Antimicrobial properties of Kalanchoe blossfeldiana: a focus on drug resistance with particular reference to quorum sensing-mediated bacterial biofilm formation.

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Research Paper

Journal of Pharmacy And Pharmacology

Antimicrobial properties of Kalanchoe blossfeldiana: a focus on drug resistance with particular reference to quorum sensing-mediated bacterial biofilm formation Ratul Sarkara,b, Chaitali Mondala, Rammohan Berac, Sumon Chakrabortya, Rajib Barikc, Paramita Roya, Alekh Kumara, Kirendra K. Yadava, Jayanta Choudhuryd, Sushil K. Chaudharyb, Samir K. Samantae, Sanmoy Karmakara,b, Satadal Dasf, Pulok K. Mukherjeea,b, Joydeep Mukherjeed and Tuhinadri Sena,b a Division of Pharmacology, bDepartment of Pharmaceutical Technology, School of Natural Product Studies, dDepartment of Environmental Studies, School of Environmental Studies, Jadavpur University, cTCG Life Sciences Ltd, fMicrobiology and Immunology Department, B.K. Roy Research Center, Peerless Hospital, Kolkata, eCalcutta Institute of Pharmaceutical Technology, Uluberia, West Bengal, India

Keywords acyl homoserine lactone; anticytokine; biofilm; Kalanchoe blossfeldiana; quorum sensing Correspondence Tuhinadri Sen, Department of Pharmaceutical Technology, Jadavpur University, Raja SC Mallik road, Kolkata 700032, India. E-mail: [email protected] Received September 16, 2014 Accepted January 6, 2015 doi: 10.1111/jphp.12397

Abstract Objectives This study attempts to investigate the antimicrobial properties of Kalanchoe blossfeldiana with a particular reference to quorum sensing (QS)mediated biofilm formation. Methods The methanol extract of K. blossfeldiana leaves (MEKB) was evaluated for antimicrobial properties including QS-controlled production of biofilm (including virulence factor, motility and lactone formation) in Pseudomonas aeruginosa. Methanol extract of K. blossfeldiana was also evaluated for anticytokine (tumour necrosis factor-alpha, interleukin-6 and interleukin-1 beta) properties in peripheral blood mononuclear cells (PBMC). Key findings Methanol extract of K. blossfeldiana exhibited antimicrobial effect on clinical isolates, as well as standard reference strains. Pseudomonas aeruginosa exposed to MEKB (subminimum inhibitory concentration (MIC)) displayed reduced biofilm formation, whereas supra-MIC produced destruction of preformed biofilms. Methanol extract of K. blossfeldiana reduced the secretion of virulence factors (protease and pyoverdin) along with generation of acyl homoserine lactone (AHL). Confocal laser scanning microscopy images indicate reduction of biofilm thickness. The extract also reduced cytokine formation in lipopolysaccharide-stimulated PBMC. Conclusions Kalanchoe blossfeldiana was found to interfere with AHL production, which in turn may be responsible for downregulating QS-mediated production of biofilm and virulence. This first report on the antibiofilm and anticytokine properties of this plant may open up new vistas for future exploration of this plant for combating biofilm-related resistant infections.

Introduction Infectious diseases still remain as one of the major cause of mortality, and surprisingly, it is also observed in developed countries.[1] The emergence of antibiotic resistance, a consequence of Darwinism has now become a serious health hazard.[2] According to the reports, antibiotic-resistant nosocomial infections are a major cause of concern, particularly in the intensive care unit of the hospitals.[3]

Epidemiological outcome studies have shown that antibiotic-resistant nosocomial pathogens are associated with increased morbidity, mortality, need for surgical intervention, enhanced hospitalisation including escalation of treatment costs[4,5] It has now been well established that the excessive and indiscriminate prescribing of antibiotics (to combat bacterial infections) has led to the emergence of

© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

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Antibiofilm properties of K. blossfeldiana

