Effect of macrophage secretory products on elaboration of virulence factors by planktonic and...

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).

http://www.elsevier.com/locate/cimid

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


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


(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.


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


(serotype)

Pyochelin production

(OD at 510 nm)

Pyoverdin production

(RF at 460 nm)


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




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


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



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




Table 5

Characterization of MSPs collected after interaction of murine peritoneal macrophages with planktonic and biofilm cells of P. aeruginosa


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%).

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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,


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.


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