Effect of macrophage secretory products on
elaboration of virulence factors by planktonic and
biofilm cells of Pseudomonas aeruginosa
Rahul Mittal, Saroj Sharma, Sanjay Chhibber, Kusum Harjai *
Department of Microbiology, Panjab University, BAMS Block, Chandigarh 160014, India
Accepted 14 November 2005
Abstract
Macrophages, which constitute the first line of defense, pour their secretions in the mileu
following stimulation with pathogens. These secretory products, referred to as macrophage secretory
products (MSPs), can influence ultimate outcome of an infection. In the present investigation, it was
observed that different strains of Pseudomonas aeruginosa vary in their ability to stimulate
macrophages leading to variability in generation of macrophage secretory products. Cytokine levels,
reactive nitrogen intermediates and protein content of macrophage secretory products generated with
biofilm cells of P. aeruginosa was found to be more as compared to their planktonic counterparts.
The effect of macrophage secretory products produced in response to interaction of macrophages
with P. aeruginosa on elaboration of virulence factors produced by planktonic and biofilm cell forms
of this pathogen was assessed. Significant enhancement in growth and elaboration of all the virulence
determinants by both the cell forms was observed when P. aeruginosa was grown in presence of
supernatants with macrophage secretory products. Implications of these findings in relation to
urinary tract infections induced by P. aeruginosa have been discussed.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Macrophage secretory products; Pseudomonas aeruginosa; Biofilm cells; Virulence factors
Resume
Les macrophages qui constituent la premiere ligne de defense contre l’infection, excretent dans le
milieu des cytokines apres stimulation par les pathogenes. Ces produits de secretions references
Comparative Immunology, Microbiology
& Infectious Diseases 29 (2006) 12–26
www.elsevier.com/locate/cimid
0147-9571/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cimid.2005.11.002
* Corresponding author. Tel.: C91 172 2534142; fax: C91 172 2541770.
E-mail address: [email protected] (K. Harjai).
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–26 13
comme «macrophage secretory products (MSPs)» peuvent influence le devenir d’une infection. Dans
le present travail, il a ete observe que differentes souches de Pseudomonas aeruginosa varient dans
leur capacite a stimuler les macrophages permettant ainsi une variabilite dans l’excretion des
produits elabores macrophages. La quantite de cytokines elaboree par les macrophages a ete plus
importante a partir de P. aeruginosa en biofilm par rapport aux formes de la meme bacterie en
culture.
L’effet des substances elaborees par les macrophages, sur les facteurs de virulence de P.
aeruginosa a ete teste selon les formes de presentation de la bacterie (biofilm ou culture). Une
augmentation significative dans l’elaboration de tous les determinants de la virulence a ete observee
dans les deux formes de P. aeruginosa en presence de surnageants des produits de secretion de
macrophages. L’implication de ces constatations en relations avec les infections du tractus urinaire
par P. aeruginosa a ete discutee.
q 2005 Elsevier Ltd. All rights reserved.
Mots cles: Produits de secretion des macrophages; Pseudomonas aeruginosa; Cellules constituant les biofilms;
Facteurs de virulence
1. Introduction
Potential of an opportunistic pathogen like Pseudomonas aeruginosa to establish in a
host depends on its ability to overcome innate defense mechanisms [1]. Resident
macrophages residing in tissues and phagocytes migrating from blood to the site of
infection constitute the primary line of innate defense against most bacterial pathogens [2–
4]. At the site of infection both the virulence factors of organism and host factors come
into play and the invading organism is exposed to phagocytes, principally macrophages
[5,6]. Nathan [7] reviewed that macrophages of tissues congregate in most acute and
chronic inflammatory reactions. They respond to antigenic stimuli and secrete a range of
over 100 substances, which vary in their biological activities ranging from induction of
cell growth to cell death. Secretory products of macrophages include peptide hormones,
complement components, enzymes, bioactive oligopeptides and lipids, reactive oxygen
and nitrogen species and other biological substances [7]. The principal constituents of
macrophage secretory products (MSPs) are cytokines, which include mainly tumor
necrosis factor (TNF)-a, TNF-b, Interleukin (IL)-1, Interferon (IFN)-a, IL-12, IL-16 and
granulocyte-macrophage colony-stimulating factor (GM-CSF).