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drug resistant strains. Antibiotic resistance is also a clinical reality and is a serious health concern that is being faced by medical practitioners around the globe.[6] According to a report published by the National Institute of Health (NIH, USA), about 80% of all microbial infections are brought about by bacteria, which proliferate within quorum sensing (QS)-mediated biofilms.[7–9] Biofilms are communities of microorganisms attached to a biotic and abiotic surface, which entrench themselves in a self-produced extracellular matrix of exopolysaccharide along with proteins and micromolecules such as deoxyribonucleic acid.[10] The extracellular polymeric substance (EPS) accounts for the majority of the dry mass of the biofilm[11] and acts as a barrier thereby diminishing the effectiveness of various antibiotics.[12] In addition, popular antibiotics (aminoglycosides, fluoroquinolones and tetracycline) are often found to be ineffective against biofilms.[13,14] The emergence of antibiotic resistance necessitates the need for novel therapeutic approaches to combat this global problem.[15] According to researchers, targeting the QS citadel, without necessarily destroying the microbial strains could be a useful approach against the emerging threat of escalating antibiotic resistance.[16] N-acyl homoserine lactone (AHL), an important signal molecule secreted by a wide array of Gram-negative pathogenic bacteria,[14,15] is known to coordinate the expressions of genes responsible for diverse biological functions that include virulence development and biofilm formation.[16] All living organisms (from microorganisms to plants and mammals) have evolved to actively defend themselves against pathogen attack for their survival.[17] Plants known to produce secondary metabolites (structurally and functionally diverse compounds), which enjoy selectional advantages over resistant organisms, are effectively utilised by the hosts for combating the plant pathogens.[18,19] Hence, the chemical diversity of the plant kingdom is considered to be a rich source of novel bio-molecules that could be utilised for neutralising the threat posed by various drugresistant pathogens.[20–22] Plant-derived compounds have long been in use to treat microbial infections.[23] Recently, these phytochemicals have attracted increased scientific attention as they may also serve as a useful source of anti-QS compounds.[24] According to reports, Capparis spinosa Linn (caper),[25] Quercus infectoria Olivier (aleppo oak),[26] Curcuma longa L. (turmeric),[27] Zingiber officinale Rosc. (ginger), Cuminum cyminum L. (cumin)[25] and Allium sativum L. (garlic)[28] have been found to display significant effectiveness against QS-mediated biofilm formation. Similarly, essential oils from Cinnamomum verum Pres (cinnamon)[29] and Syzygium aromaticum (L.) Merrill & Perry (clove)[30] are also known to inhibit QS in Gram-negative bacteria. Several plants have also been found to reduce bio-fouling by inter2

fering with QS.[31] The available reports also indicate the inhibitory role of plant-derived compounds towards AHL, an important signalling molecule of the QS pathway.[32] Kalanchoe, a genus consisting of approximately 125 species, is native to tropical Africa. Today, this plant is also distributed in the tropics (India, Thailand, Malaysia, Brazil, Australia, Madagascar).[33,34] Traditionally, the plants of genus Kalanchoe were used for the treatment of wounds, boils, arthritis, gastric ulcers,[35] dysentery,[36] fever, coughs[37] and urinary diseases.[38] Kalanchoe blossfeldiana Poelln (Flaming Katy) is a shrub belonging to this genus, widely distributed throughout tropical regions of India. Phytochemical analysis of the plant revealed of the presence of quercetin, quercetin 3-O-β-D-glucoside, quercetin 3-O-(2″-O-β-D glucopyranosyl-α-L-rhamnopyranoside,[39] squalene, 4,4dimethyl sterols, 4α-methyl sterols, sterols[40] and gallic acid.[41] Kalanchoe blossfeldiana is known to possess high virus neutralising activity.[42,43] In this study, we explored the effect of a methanolic extract of K. blossfeldiana (MEKB) against Gram-positive and Gram-negative bacteria including drug-resistant and clinical isolates. According to our survey of literature, the present findings would be the first report on antibiofilm properties (biofilm formation and preformed biofilm) of K. blossfeldiana. Moreover, this study will further assist in enhancing our knowledge on the role of ornamental plants against biofilms, produced by Gram-negative microorganisms. In this study, we have demonstrated the effect K. blossfeldiana on both planktonic cells as well as EPSentrapped cells (biofilm), along with an attempt to evaluate the probable mechanism of its antibiofilm property with a focus on QS-mediated AHL generation and production of various virulence factors at subminimum inhibitory concentration (MIC) concentrations.

Materials and Methods Plant material and extract preparation Kalanchoe blossfeldiana (Family: Crassulaceae) was collected from Jadavpur university campus, and authentication was performed by the Central National Herbarium, Botanical Garden, Howrah, West Bengal (No.-CNH/54/2012/Tech.II/ 847). The 500-g fresh leaves were extracted with 1L methanol by cold maceration for 48 h, and the extract was filtered using clean white muslin cloth. Methanol extract of K. blossfeldiana was centrifuged at 6000 rpm to remove particulates matter and concentrated under reduced pressure at 45–50°C. The yield of MEKB was found to be 3.5%.

Liquid chromatography–mass spectrometry standardisation of MEKB Methanol extract of K. blossfeldiana was standardised using high performance liquid chromatography-


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electrospray tandem mass spectrometry (HPLC-ESI-MS)[39] using quercetin and quercetin 3-O-β-D-glucoside as standards. High performance liquid chromatography-mass spectrometry experiments were performed on a Shimadzu HPLC-2010AHT system (Shimadzu Corp., Kyoto, Japan) consisting of an autosampler. The HPLC was connected with a source of electro spray ionisation and atmospheric pressure ionisation (ESI/API-3000, AB Sciex). Ionisation was achieved in both negative and positive-ion modes with ion spray voltage set at 5 kV. Nitrogen (heated to 450°C) was used as the nebulising gas, at a flow rate of 0.8 ml/min. Mass spectrometry signals were collected in the scan mode between 100 and 600 m/z for identification of chemical components. The column used was a Phenomenax (C18, 5 μm) combination of solvent A (0.1% aqueous formic acid v/v) solvent B (80:20 acetonitrile : water) was used as the mobile phase. Initially, gradient elution was performed with solvent A (72 s), and for the next 48 s, a gradient was produced with solvent B (increased up to 100% B), and it was continued for next 60 s (total run time of 3.5 min). The column eluent was analysed by mass spectrometer, and the data were analysed with Analyst 1.4.2 LC–MS Software (Shimadzu Corp., Kyoto, Japan).

