results of siderophore produced by commensals & clinical isolates and co-relate it ... Conclusion: One cannot use siderophore production as a determinant of ...
Karuna Gokarn et al., j Biosci Tech, Vol 1 (3),2010,127‐134
Siderophores and Pathogenecity of Microorganisms R. B. Pal, Karuna Gokarn Department of Microbiology,Sir Hurkisondas Nurrotumdas Medical Research Society, R.R Road, Mumbai 400 004 E.mail: ram prasad_pal@hnhospital. com E.mail: karuna gokarn@gmail. com Abstract Introduction: The ability of pathogenic microorganisms of obtaining iron from host is essential for its survival. Microorganisms require iron for a variety of metabolic processes, so they synthesize and secrete small organic molecules called siderophores that actively chelate iron. The study was carried out to compare the results of siderophore produced by commensals & clinical isolates and co-relate it to pathogenecity. Materials & Methods: Detection of microbial siderophore production was carried out qualitatively by the chrome azurol sulfonate (CAS) plate assay. Spectrophotometric assay (Liquid CAS assay) was used for quantitative estimation of siderophore produced by the organisms. Qualitative assay for determining type of siderophore was also carried by Arnow’s & Csaky’s methods. Results: The commensals & the isolates from clinical samples did not show any significant difference in the production of siderophore. It was also observed that the optimum condition for siderophore production by both the commensals & the clinical isolates was at 370C for 24 hours with aeration in an iron deficient medium. Conclusion: One cannot use siderophore production as a determinant of virulence of an organism. Thus, it is concluded that siderophore production may be a necessary feature of a bacterium, but not a determinant of virulence. The siderophore producing potential of a pathogen in vivo cannot be decided on basis of in-vitro assays. But applications of siderophores could be many if well studied.
1. Introduction: A Siderophore (Greek for iron carrier) is a low molecular weight (500-1000daltons), high affinity ferric iron-chelating compound secreted by organisms. Is it one of the determinants for virulence of a pathogen. In iron-limited environment, siderophore production is derepressed and excreted extracellularly. The siderophore then acts to sequester and solubilize the iron. The amount of free iron available in -18 the human host is extremely low (10 M), which is insufficient for bacterial growth. The ability of obtaining iron from the host is essential for the survival of microorganisms. Microorganisms require iron for a variety of metabolic processes, so they synthesize and secrete siderophores that actively chelate iron and remove it from eukaryotic iron-binding proteins like lactoferrin & transferring.
Keywords: Catecholate, Chrome azurol sulfonate, hydroxamate, Siderophores
Thus, iron is a key element of bacterial pathogenesis. [4] Therefore studies were carried out 1. To evaluate the pathogenecity of different clinical isolates 2. To compare the results of siderophore produced by commensals & clinical isolates. 2. Materials and Methods: 2.1. Collection of the cultures: Culture isolates from clinical specimens were collected from the Pathology Department of Sir H. N. Hospital and Research Centre, Mumbai, India of which there were 17 strains E. coli, 6 Acinetobacter spp., 8 Klebsiella spp, 2 Branhamella catarrhalis & 4 Proteus spp. E. coli (11) and Acinetobacter (5) isolates from the stool samples and throat swabs respectively from healthy individuals were also collected to compare the siderophore 127