P. aeruginosa is the third most common pathogen causing nosocomial catheter
associated urinary tract infections (UTIs) [8]. It has a tendency to form biofilms on surface
of indwelling urinary catheters, which exhibit greater resistance to phagocytosis as
compared to their planktonic counterparts [9]. Biofilms are difficult to eradicate and are
considered as an important cause of chronicity and recurrence of infection [10]. P.
aeruginosa possess a wide arsenal of weapons like exotoxins, alginate, elastase,
phopholipase C and outer membrane proteins. It has the ability to resist phagocytosis
mainly operative through macrophages [11–14]. Following interaction of P. aeruginosa
with macrophages, secretory products are produced. This host factor may have some
influence on the virulence factors elaborated by the invading organism and on ultimate
outcome of an infection. In the present investigation effect of macrophage secretory
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–2614
products obtained after interaction of macrophages with P. aeruginosa has been studied in
relation to elaboration of virulence factors by planktonic and biofilm cells of
P. aeruginosa. Extrapolation of available information may provide an insight into the
outcome of urinary tract infections caused by P. aeruginosa.
2. Material and methods
2.1. Organisms
Fifty strains of P. aeruginosa isolated from urine samples of patients with complicated
urinary tract infections were screened for the elaboration of virulence factors. Out of these,
five uroisolates of P. aeruginosa PA1, PA2, PA3, PA4 and PA5 used in earlier studies [15,
16] having serotypes O4, O3, O6, O7/8 and O11, respectively (as serotyped by Laboratory
of Healthcare Associated Infection, London) were selected on the basis of maximum
elaboration of virulence factors like alginate, protease, elastase, phopholipase C,
hemolysin, pyochelin and pyoverdin. In addition, a genetically characterized strain of
P. aeruginosa, PAO (obtained from Dr B.H. Iglewski, University of Rochester, New York,
USA), expressing exotoxin A, exoenzyme S, alginate, protease, pyochelin, pyoverdin,
hemolysins, phospholipase C and rhamnolipids, was used as a reference strain in the
present study. Strains were stored at K80 8C in 20% glycerol.
2.2. Generation of planktonic cells and growth profile studies
For generation of planktonic cells of P. aeruginosa, an aliquot of frozen cultures was
inoculated on 1% cetrimide agar plates and colonies were suspended in phosphate buffer
saline (PBS, pH 7.0). About 100 ml aliquots of adjusted culture (A540 0.4) was used to
inoculate RPMI medium containing 30% of macrophage secretory products collected
from unstimulated macrophages which served as control and macrophages stimulated with
planktonic cells of P. aeruginosa which served as test. These were incubated at 37 8C and
sampled at 2, 4, 6, 8 and 10 h. Viable log counts per milliliter (cfu/ml) was then calculated.
2.3. Generation of biofilms and growth profile studies
For generation of biofilms method of Mittal et al. [15] was employed. Foley’s catheter
(Bardia) was cut into 1.0 cm pieces and put in flasks having 100 ml RPMI-1640 medium
containing 30% of macrophage secretory products collected from unstimulated
macrophages (control) and macrophages stimulated with biofilm cells of P. aeruginosa
(test). About 100 ml of overnight washed adjusted bacterial culture (A540 0.4) was
inoculated separately in each flask and incubated at 37 8C. After every 24 h, catheter
pieces were removed from each flask and transferred to the new flask containing nutrient
broth with MSPs. Incubation was done till day 4. To study growth profile, catheter pieces
were rinsed three times with PBS (pH 7.4). Cells were removed from the surface of
catheter pieces by scrapping the inner surface with sterile scalpel blade. Dispersed sample
was then centrifuged and the biofilm cells were suspended in 1 ml PBS. Biofilm cells were
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–26 15
sampled at 1, 2, 3 and 4 days. Log colony forming units per milliliter (cfu/ml) was
calculated.
For each bacterial strain, experiments were done in triplicates, i.e. three independent
experiments were carried out for each parameter.
2.4. Isolation of peritoneal macrophages
Swiss Webster strain of mice (LACA), 6–8 weeks old, weighing 25G5 g obtained from
Central Animal House, Panjab University, Chandigarh, India was used for collection of
macrophages. Peritoneal macrophages were isolated from mice by the method of Harjai
et al. [16]. Briefly peritoneal cavities of mice were exposed without disrupting blood
vessels. About 8–10 ml RPMI-1640 medium was injected into the cavity and the abdomen
was massaged for 1–2 min. The peritoneal lavage was sucked back and added to a sterile
glass petriplate. The glass petriplates were incubated at 37 8C for 1 h in a CO2 incubator to
allow sticking of macrophages. The macrophages were washed twice with RPMI-1640
and cell density was adjusted to 105 cells/ml in RPMI-1640.