Bacterial strains Reference strain (Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC 441, Escherichia coli MTCC 2939 and Pseudomonas aeruginosa MTCC 2453), clinical Isolates (Enterococcus faecalis OP36936, E. coli IP/12/5229, Klebsiella pneumonia IP Ex15252, S. aureus IP00025) and Metallo-βlactamase producing P. aeruginosa (MBL 8114, MBL 05/428) were used for Bacterial susceptibility assay. Pseudomonas aeruginosa MTCC 2453 was used in rest of the experiment. The strains were maintained on nutrient agar (NA) plate and stored at 4°C. A single colony was transferred to Mueller Hinton broth (MHB) and incubated at 37°C. Density of the broth (containing the suspended organisms) was adjusted to 0.5 McFarland standard.[44]

Bacterial susceptibility assay Stock solutions of MEKB (100 mg/ml) were prepared in dimethyl sulfoxide (DMSO). The stock solution was diluted with sterile Milli-Q for further use. Minimum inhibitory concentration was determined using the broth microdilution method. Briefly, a standardised test inoculums (10 μl of a 1–5 × 105 colony-forming unit (CFU)/ml suspension) added to the wells of 96 well microtitre plate, containing 100 μl of twofold serially diluted MEKB in MHB (final concentrations ranging from 0.015 to 2 mg/ml), and were then incubated (100 rpm, 37°C) for 18 h. Dimethyl sulfoxide (0.1%) was used as the negative control. Minimum inhibitory concentration of the


sample was detected following addition of 20 μl of 0.2 mg/ml p-iodonitrotetrazolium chloride (INT). Viable bacteria produce a pink colour by reducing INT to INTformazan. The MIC value is defined as the lowest concentration where no viability was observed after 18 h.[45] Minimum bactericidal concentration (MBC) was measured by using the colony count technique (NA plates), using 20 μl of broth, collected from the clear wells (signifying no visible growth) of MHB containing microtitre plates (as described earlier). The plates were incubated at 37°C for 24 h. The minimum concentration of sample at which the visual bacterial colony was reduced to 99.9% was considered as the MBC.[46] All further experiments in this study were performed only at sub-MIC concentrations of MEKB.

Effect of methanol extract of Kalanchoe blossfeldiana on biofilm formation The effect of MEKB on biofilm formation was performed in 96-well polystyrene plates. A standardised inoculum (5 μl of a 1–5 × 105 CFU/ml suspension) was inoculated with 100 μl of fresh MHB in presence or absence (non-treated control) of MEKB (15, 30, 62.5, 125 μg/ml; equivalent to onesixteenth, one-eighteenth, one-fourth and one-half of MIC values of MEKB against P. aeruginosa). Following incubation (24 h), non-adherent bacteria were removed by washing with sterile phosphate buffer saline (PBS; pH 7.2). Biofilms were stained with 1% crystal violet solution. The absorbance of the crystal violet solution (stain bound to biofilm was removed from each well employing 33% glacial acetic acid) was measured at 492 nm (Spectramax M5, Molecular Device). Wells containing medium and extract were used as blanks.[46] Ciprofloxacin at sub-MIC concentration (0.031, 0.0625, 0.125, 0.25 μg/ml; equivalent to onesixteenth, one-eighteenth, one-fourth and one-half of MIC values of ciprofloxacin against P. aeruginosa) was employed as the positive control. At the above-referred concentrations, ciprofloxacin is able to inhibit the biofilm formation without altering the bacterial growth.[7] The percentage of inhibition of biofilm formation: (1 − optical density at 492nm of the test sample/optical density at 492 nm of non-treated control) × 100.

Effect of MEKB against preformed biofilms Standardised inoculum (5 μl of a 1-5 × 105 CFU/ml suspension) in 100 μl of MHB was incubated for 24 h. Planktonic bacteria were removed by washing with PBS (pH 7.2). Preformed biofilms were then exposed to MEKB (0.25, 1.25, 2.5 mg/ml equivalent to 1, 5 and 10 × MIC). Ciprofloxacin was used as a positive control (0.5, 2.5, 5 μg/ml equivalent to 1, 5 and 10 × MIC values of ciprofloxacin) The plates were again incubated (24 h at 37°C). Cells entrapped in the


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biofilm were isolated and subsequently mixed with 100 μl of 0.25% trypsin EDTA (5 min). The bacterial count was determined.[46]