Karuna Gokarn et al., j Biosci Tech, Vol 1 (3),2010,127‐134 production between the commensals & clinical isolates. (Fig: 1&2) 2.2. Charaterization of Isolates: The microorganisms isolated from the clinical samples were identified on basis of their morphology, colony characteristics and standard biochemical reactions. 2.3. Detection of microbial siderophore production: It was carried out by qualitative and quantitative methods 2.3.1. Qualitative detection of siderophore (plate assay) The chrome azurol sulfonate (CAS) assay – [universal assay – Schwyn & Neilands 2] was used since it is comprehensive, exceptionally responsive, and most convenient. The chrome azurol sulfonate assay agar was used. For the qualitative assay cultures were spot inoculated onto the blue agar and incubated at 370C/2448hours.The results were interpreted based on the color change due to transfer of the ferric ion from its intense blue complex to the siderophore. The sizes of yelloworange haloes around the growth indicated total siderophore activity. 2.3.2. Quantitative spectrophotometric assay for siderophore production (Liquid assay): Cultures were grown in a minimal medium (T medium) at 370C/24 hours under static conditions as well as under shaker conditions (100 rpm) at 370C/24 hours. The cells were removed by centrifugation at 3000 rpm for 15 mins. 0.5 ml of the culture supernatant was then mixed with 0.5 ml CAS solution and 10µl shuttling solution (sulfosalicylic acid). The color obtained was determined using the spectrophotometer at 630 nm after 20 mins of incubation. Necessary blank (minimal
medium) & reference solution (minimal medium + CAS dye + shuttle solution) were used during the determination. [2] 2.3.3. Qualitative assay for determining type of siderophore: To check whether the siderophore produced by E. coli isolated from gut & clinical samples are of catecholate type or hydroxamate type. [13] 2.3.3.1. Csaky’s assay For detection of hydroxamates like aerobactin of E. coli 1 ml supernatant of culture was hydrolysed with 1 ml of 6N H2SO4 in a boiling water bath/6hrs or 1300C/30mins. To this was added -3ml Na-acetate for buffering, 1 ml sulfanilic acid & then 0.5 ml iodine soln. After 35mins, excess iodine is destroyed with 1 ml of Na-arsenite soln. 1 ml of alpha naphthylamine was then added and water was used to make up vol to 10 ml. Color was allowed to develop for 20-30mins. Absorbance was measured with the help of uv-vis spectrophotometer at 526nm. [13] 2.3.3.2. Arnow’s assay For detection of catheclates like enterobactin of E. coli 1 ml culture supernatant was mixed after each orderly addition.1 ml HCl followed by 1 ml nitrite-molybdate (catechols prod yellow color) & then 1 ml NaOH (color changes to red). Color was stable for 1hour & absorbance was measured at 510 nm using a uv-vis spectrophotometer. [13] 3. Results: 3.1. Plate assay (Qualitative test) All the cultures (clinical isolates, gut flora and the ATCC strain) were siderophore positive. The intensity of the orange zones was varying indicating different amounts of siderophore being produced. To
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Karuna Gokarn et al., j Biosci Tech, Vol 1 (3),2010,127‐134
Image -1
Image-2
Microorganisms isolated from the clinical samples
CLINICAL SAMPLES
12%
7%
5%
16%
s tool
9%
44%
sputum
7%
w ound
46%
urine
pus
7%
28%
tracheal secretions
1 2 3 4 5
19%
Figure – 1
Effect of aeration on siderophore production by E.coli % siderophore produced
51
63.54
92.76 93.05 92.96
78.19
92.07
75.22 93.85
93.55 1
2
3
4
5
6
7
Acinetobacter Proteus Branhamel la
Figure – 2
% Siderophore produced by E.coli from the gut of healthy individuals
84.5
E.coli Klebsi ell a
100 80
% siderophore produced without aeration
60 40
% siderophore produced with aeration
20 0 1
8
9
10
11
3
5
7
9
11
E.coli from clinical samples
Figure - 3
Figure – 4
% Siderophore produced by E.coli from clinical isolates 80.73 81.51 92.44
69.57 52
89.3
43.91
82.25
75.15
46.19
81.5
77.58 1
2
3
4
5
6
7
8
9
10
11
12
Figure - 5
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Karuna Gokarn et al., j Biosci Tech, Vol 1 (3),2010,127‐134 Table – 1 uv-vis spectrophotometer readings using cultures grown at 370C/24 hours under static conditions of clinical isolates only (12 E. coli, 8 Klebsiella spp & 3 Acinetobacter spp were used for this study) Absorbance at 630nm 0.621 0.0.798 0.792 0.726 0.688 0.835 0.829 0.702 0.692 0.677 0.792 0.575
Reference at 630nm 0.0867 0.0867 0.0867 0.0867 0.0867 0.0867 0.0867 0.0867 0.0867 0.0867 0.0867 0.0867
% Siderophore produced 28.37 7.95 8.65 16.26 20.64 3.69 4.38 19.03 20.18 21.91 8.65 33.67
Acinetobacter 1 Tracheal secretion 2 urine 3 urine
0.564 0.771 0.718
0.867 0.867 0.867
34.94 11.07 17.53
Klebsiella 1 urine 2 urine 3 urine 4 urine 5 swab 6 sputum 7 sputum 8 Tracheal secretion