2.5. Preparation of macrophage secretory products (MSPs)
Twenty milliliters of macrophages (105 cells/ml) were interacted with 2 ml of each
bacterial strain (1!108 cells/ml) separately in planktonic and biofilm cell mode. These
were kept at 37 8C for 20 min. This time period allowed optimal phagocytosis of bacteria
by macrophages. The macrophages were then repeatedly washed with RPMI-1640 to
remove any unphagocytosed adherent bacteria. After resuspending in 100 ml serum and
antibiotic free RPMI-1640, the macrophages were kept at 37 8C in 5% CO2 environment.
Supernatant containing macrophage secretory products (MSPs) was collected at 18 h post-
interaction time period. This was then passed through 0.2 mm filter and stored at K80 8C
prior to experiment [17]. All the experiments were carried out from same sample of
macrophage secretory products to avoid batch to batch variability. Sterility of macrophage
secretory products was checked each time before putting experiment by plating it on
nutrient agar plates. Secretory products collected from unstimulated macrophages served
as control to rule out non-specific stimulation of macrophages during handling procedures.
2.6. Characterization of macrophage secretory products (MSPs)
Macrophage secretory products (MSPs) were characterized in terms of cytokines,
reactive nitrogen intermediates (both nitrates and nitrites) and protein content. Cytokines
namely TNF-a, TNF-b, GM-CSF, IFN-g, IL-1a, IL-1b, IL-2, IL-4, MIP-2, IL-6, IL-10,
IL-12 and IL-18 were measured using ELISA kits (R & D Systems, Minneapolis;
Chemikon, USA and Beckton and Dickinson, USA). ELISA was performed according to
manufacturer’s guidelines. Cytokine levels in MSPs were calculated by plotting standard
curve and results were expressed as picogram per ml. Reactive nitrogen intermediates
were measured using method of Rockett et al. [18]. Briefly, for nitrite estimation 100 ml ofsample was mixed with 200 ml of Griess reagent (Sigma, USA) and for nitrate estimation
100 ml of sample was mixed with 15 ml of NADPH (Sigma, USA), 5 ml of nitrate reductase
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–2616
(Sigma, USA) and 200 ml of Griess reagent followed by addition of 100 ml of 10%
trichloroacetic acid and incubated for 20 min at room temperature. After centrifugation,
optical density of supernatants was read at 545 nm. The amount of nitrite and nitrate was
determined using standard curves of sodium nitrite and sodium nitrate, respectively.
Protein content of MSPs was determined following method of Lowry et al. [19].
2.7. Preparation of cell free supernatant (CFS)
A540 of each bacterial strain was measured and adjusted to 1.5 with sterile culture
medium. Culture supernatants of all the uroisolates as well as standard strain PAO growing
in planktonic mode and biofilm cell mode (4 day old) in presence of supernatants with
their respective MSPs (30%) were collected for estimation of virulence factors.
2.8. Alginate determination
Alginate in the culture supernatant was precipitated with an equal volume of 2% (w/v)
cetylpyridinium chloride followed by centrifugation at 8000 g for 20 min at room
temperature. Alginate pellet was suspended in 5 ml of sodium chloride (1 M),
reprecipitated with 5 ml of cold 2-propanol and centrifuged at 8000 g for 10 min. Final
alginate pellet was resuspended in 1 ml of normal saline. Amount of alginate was
determined using a borate/carbazole method with D-mannuronate lactone used to calibrate
a standard curve according to the method of Mathee et al. [20].
2.9. Cell surface hydrophobicity
Cell surface hydrophobicity was measured by using the method of phase separation
with p-xylene. Initial optical density and optical density of the aqueous phase was taken
and surface hydrophobicity was expressed as percentage according to method of
Rosenberg et al. [21].
2.10. Pyochelin estimation
Cell free supernatant (CFS) was extracted with ethyl acetate in the ratio of 5:2 and was
used for pyochelin estimation following the method of Yadav et al. [22]. Briefly, 1 ml of
culture supernatant was mixed with 1 ml each of 0.5 N HCl, nitrite molybdate reagent and
1 N NaOH. Final volume was made to 5 ml with double distilled water and absorbance
was read at 510 nm.