In-situ visualisation of biofilm Light microscopic analysis For visualisation of the biofilms, the cell were allowed to grow on coverslips (placed in 12-well polystyrene plates containing MHB; in presence or absence of MEKB), incubated for 24 h at 37°C. The coverslips were stained with 0.4% crystal violet and visualised with an inverted phase contrast microscope (Model: OLYMPUS IX70, Olympus Optical Co. Ltd.) at a magnification of ×200. Visible biofilms were documented with a digital camera (Olympus, Inc. Japan).[10] Confocal laser scanning microscopic (CLSM) analysis The effect of MEKB on biofilm formation and preformed biofilm was monitored under a confocal laser scanning microscope (CLSM; Andor spinning disk confocal microscope) by two different experiments. For determining the effect of MEKB on biofilm formation, the cell were allowed to grow on glass slides (placed in 12-well polystyrene plates containing MHB; in presence or absence of MEKB), incubated at 37°C. After 24 h, biofilm formed on the glass slides were washed with PBS and stained with 20 μl of 1% acridine orange (0.02%, w/v for 15 min at 4°C in dark place). The excess stain was washed, and the stained glass slides were visualised to determine the thickness of the biofilm using CLSM.[22] In the second experiment, the effect of MEKB was studied on preformed biofilms produced on glass slides. The glass slides were washed with PBS and immersed in MH broth containing MEKB (MEKB was not used in the negative control). After 24 h, glass slides were washed with PBS and stained with SYTO9 and propidium iodide (PI). Cells containing intact membranes were stained fluorescent green, whereas with disrupted membranes, stained fluorescent red.[47] Swarming motility assay Motility assays were performed in six-well polystyrene plates. Five microlitres of bacterial broth culture representing approximately 105 CFU/ml were inoculated at the centre of the well containing a medium consisting of 1% peptone, 0.5% NaCl, 0.5% agar and 0.5% D-glucose, containing various concentrations of MEKB (15, 30, 62.5, 125 μg/ml; equivalent to one-sixteenth, one-eighteenth, one-fourth and one-half of MIC). Ciprofloxacin (0.125 μg/ml) was used as a positive control. The diameters of the swarming zones were measured after incubation for 24 h (37°C).[48] 4

Pyoverdin assay Methanol extract of K. blossfeldiana (15, 30, 62.5, 125 μg/ ml; equivalent to one-sixteenth, one-eighth, one-fourth and one-half of MIC) was incubated for 24 h with a 10% culture of P. aeruginosa. The cells were centrifuged, and the cell-free supernatant was used for pyoverdin assay. The pyoverdin concentration in the supernatant was measured fluorometrically (excitation – 405 nm; emission – 465 nm) in a multimode microplate reader (Spectramax M5; Molecular Device).[15] The activity was expressed in relative fluorescence units. Azocasein-degrading proteolytic activity Bacterial culture was grown in MHB in presence or absence of MEKB. The proteolytic activity (azocasein degradation) was determined in the cell-free supernatant of P. aeruginosa.[49] To evaluate the direct inhibitory effect of MEKB on pseudomonal protease, the cell free supernatant was incubated with different concentration of the extract (62.5, 125 μg/ml; equivalent to one-fourth and one-half of MIC) for 1 h; 37°C. Thereafter, azocasein-degrading activity was assessed.[50] Determination of extracellular polymeric substance and lactone type quorum-sensing compounds To determine EPS and lactone-type QS compounds, a standardised inoculum (100 μl of a 1-5 × 105 CFU/ml suspension) was added to MHB containing MEKB (15, 30, 62.5, 125 μg/ml; equivalent to one-sixteenth, oneeighteenth, one-fourth and one-half of MIC). After 24 h, the culture broth was centrifuged (1000 rpm) to separate the cell-free supernatant from the biofilm. Biofilm was used for determination of EPS, whereas the cell free supernatant was used for detection of lactone (AHLs). Biofilms were dried at 40°C and resuspended in NaOH (1 N). The sugar and protein concentrations in the EPS in the supernatant were determined according to Mitra et al., 2011.[51] The cell-free supernatant was extracted twice with equal volumes of ethyl acetate, dried, and the residues were dissolved in distilled water. Thereafter, it was mixed with equal volumes of hydroxyl amine (2 M) and NaOH (3.5 M). Subsequently, a 1:1 mixture of ferric chloride (10% in 4 M HCl): 95% ethanol was added. Lactone-type compounds were determined at 520 nm using γ-valerolactone (Sigma-Aldrich) as the standard.[51] Effects on production of cytokines The test compounds were preincubated with human peripheral blood mononuclear cells for 30 min at 37°C and


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then stimulated with lipopolysaccharide (E. coli: B4; 1 μg/ml) for 18 h (37°C; 5% CO2). Cytokine (tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β)) concentration was estimated using ELISA kits (Cayman Chemical, Ann Arbor, USA).[52] Statistical analysis All the reported values represent the average of three independent experiments in duplicate. Statistical analysis was performed with one-way analysis of variance followed by post-hoc Dunnett’s test. Unless otherwise mentioned, ‘P’ values less than 0.05 was considered to be statistically significant.

Results Standardisation of Methanol extract of K. blossfeldiana Liquid chromatography-electrospray tandem mass spectrometry analysis (Figure 1) confirmed the presence of quercetin (m/z 301) and quercetin 3-O-β-D-glucoside (m/z 463). Quercetin and its glucoside could be detected in the positive ionisation mode, and the adduct ions [M + H]+ were detected.

