24 h.
These data suggest that azurechelin can compete with
transferrin for iron.
To confirm that P . cepacia strains could use azure
chelin to transport iron, their ability to accumulate
ferri-azurechechelin was determined and the uptakes
of ferri-azurechelin and ferri-pyochelin were compared (fig. 3). Strains Pc275c and H1721 were grown
in M9 minimal medium, in which pyochelin production is not detectable and azurechelin production is
’9
A
I
E
C
Fig. 2. Effects of azurechelin and pyochelin on growth of P. cepacia strains in M9 medium containing transferrin 200 mg/L: A, strain K45-1;
B, strain H1721;C, strain Pc275c; a,no siderophore added; 0 ,azurechelin 10 mg/L added; m, pyochelin 10 mg/L added.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:48:43
188
P. A. SOKOL, C. J. LEWIS A N D J. J. DENNIS
Table 111. Iron-binding activity of azurechelin in equilibrium dialysis
Percentage of 59Fereleased from 59[Fe]-transferrin
Buffer pH
7.4
6.0
5.0
6.0
6-0
6.0
Time (h)
24
24
24
2
24
120
without azurechelin
with azurechelin
0
0
0
1-5
5.6
7.5
0
56.8
50.5
5.3
44.6
49.7
59[Fe]-labelledtransferrin was prepared by the method of Simonson et
The assay consisted of
20 pg of azurechelin on one side of the dialysis membrane and s9[Fe]-transferrin (6 mg/L, 100%
saturated) on the opposite side. Apotransferrin (100 mg/L) was included on both sides with 100 PM
MOPS buffer, 20 m M sodium bicarbonate and 10 mM pyophosphate as the diluent; c. 56 000 cpm of
59Fewere available for equilibrium in the first experiment; 34 500 cpm in the second.
30
-
at 210, 250 and 310nm, whereas those of pyochelin
are at 240 and 310 nm and those of cepabactin are
25 reported to be 330 and 440 nm.” Moreover azurechelin reacted only with FeC13; whereas pyochelin
n 20and cepabactin react also with the phenolate detection
spray reagent.
Azurechelin promoted the growth of P. cepacia
strains in a medium containing transferrin. This effect
was most dramatic in the two siderophore-negative
strains examined (K45-1 and H1721; fig. 2). One
strain, K45-1, was able to grow in the iron-limited
0
2
4
6
8
1 0 1 2 1 4 1 6 medium without the addition of azurechelin or
pyochelin although the lag phase was quite long. This
Time (min)
suggests that there may be an alternative iron
Fig. 3. Uptake of s9Fe by P.cepueiu strains H1721 ( 0 , O )and
acquisition mechanism in this strain. This strain may
Pc275c (W, 0).
Uptake assays were initiated by the addition of
produce the siderophore cepabactin, although this
either s9[Fe]-azurechelin(closed symbols) or s9[Fe]-pyochelin(open
possibility was not investigated. Another possibility is
symbols); I-ml samples were removed at intervals and the amount
of s9Feaccumulated was determined.
that this strain produces a protease which we have
shown degrades transferrin (Sokol and Lewis, unpublished observations).This degradation may relieve the
iron limitation.
very low (table I). When the cultures reached a density
In an equilibrium dialysis assay, azurechelin reof lo8 cfu/ml, the cells were washed and either 59[Fe]moved radiolabelled iron from transferrin at pH 6.0
pyochelin or 59[Fe]-azurechelin was added to the
and 5.0, though not at pH 7-4. Sriyosachati and
cultures and the amount of 59Feaccumulated by the
Cox’ previously reported similar pH-dependent iron
cells was determined at selected intervals, Both strains
accumulated 59[Fe]-pyochelinand 59[Fe]-azurechelin. sequestration results for pyochelin and pyoverdin from
P. aeruginosa. Therefore, azurechelin appears similar
Strain H 1721, which did not produce detectable levels
of either siderophores, initially bound more 59[Fe]- to these other Pseudomonas siderophores in that it is
more effective at acidic pH. Acid pH levels have been
siderophore than did strain Pc275c, which produced
reported in inflammatory exudates. 5 9 These siderboth siderophores. However, rates of uptake were
ophores may be most effective at obtaining iron from
very similar for both strains.
transferrin in sites of inflammation.
Both azurechelin and pyochelin promoted iron
uptake by P. cepacia. Strain H1721, which did not
Discussion
produce detectable levels of siderophores, bound more
59[Fe]-azurechelin and 59[Fe]-pyochelin than did
We investigated the characteristics of a novel
strain Pc275c, which produced both siderophores. It
siderophore produced by respiratory isolates of P .
is possible that strain H1721 may produce larger
cepacia. This compound, given the trivial name
amounts of the receptors for these siderophores since
azurechelin, had properties common to other sideroit would be deficient at acquiring iron as compared to
phores. In particular, production of azurechelin was
siderophore-positive strains. This has not yet been
regulated by the iron concentration of the culture
investigated. Although the rates of pyochelin and
medium. Azurechelin was distinct from the other
azurechelin uptake appeared similar for these strains,
siderophoresof P . cepacia, having absorbance maxima
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:48:43
SIDEROPHORE FROM P . CEPACIA
189
direct comparison is difficult because there may be a
difference in expression of the respective siderophore
receptors in the M9 medium.