0.839 0.834 0.634 0.763 0.471 0.697 0.496 0.657
0.867 0.867 0.867 0.867 0.867 0.867 0.867 0.867
3.22 3.80 26.87 11.99 45.67 19.6 42.79 24.22
E.coli
Sample
1 2 3 4 5 6 7 8 9 10 11 12
urine urine stool urine stool stool urine urine urine stool urine urine
Table – 2 uv-vis spectrophotometer readings using cultures grown at 370C/24 hours under shaker conditions at 100 rpm. . (E. coli (i & ii) & Acinetobacter (iii & iv) spp were used for this study) i) Gut flora E.coli isolated from stool samples of healthy individuals Absorbance Reference % Siderophore E.coli at 630nm at 630nm produced 1 0.461 0.939 51.00 2 0.420 1.152 63.54 3 0.080 1.152 93.05 4 0.22 1.009 78.19 5 0.25 1.009 75.22 6 0.062 1.009 93.85 7 0.065 1.009 93.55 8 0.08 1.009 92.07 9 0.071 1.009 92.96 10 0.073 1.009 92.76 11 0.145 0.939 84.50
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Karuna Gokarn et al., j Biosci Tech, Vol 1 (3),2010,127‐134 Siderophore produced by Acinetobacter from throat swab/sputum of healthy individuals
% Siderophore produced
100 90 80 70 60 50 40 30 20 10 0
Siderophore produced by Acinetobacter from clinical isolates
90.08
86.97
100 44.49 40.53
39
% Siderophore produced
50
13.19
46.75 42.53
44.6 19.06 25.45
0 1
1
2
3
4
2
5
Acinetobacter isolates
Figure – 6
4
5
6
Figure - 7
0.3 0.2
Enterobactin Aerobactin
0.1 0
Detection of Enterobactin & Aerobactin in clinical isolates of E.coli S pe c trophotom e tric a bs orba nc e
Detection of Enterobactin & Aerobactin in normal flora E.coli Spectrophotometric absorbance
3
Acinetobacter isolates
0.25 0.2 0.15 0.1 0.05 0
1 2 3 4 5 6 7 8 9 10
Enterobactin Aerobactin
1
E.coli isolates
3
5
7
9
11 13
E.coli isolates
Figure - 8
Figure - 9
Table – 3 ii) E.coli isolated from various clinical samples E.coli
Sample
1 2 3 4 5 6 7 8 9 10 11 12
urine urine stool urine stool stool urine urine urine stool urine urine
Absorbance at 630nm 0.213 0.087 0.182 0.175 0.455 0.221 0.277 0.245 0.553 0.521 0.300 0.190
Reference at 630nm 1.152 1.152 0.986 0.986 0.986 0.986 0.986 0.986 0.986 0.986 0.986 0.986
% Siderophore produced 81.51 92.44 89.30 82.25 46.19 77.58 81.5 75.15 43.91 52 69.57 80.73
Table – 4 iii) Throat flora Acinetobacter isolated from sputum & throat swabs of healthy individuals Absorbance Reference read % Siderophore Acinetobacter at 630nm at 630nm produced 1(throat swab) 1.0 1.152 13.19 2(throat swab) 0.1 1.009 90.08 3(throat swab) 0.56 1.009 44.49 4(throat swab) 0.6 1.009 40.53 5 (sputum) 0.573 0.939 39.00
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Karuna Gokarn et al., j Biosci Tech, Vol 1 (3),2010,127‐134 Table – 5 iv) Acinetobacter isolated from clinical samples Acinetobacter
Sample
1 2 3 4 5 6
tracheal secretion tracheal aspirate wound discharge pus urine tracheal secretion
Absorbance at 630nm 0.52 0.76 0.70 0.58 0.662 0.15
Organism E.coli (clinical isolates)
Blank 1 2 3 4 5 6 7 8 9 10 11 12 13 Organism E.coli (gut flora) 1 2 3 4 6 7 8 9 10 11 Blank Organism Acinetobacter (clinical isolates)
Blank 1 Organism Acinetobacter (commensal)
1 2 3 4 5
Reference at 630nm 0.939 0.939 0.939 0.939 1.152 1.152
% Siderophore produced 44.60 19.06 25.45 46.75 42.53 86.97
Table – 6 Arnow (Enterobactin) 510 nm 0.000 0.209 0.124 0.115 0.134 0.163 0.083 0.158 0.169 0.022 0.158 0.180 -------0.134
Csaky (Aerobactin) 526 nm 0.000 0.058 -----0.005 0.031 0.081 --------------0.009 0.006 0.050 0.091 -------0.020
Arnow (Enterobactin) 510 nm ------0.147 0.186 0.168 0.038 0.155 0.237 0.189 0.173 0.284 0.000 Arnow (Enterobactin) 510 nm 0.000 0.130 Arnow (Enterobactin) 510 nm --------0.331 0.022 0.158 0.180
Csaky (Aerobactin) 526 nm ------------0.038 0.077 -----0.094 0.083 0.083 0.074 0.116 0.000 Csaky (Aerobactin) 526 nm 0.000 0.171 Csaky (Aerobactin) 526 nm ------------------0.006 0.050 0.091