Bacterial susceptibility assay The antibacterial activity was tested in in vitro against different multidrug-resistant clinical isolates and reference bacterial strains. Methanol extract of K. blossfeldiana was found to exhibit strain specific antibacterial activity against clinical isolates, as well as different standard reference strains (Gram-positive and Gram-negative) at concentrations ranging 0.0625–0.5 mg/ml (Table 1).

Effect of Methanol extract of K. blossfeldiana on biofilm formation Effect of MEKB on biofilm formation was evaluated by crystal violet assay. A decrease in biofilm formation was

observed with MEKB. From Figure 2, the viability of P. aeruginosa biofilm was found to be reduced by MEKB in a concentration dependent manner (84.37%, 69.22%, 37.9%, 11.82 at one-sixteenth, one-eighteenth, one-fourth and one-half of MIC, respectively). The images obtained from CLSM (Figure 3) substantiate our claim with MEKB, where the extract preincubation was found to produce substantial reduction in biofilm thickness (one-fourth MIC – 12 mm; one-eighth MIC – 18.47 mm) as compared the negative control where an average thickness of 26.85 mm was observed.

Effect of methanol extract of Kalanchoe blossfeldiana against preformed biofilms Methanol extract of Kalanchoe blossfeldiana was also found to be very effective in disrupting the preformed (mature) biofilms produced by P. aeruginosa. When the mature biofilms were treated with MEKB (1, 5 and 10 MIC) the viable cell count decreased by 46.88%, 72.82% and 91.77%, respectively (Figure 4).

Swarming motility assay Swarming motility is a flagella-mediated movement that facilitates the colonisation of the cells in a nutrient-rich environment, and aids in surface adherence leading to biofilm formation. In this study, MEKB at one-sixteenth of MIC (15 μg/ml) produced significant reduction of (15.23 ± 1.19 mm) flagella-mediated swarming motility of P. aeruginosa (control; 21.89 ± 1.17 mm). Pretreatment with ciprofloxacin (used as a standard) also produced significant reduction of swarming (9.08 ± 1.23 mm) activity at onefourth of MIC (0.125 μg/ml).

Pyoverdin assay Pyoverdin is one of the virulence factors, which is regulated by QS, and it competes with mammalian transferrin for iron. This assay was carried out to evaluate the effect of

Table 1 Assessment of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Kalanchoe blossfeldiana leaf extract against different bacterial strains

Reference strain

Clinical isolates

Metallo-β-lactamase producing P. aeruginosa

Escherichia coli MTCC 2939 Pseudomonas aeruginosa MTCC 2453 Bacillus subtilis MTCC 441 Staphylococcus aureus MTCC 96 E. coli IP/12/5229 Klebsiella pneumoniae IP Ex15252 S. aureus IP00025 Enterococcus faecalis OP36936 MBL 8114 MBL 05/428


MIC (mg/ml)

MBC (mg/ml)

0.25 0.25 0.5 0.25 0.25 0.062 0.125 0.25 0.5 0.5

1 1 1 0.5 1 0.25 0.25 0.5 1 1

5

Figure 1 Offline electrospray ionisation mass spectrometry (ESI-MS) scan of a methanol extract of K. blossfeldiana extract using Shimadzu HPLC-2010AHT system. Liquid chromatographyelectrospray tandem mass spectrometry analysis confirmed the presence of quercetin (m/z 301) and quercetin 3-O-β-D-glucoside (m/z 463).


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100 90 80 70 60 50 40 30 20 10 0 Cipro MEKB Cipro MEKB Cipro MEKB Cipro MEKB

40 30 20 10 0

Cipro

MEKB MIC

Cipro MEKB 5 MIC

Cipro MEKB 10 MIC

1/16 MIC

(b)

eg N

(1 B M

EK

.C

/1

on

6M

tr

IC

ol

)

) IC M /8 (1 B

B EK M

M

EK

B

(1

(1

/2

/4

M

M

IC

(a)

)

MEKB

Figure 4 Effect methanol extract of K. blossfeldiana and ciprofloxacin (Cipro) at lethal concentrations (minimum inhibitory concentration (MIC), five MIC and 10 MIC) on preformed biofilm of P. aeruginosa. The results are expressed as a percentage of the biofilm viability, assessed by colony counting, with respect to untreated control taken as 100%. Values are expressed as mean ± SEM.; n = 6.

IC

Figure 2 Effect of methanol extract of K. blossfeldiana and ciprofloxacin (Cipro) on biofilm formation. Biofilm formation was assessed by colorimetric technique using crystal violet. The results are expressed as percentage of biofilm inhibition with respect to untreated control. Values are expressed as mean ± SEM.; n = 6.

Neg. Control

50

EK

1/8 MIC

60

M

1/4 MIC

70

)

1/2 MIC

Biofilm viability (% of Control)


RFU (EX-405; EM-465nm)

% of Biofilm inhibition

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Figure 5 Pyoverdin production in Pseudomonas aeruginosa, grown in presence or absence of methanol extract of K. blossfeldiana. The fluorescence emission spectrum was recorded by exciting at 405 nm. The activity was expressed in relative fluorescence units. Values are expressed as mean ± SEM.; (n = 6); *P < 0.05 (vs control).