2.11. Pyoverdin estimation
Pyoverdin estimation in the cell free supernatant was carried out following the method
of Yadav et al. [22]. Briefly, CFS was adjusted to pH 2.0 and extracted with ethyl acetate in
the ratio 5:2. Aqueous phase was diluted with 50 mM Tris HCI (pH 7.4) and fluorescence
was measured at 460 nm, while the samples were excited at 400 nm in a Gibson Spectro
Gloflourometer.
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–26 17
2.12. Protease production
For proteolytic activity, culture supernatantswere diluted in 10 mMTris (pH 7.5) and 3 ml
of diluted supernatant was incubated with 15 mg hide powder azure (Sigma Chemical
Company, USA) at 37 8C for 1 h, while being shaken vigorously as described byWoods et al.
[23]. Unsolubilized substrate was removed by centrifugation (3000 g, 10 min). Protease
activitywas determined in the supernatants spectrophotometrically at 595 nmwavelength and
units per liter (U/l) were calculated according to the method of Visca et al. [24].
2.13. Elastase production
Elastolytic activity was measured using elastin-congo red (Sigma Chemical Company,
USA) as substrate. Five milligrams of elastin-congo red were suspended in 1 ml of
100 mM Tris-succinate buffer (pH 7.0), 1 mM CaCl2 supplemented with 1 ml of culture
supernatants and incubated at 37 8C for 1 h under vigorous stirring. Reaction was stopped
by adding 1 ml of 0.7 M sodium phosphate buffer (pH 6.0). Unsolubilized substrate was
removed by centrifugation (3000 g, 10 min) and elastase activity was measured by reading
optical density of supernatants at 495 nm. Elastolytic activity was expressed in units per
liter (U/l) according to the method of Visca et al. [24].
2.14. Phopholipase C (PLC) production
PLC activity was determined according to the method of Visca et al. [22] using
p-nitrophenylphosphorylcholine (PNPC, Sigma Chemical Company, USA) as substrate.
Two millilitres of 10 mM PNPC in 250 mM Tris–HCl, 1 M ZnCl2, 60% glycerol (pH 7.2)
were added to 0.25 ml of culture supernatants and incubated at 37 8C for 1 h. Enzyme
activity was measured spectrophotometrically at 405 nm wavelength and expressed in
units (U/l) according to the method of Woods et al. [25].
2.15. Hemolysin production
Quantitative determination of both cell free and cell bound hemolysin was done
following the method of Linkish and Vogt [26].
2.16. Cell free hemolysin assay
About 1.5 ml of 2% suspension of washed human erythrocytes was added to 1.5 ml of
cell free supernatant. The mixture was incubated at 37 8C for 2 h and centrifuged (5000 g
for 5 min). Absorbance of supernatant was read at 545 nm.
2.17. Cell bound hemolysin assay
About 1.5 ml of 2% suspension of washed human erythrocytes was added to 1.5 ml of
adjusted bacterial culture (A540 0.4). The mixture was incubated at 37 8C for 2 h and assay
mixture was centrifuged at 8000 g for 15 min. The supernatant was collected and
Table 1
Alginate and cell surface hydrophobicity of planktonic and biofilm cells of Pseudomonas aeruginosa in presence
of supernatants with and without MSPs
P. aeruginosa strains
(serotype)
Alginate concentration
(mg/ml)
Cell surface hydrophobicity
(percentage)
Controla Testb Controla Testb
PA1 (O4) A 550G3.90 710G4.93 20.02G0.80 41.28G0.96
B 630G2.93 960G3.90 39.10G0.83 67.61G0.98
PA2 (O3) A 527G2.95 705G3.90 23.41G0.80 45.54G0.85
B 665G3.91 970G5.91 41.02G0.86 66.32G0.90
PA3 (O6) A 510G2.91 700G6.95 22.56G0.76 47.71G0.86
B 685G4.93 950G6.94 33.27G0.76 69.91G0.84
PA4 (O7/8) A 516G5.96 700G3.95 24.67G0.70 45.39G0.91
B 625G3.90 950G4.94 43.19G0.78 68.89G0.97
PA5 (O11) A 521G3.69 725G5.92 19.87G0.85 44.14G0.96
B 660G2.92 970G6.98 46.21G0.72 65.42G0.99
PAO (PT) A 541G3.92 750G5.95 21.18G0.88 46.08G0.93
B 680G3.98 970G6.90 45.09G0.70 68.98G0.99
Data represents meanGstandard deviation; PT, polytypable; A, Planktonic cells; B, Biofilm cells; p values
Control vs Test p!0.001.a In presence of supernatant without MSPs.b In presence of supernatant with MSPs (30%).