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Karuna Gokarn et al., j Biosci Tech, Vol 1 (3),2010,127‐134 measure the amount of siderophore production spectrophotometric assay was carried out for all the cultures. (Image-1) 3.2. Liquid Assay - Spectrophotometric Assay: The change in color of the blue dyechrome azurol sulphonate assay solution to purple-orange indicates the presence of siderophore (Image-2). The reference solution showed greatest absorbance (blue color) as all the blue color is measured (Ar). Samples (i.e culture supernatant taken after growing the cultures at 370C/24 hours under static & shaker conditions) showed lower readings as siderophore removes the iron from the dye complex (As). The values of the siderophore excreted were determined 100 which using the formula gives percent siderophore units. (Fig: 3-7) 3.3. Qualitative test readings to check the type of siderophore: Whether aerobactin or enterobactin was produced. Only E. coli & Acinetobacter spp were used for this study (Fig: 8 & 9) 4. Discussion: The CAS assay used showed orange color around siderophore producing colonies within 24-48 hours. The sizes of yelloworange haloes around the growth indicated total siderophore activity. To quantitate the siderophore produced by the cultures CAS liquid assay was used. The variable parameter for the clinical isolates was aeration, so studies were carried out using growth of the cultures under static and shaker conditions. It was observed that aeration enhanced the production of siderophore to a very great extent. This indicates that in vitro, aeration provides the optimum conditions for siderophore production. The same conditions (ie.with aeration) which were used to check
siderophore production by the commensals also proved the same. Thus, there was no significant difference in the siderophore production in the commensals as well as the clinical isolates of E. coli. In addition assays carried out to determine the type of siderophore produced also did not give any striking difference between the commensals and the clinical isolates. The results of this study is supported by studies carried out by A.Miles et al [1], which showed that siderophores (enterobactin) cannot be used as a determinant of virulence in an organism belonging to the Enterobacteriaceae family. Contrary to this, Vagrali et al [12] carried out studies that showed siderophores could be considered as urovirulence markers of uropathogenic E.coli though the paper does not elaborate on the methodology of siderophore detection. In this study enterobactin was found to be produced by commensals & clinical isolates, therefore no association between virulence and the ability to synthesize cathecholates enterobactin was found. Hydroxamates like aerobactin was not produced by all the E.coli under study, again which cannot be related to pathogenecity. Perhaps other types of siderophores like salmochelin & yersiniabactin need to be tested, though they not are produced in large quantities like enterobactin. These siderophores may also require sophisticated purification methods. It may be possible for an opportunistic organism to trigger a particular type of siderophore. Eg: when E.coli leaves its normal site (gut) & enters a different site like the urinary tract. Therefore, one
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Karuna Gokarn et al., j Biosci Tech, Vol 1 (3),2010,127‐134 cannot use any siderophore produced as a determinant of virulence for an organism. Any bacteria will produce sufficient quantities of siderophore when grown in an iron deficient environment & incubated at its optimum growth conditions. 5. Conclusion: From the quantitative & qualitative assay results; we can conclude that there was no significant difference in the production of siderophores by the commensals & isolates from clinical samples. Therefore, one cannot use siderophore production as a determinant of virulence of an organism. Any bacteria will produce sufficient quantities of siderophore when grown in an iron deficient environment when incubated at its optimum growth conditions. This suggests that siderophore production may be a necessary feature of a virulent bacterium but not a determinant of virulence. [1] The siderophore producing potential of a pathogen in vivo cannot be decided on basis of in-vitro assays. The types of siderophore & the amount of siderophore produced inside the body cannot be related to the types & production carried out in a tube. Other research avenues of siderophores can be explored for further studies.
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