(c)

Figure 3 Microscopic images of Pseudomonas aeruginosa biofilm grown in presence or absence of methanol extract of K. blossfeldiana. (a) Light microscopy image; (b) CLSM image of biofilm formation stained with acridine orange; (c) CLSM image of preformed biofilms treated with methanol extract of K. blossfeldiana (five minimum inhibitory concentration), stained with Syto-9 and PI. Biofilm with intact cell membranes are stained fluorescent green, whereas biofilm with damaged cell membranes are stained fluorescent red. The overlap of the red and green areas is orange.

MEKB on pyoverdin production by P. aeruginosa. Pretreatment with MEKB (one-sixteenth, one-eighteenth, onefourth and one-half of MIC, respectively) produced significant reduction of pyoverdin concentration (21.61%, 30.61%, 60.14% and 79.02%) in a dose-dependent manner (Figure 5).

Azocasein-degrading proteolytic activity The protease activity (azocasein breakdown) was measured in the culture supernatants, when the cells were incubated in presence and absence of the test substances. Significant and dose-dependent decrease (30.78%, 54.65%, 76.13% and 92.84%) in total protease production (culture supernatant) was observed in P. aeruginosa when preincubated with


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Effects on production of cytokines

Optical Density (OD at 400nm)

0.5

Methanol extract of K. blossfeldiana significantly inhibited the secretion of different proinflammatory cytokines namely TNF-α, IL6 and IL-1β in lipopolysaccharide (LPS)stimulated human peripheral blood mononuclear cells (Table 3). The cytokine levels in the cell culture supernatant of uninduced cells were negligible, whereas LPS-treated cells produced significantly higher level of cytokines. However, when the cells were preincubated with MEKB before stimulating with LPS, a dose-dependent reduction of cytokine production was observed. Methanol extract of K. blossfeldiana (50 μg/ml) decreased the production of the TNF-α, IL6 and IL1β (43.31 ± 2.7%, 87.06 ± 5.32% and 78.48 ± 4.71%) (Table 3).

0.4 0.3 0.2 0.1

tr ol

IC )

on N

eg

.C

/1 6 M

EK B

(1

(1 M

EK B

(1 EK B M

M

/8 M

IC ) /4 M

IC ) /2 M (1 EK B M

IC )

0.0

Figure 6 Effect of methanol extract of K. blossfeldiana (MEKB) on the production of protease (in the cell-free supernatant) by Pseudomonas aeruginosa (cells were preincubated with MEKB). The proteolytic activity was determined from the breakdown of azocaesin. Values are expressed as mean ± SEM; (n = 6); *P < 0.05 (vs control).

one-sixteenth, one-eighteenth, one-fourth and one-half of MIC, respectively of MEKB (Figure 6). Another study was conducted to understand the possibility of any direct interaction between MEKB with the protease present in the cell free supernatant. On direct incubation of the cell free supernatants (grown in absence of the test samples) with MEKB, there was no significant alteration of protease activity. Therefore, from this study it may be suggested that preincubation of P. aeruginosa cells with MEKB affects protease production, and this effect may be mediated through alteration of AHL-mediated QS.

Estimation of exopolysaccharide Production of EPS is essential for biofilm formation, stability and architecture. Exopolysaccharide of the biofilms contained much higher proportion of sugars than proteins. Preincubation with MEKB produced significant inhibition of EPS production (concentration dependent) by P. aeruginosa as evident from the reduced content of carbohydrate and protein in the EPS (Table 2).

Estimation of lactone type quorum-sensing compounds Acyl homoserine lactone molecule is a widely used QS molecules by the Gram-negative organism. The concentration of the lactone type QS compounds was measured in the cell free supernatant. The concentration of this lactone-like signalling compound in MEKB treated cell-free supernatant was significantly lower as compared with that of untreated control (Figure 7). 8

Discussion As evident from numerous reports, extensive as well as nonjudicious utilisation of antibiotics, both in human and veterinary medicine, along with the widespread application in agriculture have resulted in the development and spread of resistant microbes across the globe.[53–55] The conventional approach of developing newer antibiotics against resistant bacterial strains has failed to curb the ever-growing hazard posed by antibiotic resistance. Therefore, intensive research is urgently necessary for developing novel strategies for combating the threat of emerging drug resistance. Based on the abovementioned observation, MEKB was screened for antibacterial activity (in vitro). In this study, MEKB was found to display varying degrees of strainspecific antibacterial activity against different multidrugresistant clinical isolates, as well as different standard reference strains (Gram-positive and Gram-negative). It will be important to mention that MBC values of MEKB against all of the organisms investigated, were either two or at most fourfold higher than the corresponding MIC values, thereby indicating a bactericidal property.[52] Bacterial cells growing in biofilm encase themselves in a self-produced matrix of extracellular polymeric substance, which enhances the antibiotic resistance by several folds by protecting the entrapped bacteria from the outer environment.[56] According to reports, the negatively charged polymers present in the biofilm matrix are known to interact with positively charged antibiotics (e.g. aminoglycoside), thereby limiting/slowing the penetration of such antibiotics.[57] During the initial stage of biofilm formation, the planktonic cells adhere to a solid surface, and this results in rapid growth of bacteria. During the initial hours, surface adhesion is known to be reversible.[58] Hence, preventing bacterial adhesion during the initial stages may considerably reduce the possibility of biofilm formation and subsequent development of resistance. In our study, sub-MIC