Table 2
Siderophore production by planktonic and biofilm cells of Pseudomonas aeruginosa in presence of supernatants
with and without MSPs
P. aeruginosa strains
(serotype)
Pyochelin production
(OD at 510 nm)
Pyoverdin production
(RF at 460 nm)
Controla Testb Controla Testb
PA1 (O4) A 0.042G0.005 0.100G0.010 234G1.11 502G3.15
B 0.090G0.003 0.167G0.012 492G2.15 964G3.11
PA2 (O3) A 0.045G0.004 0.098G0.009 205G1.01 436G2.21
B 0.045G0.008 0.161G0.008 380G2.12 843G3.16
PA3 (O6) A 0.048G0.006 0.095G0.009 200G3.07 450G2.98
B 0.100G0.004 0.170G0.015 480G2.18 975G4.20
PA4 (O7/8) A 0.040G0.007 0.083G0.008 210G1.98 428G2.12
B 0.105G0.009 0.159G0.011 462G4.16 951G3.10
PA5 (O11) A 0.037G0.004 0.072G0.009 227G3.65 461G3.04
B 0.095G0.007 0.140G0.014 465G2.97 962G3.23
PAO (PT) A 0.040G0.002 0.080G0.013 200G3.44 405G4.04
B 0.090G0.010 0.170G0.016 411G4.14 939G4.98
Data represents meanGstandard deviation; PT, polytypable; A, Planktonic cells; B, Biofilm cells; p values
Control vs Test p!0.001.a In presence of supernatant without MSPs.b In presence of supernatant with MSPs (30%).
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–2618
Table 4
Hemolysin production by planktonic and biofilm cells of Pseudomonas aeruginosa grown in presence of
supernatants with and without MSPs
P. aeruginosa
strains (serotype)
Hemolysin (hemoglobin released, mg/ml)
Cell bound Cell free
Controla Testb Controla Testb
PA1 (O4) A 0.45G0.04 2.52G0.11 0.76G0.06 2.12G0.15
B 1.10G0.05 4.00G0.25 1.40G0.08 4.25G0.31
PA2 (O3) A 0.62G0.07 2.45G0.18 0.85G0.05 2.29G0.22
B 1.40G0.08 4.25G0.31 1.55G0.09 4.30G0.37
PA3 (O6) A 0.50G0.04 1.81G0.15 0.61G0.05 1.89G0.12
B 1.45G0.09 3.50G0.22 1.60G0.10 4.00G0.20
PA4 (O7/8) A 0.56G0.05 1.67G0.10 0.90G0.08 2.36G0.13
B 1.40G0.07 3.75G0.37 1.65G0.07 4.20G0.35
PA5 (O11) A 0.53G0.03 1.80G0.16 0.42G0.04 2.44G0.18
B 1.05G0.05 3.75G0.39 1.25G0.12 4.27G0.41
PAO (PT) A 0.42G0.04 2.00G0.28 0.59G0.04 2.10G0.11
B 1.10G0.08 4.10G0.32 1.20G0.09 4.30G0.30
Data represents meanGstandard deviation; PT, polytypable; A, Planktonic cells; B, Biofilm cells; p values
Control vs Test p!0.001.a In presence of supernatant without MSPs.b In presence of supernatant with MSPs (30%).