There are three general classes of siderophores,
hydroxamates, catechols and miscellaneous.1 9 7 2o
Azurechelin does not have the characteristics of either
the catechol or hydroxamate siderophores, giving
negative results in the standard assays for these
compounds. ' 3-1 Catechol siderophores usually have
three absorption maxima, at 320nm, 250nm and
210 nm, with the most prominent occurring at
210 nrn.l9 However, azurechelin had two peaks of
approximately equal prominence around 240 nm and
3 10 nm. Until its structure is determined, azurechelin
must be placed in the miscellaneous class of siderophores described by Neilands. l 9 Rhizobiurn rneliloti
also produces a siderophore, rhizobactin, which has
been characterised structurally and which is neither a
catechol nor a hydroxymate.*l Siderophores in the
miscellaneous class can be assayed only by functional
or biological techniques' such as the Chrome Azurol
S assay, which was used here. Azurechelin required a
shuttle reagent, 5-sulphosalicylic acid, to accelerate
the reaction. Similar properties are reported for
rhizobactin.' *
It is interesting that strains of P . cepacia express at
least three siderophore-mediated iron-transport systems, pyochelin, cepabactin, and azurechelin. Although previous studies have implicated pyochelin as
a virulence factor in P . cepacia respiratory infections,6T the contribution of azurechelin and cepabactin to the pathogenesis of P. cepacia infections has not
yet been determined. It would be of interest to compare
mutants deficient in each siderophore systems to
determine their importance to iron acquisition and
pathogenicity .
References
12. Arnow LE. Colorimetric determination of the components of
3,4-dihydroxyphenylalanine-tyrosinemixtures. J Biol
Chem 1937; 118: 531-537.
13. Rioux C, Jordan DC, Rattray JBM. Colorimetric determination
of catechol siderophores in microbial cultures. Anal
Biochem 1983; 133: 163-169.
14. Csiky 2. On the estimation of bound hydroxylamine in
biological materials. Acta Chem Scand 1948; 2 : 45M54.
15. Sriyosachati S, Cox CD. Siderophore-mediated iron acquisition
from transferrin by Pseudomonas aeruginosa. Infect Immun
1986; 52: 885-891.
16. Simonson C, Brener D, DeVoe IW. Expression of a highaffinity mechanism for acquisition for transferrin iron by
Neisseria meningitidis. Infect Immun 1982; 36: 107-1 13.
17. Meyer J-M, Hohnadel, D, HallC F. Cepabactin from Pseudomonas cepacia, a new type of siderophore. J Gen Microbiol
1989; 135: 1479-1487.
18. Menkin V. The role of the hydrogen ion concentration and the
cytology of an exudate. Glycolysis in inflammation. Some
aspects of the chemistry of exudates. In: Ryan EJ (ed)
Biochemical mechanisms in inflammation. Springfield, IL,
Charles C Thomas. 1956: 66-103.
19. Neilands JB. Methodology of siderophores. Structure and
Bonding 1984; 58 : 1-24.
20. Neilands JB. Microbial iron compounds. Annu Rev Biochem
1981; 50: 715-731.
21. Smith MJ, Shoolery JN, Schwyn B, Holden I, Neilands JB.
Rhizobactin, a structurally novel siderophore from Rhizobium meliloti. J A m Chem Soc 1985; 107: 1739-1743.
1. Payne SM, Finkelstein RA. The critical role of iron in hostbacterial interactions. J Clin Invest 1978; 61: 1428-1440.
2. Crosa JH. The relationship of plasmid-mediated iron transport
and bacterial virulence. Annu Rev Microbiol 1984; 38: 6989.
3. Neilands JB. Microbial envelope proteins related to iron. Annu
Rev Microbioll982; 36: 285-309.
4. Barclay R. The role of iron in infection. Med Lab Sci 1985; 42:
166177.
5. Isles A, Maclusky I, Corey R et al. Pseudomonas cepacia
infection in cystic fibrosis: an emerging problem. J Pediatr
1984; 104: 206210.
6. Sokol PA. Production and utilization of pyochelin by clinical
isolates of Pseudomonas cepacia. J Clin Microbioll986; 23:
560-562.
7. Sokol PA, Woods DE. Effect of pyochelin on Pseudomonas
cepacia respiratory infections. Microb Pathog 1988;5 : 197205.
8. McKevitt AI, Woods DE. Characterization of Pseudomonas
cepacia isolates from patients with cystic fibrosis. J Clin
Microbioll984; 19: 291-293.
9. Cox CD, Graham R. Isolation of an iron-binding compound
from Pseudomonas aeruginosa. J Bacterioll979; 137: 357364.
10. Cox CD. Iron uptake with ferripyochelin and ferric citrate by
Pseudomonas aeruginosa. J Bacterioll980; 142 : 581-587.
11. Schwyn B, Neilands JB. Universal chemical assay for the
detection and determination of siderophores. Anal Biochem
1987; 160: 47-56.
'
These studies were supported by the Canadian Cystic Fibrosis
Foundation. P.A.S is an Alberta Heritage Foundation for Medical
Research Scholar.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:48:43