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Table 2 Effect of methanol extract of Kalanchoe blossfeldiana (MEKB) on biofilm EPS, produced by Pseudomonas aeruginosa (represented as sugar and protein concentration) Treatment Constituents of EPS

MEKB (1/2MIC)

MEKB (1/4MIC)

MEKB (1/8MIC)

MEKB (1/16MIC)

Negative control

Sugar (μg/ml) Protein (μg/ml)

182.23 ± 13.31* 34.67 ± 2.60*

243.68 ± 13.69* 60.82 ± 4.05*

321.81 ± 23.09* 84.21 ± 4.85*

383.33 ± 21.72* 112.39 ± 8.67*

470.66 ± 26.71 175.57 ± 10.01

% of inhibition of Lactone production

MIC, minimum inhibitory concentration.Values are expressed as mean ± SEM; (n = 6); *P < 0.05 (vs control).

90 80 70 60 50 40 30 20 10 0 MEKB (1/2MIC)

MEKB (1/4MIC)

MEKB (1/8MIC)

MEKB (1/16MIC)

Figure 7 Effect of methanol extract of K. blossfeldiana on quorum sensing-mediated acyl homoserine lactone production by Pseudomonas aeruginosa. The lactone was quantified using γ-valerolactone as the standard. Values are expressed as mean ± SEM; n = 6.

concentration of MEKB significantly reduced the biofilmforming ability of the organisms. However at this concentration, MEKB did not affect the growth of planktonic cells. Biofilm inhibition assay and microscopic observations clearly indicate that MEKB effectively produced significant reduction of colony formation along with reduced dispersion of microcolonies. At supra-MIC concentration of MEKB, the viability of biofilms was also reduced significantly. Characteristics of the biofilm architecture are an additional interesting feature, particularly important for understanding the effect of drug on biofilm formation.[59] In this study, the results of CLSM analysis revealed the altered architecture of the MEKB-treated biofilms, where the matrix appeared to be slack, and the biofilm thickness was considerably reduced when compared with that of the control (Figure 4). Similar observations have been recorded with Dalea pulchra[16] or garlic,[28] which are also known to inhibit biofilm formation in P. aeruginosa. Quorum sensing-based EPS production is considered to be important for maintaining the biofilm architecture and integrity and is also known to influence microcolony for-

mation.[60] According to reports, inhibition of EPS may exert a positive influence on antibiotic activity by facilitating drug penetration thereby arresting the progression of film formation, promoting the disruption of the biofilm architecture and destruction of the entrapped cells.[61] In the present context, MEKB significantly inhibited the EPS production by P. aeruginosa, as evident from the reduction of both protein and carbohydrate in the EPS of biofilm.[51] This may also suggest that MEKB could possibly interfere with the synthesis of extracellular polysaccharides, however, this hypothesis need further confirmation. Colonisation of microbes in a nutrient-rich environment is facilitated by QS-dependent swarming motility (flagellamediated movement), and it also related to biofilm formation, pathogenesis[62] including overproduction of virulence factors.[63] From experimental evidence it has been observed that disturbance of swarming motility might have a negative influence on biofilm formation.[63] In the present context, preincubation of MEKB with P. aeruginosa significantly reduced the motility of the cells. As evident from reports, biofilm formation, virulence and pathogenicity are regulated by the QS pathway.[64] In more than 70 species of Gram-negative bacteria, QS is mediated through AHLs, which are also referred as autoinducers. These AHLs are produced by specific enzymes, and they are recognised by specific receptors. Moreover, LasIR-encoded protease, a virulence factor, is known to play a crucial role during invasion of host cell by P. aeruginosa.[49] These proteases are hydrolytic enzymes that are involved in the digestion of the structural components of the infected cells, thereby facilitating bacterial invasion and growth. Preincubation with MEKB produced concentration-dependent inhibition of protease production in the cell free supernatant (protease dependent azo-caseine degradation activity). It is also pertinent to mention that MEKB was devoid of any direct inhibitory effect on the protease activity (Figure 7). Therefore, such alteration of protease production, observed with MEKB may be attributed to the inhibitory role of MEKB on AHL mediated QS.[50] Similar to protease, pyoverdin is also a QS-regulated virulent factor (siderophore) present in various Gramnegative organisms including Pseudomonas species. Pyoverdin consist of a dihydroxyquinoline chromophore,


9


Ratul Sarkar et al.