Table 3
Exoenzyme production by planktonic and biofilm cells of Pseudomonas aeruginosa grown in presence of
supernatants with and without MSPs
P. aeruginosa
strains
(serotype)
Protease (U/l) Elastase (U/l) Phopholipase C (U/l)
Controla Testb Controla Testb Controla Testb
PA1 (O4) A 12.6G0.42 27.2G0.62 35.6G0.60 103.3G0.82 68.9G0.63 147.4G0.76
B 30.3G0.50 62.6G0.70 75.4G0.66 162.7G0.88 111.7G0.71 189.3G0.92
PA2 (O3) A 18.4G0.38 37.5G0.59 43.2G0.70 112.7G0.80 75.6G0.61 162.5G0.96
B 43.3G0.52 65.6G0.67 82.7G0.62 177.9G0.87 122.4G0.75 204.7G0.83
PA3 (O6) A 28.7G0.43 40.1G0.54 49.3G0.77 118.2G0.84 80.2G0.81 166.3G0.90
B 40.1G0.50 63.2G0.75 89.1G0.63 180.8G0.92 132.1G0.87 219.4G0.97
PA4 (O7/8) A 21.7G0.41 37.9G0.60 24.8G0.67 85.6G0.79 56.7G0.70 132.1G0.82
B 38.3G0.48 61.3G0.78 51.6G0.71 147.9G0.90 102.6G0.82 172.5G0.93
PA5 (O11) A 19.8G0.44 35.6G0.54 66.8G0.74 154.3G0.82 82.4G0.72 175.7G0.81
B 36.6G0.51 64.6G0.82 112.1G0.79 197.9G0.94 144.8G0.82 233.2G0.94
PAO (PT) A 15.2G0.47 30.3G0.69 27.2G0.64 96.4G0.85 61.4G0.71 141.8G0.80
B 33.3G0.53 61.3G0.59 62.4G0.82 150.2G0.96 107.3G0.83 184.2G0.98
Data represents meanGstandard deviation; PT, polytypable; A, Planktonic cells; B, Biofilm cells; p values
Control vs Test p!0.001.a In presence of supernatant without MSPs.b In presence of supernatant with MSPs (30%).
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–26 19
Table 5
Characterization of MSPs collected after interaction of murine peritoneal macrophages with planktonic and biofilm cells of P. aeruginosa
P. aeruginosa strains
PA1 PA2 PA3 PA4 PA5 PAO
A B A B A B A B A B A B
TNF-a 412G20 798G55 450G33 833G59 427G27 864G37 387G15 754G32 423G18 850G28 462G23 871G41
TNF-b 69G10 119G22 81G16 144G25 70G20 129G29 47G17 90G28 60G 26 137G38 98G30 166G42
GM-CSF 523G37 878G62 617G45 896G75 554G30 850G47 492G35 809G50 503G22 834G39 654G20 866G35
IFN-g 35G2 68G10 49G 11 75G9 40G5 81G8 27G9 54G13 32G5 73G9 39G7 94G15
IL-1a 52G5 97G7 70G14 148G20 62G7 136G19 36G6 79G15 87G7 155G16 95G10 170G18
IL-1b 505G30 849G70 574G50 909G66 521G24 874G39 477G20 821G35 589G17 933G67 568G14 950G29
IL-6 527G33 880G50 580G70 845G80 550G30 860G48 496G28 800G38 603G20 879G28 619G18 850G32
MIP-2 539G30 843G70 611G62 915G50 632G45 966G56 504G37 804G40 652G17 975G30 627G15 931G33
IL-12 63G7 109G15 86G14 165G30 98G10 189G12 48G8 100G14 116G10 207G21 100G12 185G19
IL-18 119G10 221G25 137G15 250G19 158G15 319G27 85G11 144G20 120G14 232G20 106G17 200G30
Nitrates
(mmol)
4.3G0.4 7.9G0.6 5.1G0.8 8.8G1 4.7G0.6 8.5G0.9 3.2G0.5 6.7G0.8 4.8G0.7 9.2G0.8 5.5G0.6 9.7G1.3
Nitrites
(mmol)
50.1G5.5 83.5G7.9 57.4G8.6 90.5G7.5 43.4G10 87.9G16 33.5G9 70.6G15 54.3G8 85.2G16 51.3G11 82.4G24
Protein
content
(mg/ml)
150G12 287G20 169G28 302G33 177G17 326G30 130G19 250G28 189G16 357G28 183G17 341G29
All cytokine levels in pg/ml; Data represents meanGstandard deviation; A, Planktonic cells; B, Biofilm cells.
R.
Mitta
let
al.
/C
om
p.
Imm
un
.M
icrob
iol.
Infect.
Dis.
29
(20
06
)1
2–
26
20
Table 6
Growth profile of planktonic cells of P. aeruginosa in presence of supernatants with and without MSPs at different time intervals
P. aeruginosa strains Log (cfu/ml)
2 h 4 h 6 h 8 h 10 h
Controla Testb Controla Testb Controla Testb Controla Testb Controla Testb
PA1 (O4) NG 1.56G0.20 1.65G0.34 3.54G0.28 3.87G0.34 5.09G0.39 4.50G0.41 5.91G0.34 4.52G0.41 5.90G0.44
PA2 (O3) 1.51G0.21 2.62G0.32 2.45G0.27 3.97G0.38 4.65G0.39 5.77G0.28 5.09G0.38 6.50G0.40 5.10G0.34 6.52G0.38
PA3 (O6) 1.57G0.27 2.71G0.24 2.46G0.26 3.92G0.24 4.90G0.29 5.98G0.37 5.27G0.30 6.61G0.44 5.25G0.37 6.60G0.32
PA4 (O7/8) NG 1.60G0.26 1.74G0.30 3.77G0.29 3.97G0.33 5.15G0.38 4.45G0.42 5.97G0.37 4.47G0.40 5.97G0.34
PA5 (O11) 1.53G0.25 2.76G0.29 2.46G0.35 3.95G0.33 4.98G0.29 6.09G0.31 5.39G0.40 6.81G0.30 5.40G0.42 6.83G0.40
PAO (PT) 1.56G0.22 2.67G0.21 2.41G0.33 3.99G0.37 4.95G0.30 6.05G0.28 5.43G0.37 6.87G0.36 5.43G0.35 6.89G0.39
Data represents meanGstandard deviation; PT, polytypable; NG, no growth; p values Control vs Test p!0.001.a In presence of supernatant without MSPs.b In presence of supernatant with MSPs (30%).
R.
Mitta
let
al.
/C
om
p.
Imm
un
.M
icrob
iol.
Infect.
Dis.
29
(20
06
)1
2–
26
21
Table 7
Growth profile of biofilm cells of P. aeruginosa in presence of supernatants with and without MSPs at different time intervals
P. aeruginosa strains Log (cfu/ml)
1 Day 2 Day 3 Day 4 Day
Controla Testb Controla Testb Controla Testb Controla Testb
PA1 (O4) 2.10G0.50 3.78G0.56 3.30G0.43 4.97G0.50 5.50G0.58 6.95G0.50 6.92G0.57 8.32G0.70
PA2 (O3) 2.52G0.57 3.91G0.52 3.46G0.54 5.54G0.55 5.72G0.53 6.90G0.64 7.19G0.68 9.27G0.66
PA3 (O6) 2.17G0.44 3.89G0.50 3.71G0.60 5.17G0.65 5.85G0.50 6.97G0.66 7.05G0.50 8.90G0.62
PA4 (O7/8) 2.21G0.40 3.96G0.61 3.42G0.64 5.33G0.52 5.11G0.54 7.07G0.61 6.52G0.55 9.50G0.64
PA5 (O11) 2.40G0.37 3.90G0.49 3.20G0.58 5.63G0.50 5.00G0.50 7.50G0.56 6.42G0.59 9.37G0.58
PAO (PT) 2.00G0.32 3.48G0.52 3.12G0.40 4.90G0.55 5.30G0.59 6.71G0.53 6.34G0.60 9.50G0.50
Data represents meanGstandard deviation; PT, polytypable; NG, no growth; p values Control vs Test p!0.001.a In presence of supernatant without MSPs.b In presence of supernatant with MSPs (30%).
R.
Mitta
let
al.
/C
om
p.
Imm
un
.M
icrob
iol.
Infect.
Dis.
29
(20
06
)1
2–
26
22
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–26 23
absorbance was read at 545 nm. The amount of hemolysin was determined using
lyophilized haemoglobin to calibrate a standard curve.
All the experiments were carried out in triplicate in two sets.
2.18. Statistical analysis
Results were statistically analyzed by applying student’s t test and Fischer two-tailed
exact test for calculating p values.
3. Results and discussion
Macrophages form one of the first line of defense on mucosal surfaces following tissue
invasion [27]. These cells pour their secretory products which include a cocktail of
biomolecules, at the site of infection. Macrophages can play a key role during the course of
urinary tract infection, not only through their phagocytic activity, but also through effects
mediated by their secretory products. In the present investigation, macrophage secretory
products (MSPs) obtained after interaction of macrophages with P. aeruginosa were
employed to assess their effect on the elaboration of virulence factors by this organism in
planktonic and biofilm cell mode. Employing secretory products of macrophages more
closely simulate the in vivo environment of host–parasite interaction. About 30%
concentration of MSPs was used since this concentration has been reported to modulate
host cell responses [17,28]. Planktonic and biofilm cells of P. aeruginosa obtained by
growing this organism in the presence of 30%MSP showed significant increase in alginate
production and cell surface hydrophobicity (p!0.001) (Table 1). Significant enhancement
in elaboration of extracellular factors like pyochelin, pyoverdin, protease, elastase,
phopholipase C and hemolysin was also observed (p!0.001) (Tables 2–4). However,
biofilm cells of P. aeruginosa produced significantly higher levels of all these virulence
traits as compared to their planktonic counterparts in presence and absence of MSPs (p!0.001).