Table 3 Effect of methanol extract of Kalanchoe blossfeldiana (MEKB) on lipopolysaccharide(LPS)-induced tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-beta (IL-1β) production in human peripheral blood mononuclear cells stimulated with lipopolysaccharide (Escherichia coli: B4; 1 μg/ml) % of inhibition TNF-α

IL-6

IL-1β

Samples

50 μg/ml

10 μg/ml

50 μg/ml

10 μg/ml

50 μg/ml

10 μg/ml

MEKB Dexamethasone

43.31 ± 2.7

11.29 ± .89 98.27 ± 7.3

87.06 ± 5.32

24.19 ± 1.26 85.34 ± 6.7

78.48 ± 4.71

19.28 ± 1.2 89.66 ± 6.2

Values are expressed as mean ± SEM of six independent experiments.

which provides one bidentate ligand for Fe (III) and is known to compete with mammalian transferrin (iron deficiency in the host cells), ultimately promoting pathogenicity through stimulation of microbial growth.[65] In the present investigation, the concentration of pyoverdin (in the cell free supernatant) was found to be decreased following pretreatment with MEKB. The autoinducer (AHLs) freely diffuses through the bacterial cell envelope, gradually accumulating in the external environment. As the cell density increases (quorum formation), AHL-like autoinducers attain a critical concentration, prompting a back-diffusion into the cells. Inside the bacteria, AHL interact with LasR and the LasR-autoinducer complex, thereby regulating a range of diverse cellular functions.[66,67] It has also been suggested that many virulent Gram-negative organisms may be rendered non-pathogenic through inhibition of AHL mediated quorum-sensing mechanism.[16] In this study, pretreatment with MEKB produced significant reduction of AHL in P. aeruginosa. According to the reports, Gram-negative organisms (both planktonic and those present within the biofilm) are known to induce inflammation.[68] In such situations, the bacterial LPS is considered as the causative factor for inducing the immune response, and this may play a predominant role particularly in sepsis-like conditions.[69] It has been observed that pathogenic conditions aggravate the release of proinflammatory cytokines (TNF-α and IL-6, IL-12) triggering a ‘cytokine storm’-like effect.[70] In the present investigation, preincubation with MEKB reduced the LPSmediated production of inflammatory cytokines (IL-6, IL-12 and TNF-α) in peripheral blood mononuclear cells. From the liquid chromatography–mass spectrometry (LC–MS) analysis (MS fragmentation), the presence of quercetin (m/z 301) and 3-O-β-D-glucoside (m/z 463)

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Conclusion On the basis of the present findings, it may be suggested that MEKB obtained from K. blossfeldiana, may provide a viable alternative for the management of infections associated with drug-resistant strains, particularly related to biofilm formation. It may also be mentioned that the current report is the first of its kind that demonstrates the antibiofilm and QS inhibition properties of K. blossfeldiana. Moreover, our observation further indicates the role of the extract on the production of pro-inflammatory cytokines, thereby opening up newer avenues for further exploration of this plant in the management of sepsis-like conditions.

Declaration Acknowledgement We are thankful to the University Grants Commission, New Delhi (DSA Phase-III and UPE-II programme of UGC) for their support.

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Defensive remodeling: How bacterial surface properties and biofilm formation promote resistance to antimicrobial peptides.

Effects of Material Properties on Bacterial Adhesion and Biofilm Formation.

Bicyclic brominated furanones: a new class of quorum sensing modulators that inhibit bacterial biofilm formation.

Inhibition of bacterial quorum sensing and biofilm formation by extracts of neotropical rainforest plants.

Roles of quorum sensing molecules from Rhizobium etli RT1 in bacterial motility and biofilm formation.

Update on Monoterpenes as Antimicrobial Agents: A Particular Focus on p-Cymene.

Bacterial strategies of resistance to antimicrobial peptides.

A bacterial volatile signal for biofilm formation.

Influence of bacterial presence on biofilm formation of Candida albicans.

Effects of different osmolarities on bacterial biofilm formation.

Hospital health care for HIV infection with particular reference to injecting drug users.

Bisphosphonates enhance bacterial adhesion and biofilm formation on bone hydroxyapatite.

Immunity to Chlamydia psittaci with particular reference to sheep.

The complex interplay of iron, biofilm formation, and mucoidy affecting antimicrobial resistance of Pseudomonas aeruginosa.

Psychodynamics of suicide, with particular reference to the young.

Agents that inhibit bacterial biofilm formation.

Some studies on the metabolism of BDH 7538 in animals and man with particular reference to its anti-inflammatory properties.

Adverse reactions to methyldopa with particular reference to hypotension.

Diseases of travel, with particular reference to tropical diseases.

Behavioural toxicity of antidepressants with particular reference to moclobemide.

Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa.

Biofilm formation and antimicrobial resistance in methicillin-resistant Staphylococcus aureus isolated from burn patients, Iran.

Stenotrophomonas maltophilia in Mexico: antimicrobial resistance, biofilm formation and clonal diversity.

Impairment of Pseudomonas aeruginosa Biofilm Resistance to Antibiotics by Combining the Drugs with a New Quorum-Sensing Inhibitor.