The characterization of MSPs revealed that it contains a variety of cytokines like
TNF-a, TNF-b, IL-1a, IL-b, GM-CSF, MIP-2, IL-6, IL-12 and IL-18 as well as
reactive nitrogen intermediates (Table 5). Cytokine levels, reactive nitrogen
intermediates and protein content was found to be more in supernatants collected
from macrophages stimulated with biofilm cells as compared to in supernatants
collected from macrophages stimulated with planktonic cells. However, strain-to-strain
variation was observed. On the contrary, none of the cytokines were detectable in
secretory products collected from unstimulated macrophages which acted as control.
Surface of variety of gram-negative and gram-positive bacteria have been shown to
possess receptors for cytokines [29]. Cytokines alone or in combination have been
reported to influence growth of pathogens [30]. Cytokines like IL-1, IL-2 and
granulocyte-macrophage colony stimulating factor (GM-CSF), which are known to be
produced in vivo following inflammation, were reported to serve as growth factors for
virulent strains of E. coli in vitro [31,32]. Similarly TNF-a was shown to augment
growth of E. coli both in vitro as well as in vivo [33]. In the present investigation,
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–2624
significant enhancement in growth of both planktonic and biofilm cells of P.
aeruginosa was observed in presence of MSPs (p!0.001) (Tables 6 and 7). Meduri
et al. [34] and Kanagat et al. [35] also observed concentration dependent specific
growth enhancement of P. aeruginosa, S. aureus and Acinetobacter in presence of
cytokines such as IL-1b, IL-6 and TNF-a in vitro and in vivo either alone or in
combination. Precise mechanisms by which bacteria utilizes cytokines as growth
factors are still unclear. Although number of reports regarding binding of cytokines
with pathogens resulting in enhancement of growth of various organisms does exist in
literature but little is known whether such binding can alter the biological properties of
these pathogens. In this context Luo et al. [36] reported alteration in the virulence
properties of Shigella flexneri following binding with TNF-a resulting in 20-fold
enhanced invasion of HeLa cells. Bacteria are known to breakdown cytokines into
biologically active fragments, which are then transported across the bacterial cell
membranes. These then act on transcription and translation of specific genes leading to
alteration in virulence properties of organism [33]. Higher production of virulence
determinants by P. aeruginosa observed in the present study may be due to binding of
cytokines present in MSPs to the surface of this pathogen and subsequent alteration in
gene expression encoding virulence traits. However, due to paucity of literature in this
regard no direct comparisons are possible.
MSPs have been shown to have potential to alter gene expression of not only bacterial
cells but also of host cells. Sharma et al. [17] observed increased mesengial mRNA
expression of TGF-b leading to stimulation of mesengial cell proliferation. It has also been
reported that MSPs at concentration varying from 30 to 50% stimulates mesengial cell
proliferation in renal tissue whereas higher concentration of MSPs (80%) when used had
suppressive effect [28]. Utilization of MSPs by P. aeruginosa for enhancement of its
growth and virulence traits can have far reaching consequences including chronicity and
recurrence of infections caused by this pathogen. Although available studies employing
pure cytokines in isolation provide explanation for growth enhancement but alteration in
virulence properties have not been reported yet. The existing studies do not provide
precise conclusions since in vivo, a cocktail of secretory products poured from
macrophages is available which will have an entirely different effect on ultimate outcome
of an infection. Since MSPs contain a diverse array of biomolecules which can act in a
complex manner amongst themselves, further in vivo studies are warranted which can
throw more light on the observations of the present investigation.
Acknowledgements
We are thankful to Judith Glover Laboratory of HealthCare Associated Infection,
London for serotyping of Pseudomonas aeruginosa. We are also thankful to Dr Barbara H.
Iglewski for providing us standard strain of P. aeruginosa. A contingency grant for this
project work by Indian Council of Medical Research (New Delhi, India) is gratefully
acknowledged.
R. Mittal et al. / Comp. Immun. Microbiol. Infect. Dis. 29 (2006) 12–26 25
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