ANALYSIS OF QUORUM QUENCHING POTENTIAL OF EUPHORBIA TRIGONA MILL.

ANALYSIS OF QUORUM QUENCHING POTENTIAL OF EUPHORBIA TRIGONA MILL.

K. Marathe ^*, N. Nashikkar, S. Bundale and A. Upadhyay

Hislop School of Biotechnology, Hislop College, Temple Road, Civil Lines, Nagpur - 440001, Maharashtra, India.

ABSTRACT: S. marcescens and P. mirabilis are gram-negative bacteria responsible for nosocomial infections. Quorum sensing regulates virulence in S. marcescens and P. mirabilis. Present study documents the potential of E. trigona extracts in inhibiting quorum sensing in both the bacterial species. Extracts of aerial part of the plant were prepared in petroleum ether, chloroform, ethyl acetate, acetone and methanol using soxhlet extraction technique. Petroleum ether extract, chloroform extract, and acetone extract were found to quench quorum in S. marcescens. These extracts were next subjected to column chromatography and assayed for its quorum quenching capacity. Later, thin layer chromatography was used to separate and identify the bioactive fraction responsible for quorum quenching. The fraction exhibiting quorum quenching capacity was subjected to GC-MS analysis. It revealed the presence of fatty acid derivatives in the bioactive fraction. A major part of the bioactive fraction was 9,12-Octadecadienoyl chloride (Z, Z), a derivative of linoleic acid, and, hence linoleic acid was also used for testing its effect on swarm motility of bacteria. Furthermore, the bioactive fraction was employed to study the inhibition of virulence factors in S. marcescens and P. mirabilis. Bioactive fraction lowered the prodigiosin production as well as reduced swarming motility and biofilm formation in S. marcescens. It was also found effective in decreasing urease synthesis, biofilm formation and swarming in P. mirabilis. Therefore, bioactive fraction isolated from E. trigona and linoleic acid both quenched quorum in S. marcescens and P. mirabilis.

Quorum Sensing (QS), Fatty acid derivatives, 9,12-Octadecadienoyl chloride (Z, Z)-, Swarming, Biofilm

INTRODUCTION: Bacterial communication involving the production of molecules called autoinducers and their detection by receptors is referred to as quorum sensing (QS). QS is a population density-dependent phenomenon responsible for regulating the behavior of bacterial cells in consortium ¹. It accounts for various attributes of bacterial behavior including swarming, luminescence, biofilm formation, sporulation, conjugation, and virulence.

Disruption of QS in bacteria, therefore, might serve as an effective strategy to deal with bacterial infections ².

Serratia marcescens is a member of the Enterobacteriaceae family of gram-negative bacteria ³. It causes nosocomial infections of respiratory tract, urinary tract ⁴, ocular region ⁵ and also septicemia and meningitis ⁶. QS regulates the production of prodigiosin, chitinase, protease, hemolysin production and virulence factors like swarming motility, swimming motility, biofilm formation ⁷. Proteus mirabilis is also a gram-negative bacterium belonging to the family of Enterobacteriaceae. This organism is primarily responsible for causing urinary tract infections comprising pyelonephritis as well as cystitis.

Besides, P. mirabilis is associated with infections of eyes, ears, respiratory tract, throat, skin burns, and wounds. P. mirabilis swarms on the surface of the agar in an extremely organized mode giving rise to the characteristic bull’s eye pattern. Swarming of P. mirabilis is concomitantly associated with its virulence including urease synthesis, biofilm formation and host cell invasion ⁸. QS regulates swarming and synthesis of virulence factors in P. mirabilis ⁹.

An alternative approach to treat S. marcescens and P. mirabilis infections is the need of the hour since many strains of these bacteria are highly resistant to antibiotics. Interference and inhibition of QS system of S. marcescens and P. mirabilis may, therefore, be a pertinent and proficient technique to reduce the pathogenicity of these bacteria.

Many species of Euphorbia (E. hirta, E. nerifolia, E. antiquourm, E. tirucalli, E. trigona) are employed in traditional ayurvedic medicines for the treatment of infections and inflammation ⁹. They are also used for conditions like rheumatoid arthritis, gout, blood disorders, jaundice, tumors, insect bite and hepato-splenomegaly ¹⁰. Euphorbia trigona Mill. is a tropical plant that yields milky latex ¹¹. Previous work in our lab has established anti-QS ⁹, anti-swarming, immunostimulatory ¹² anti-fungal and antioxidant properties ¹⁰ of E. trigona Mill. The present study aims to isolate the anti-QS compound(s) from E. trigona Mill. and utilize it to study anti-QS effects against S. marcescens and P. mirabilis.

MATERIALS AND METHODS:

Bacterial Strains and Growth Conditions: Bacterial strains and their culture conditions are described in Table 1.

TABLE 1: BACTERIAL STRAINS AND GROWTH CONDITIONS

Collection of Plant Material and Preparation of Extract: The aerial part of the plant was collected from the garden of Hislop College, Nagpur and identified by comparison with reference material (accession no. 10081) at Department of Botany, RTMNU, Nagpur. Plant material was washed and shade dried. The dried plant material was powdered and subjected to soxhlet extraction using petroleum ether, chloroform, ethyl acetate, acetone, and methanol. Extracts obtained were dried using vacuum concentrator and re-suspended in a minimum volume of a suitable solvent and stored at 4 °C for further use.

Preliminary Phytochemical Analysis: The extracts obtained from soxhlet extraction were qualitatively analyzed for the presence of phenols ¹³, tannins, saponins, flavonoids, alkaloids ¹⁴, anthraquinones ¹⁵, steroids ¹⁶ and triterpenoids ¹⁷.

Analysis of Anti-QS Activity of Crude Extracts Against S. marcescens and Isolation of Bioactive Principle: 1% of the overnight grown culture of S. marcescens was added to molten nutrient agar and plated. The agar was allowed to solidify, and wells (diameter = 0.5 cm) were punched into it. 100 μl of concentrated extracts were added to these wells and incubated overnight at room temperature against a suitable control. Plates were observed the next day for the zone of pigment inhibition. The extracts inhibiting pigment production were subjected to column chromatography.

The column for chromatography was prepared using 7 g of silica mesh 60-120 pore size. 600-800 μl of concentrated extract was adsorbed onto 0.2 – 0.3 g of silica mesh 60-120 pore size and used for chromatographic extraction. Elution was carried out at the rate of 40-60 drops per minute using different solvents and solvent systems - chloroform, chloroform: acetone: acetic acid (75: 24:1, 75: 40: 1, 75: 50: 1, 75: 60: 1 and 75: 60: 5) and methanol. Different fractions were collected, concentrated and assayed for anti-QS activity by agar well diffusion method.

Preparative TLC of Bioactive Principle: The bioactive fractions isolated using column chromatography was subjected to TLC. The bioactive fractions were dissolved in 100 μl solvent, and 10μl was used to spot on TLC plate (silica gel 60 f₂₅₄). The plate was run using ethyl acetate: methanol: water (6: 2: 1) as the mobile phase. The separated bands on TLC were extracted from silica by scraping it and extracting using small volumes of the solvent system until the silica was rendered completely colorless. The pooled extracts were concentrated, dried and then assayed for its anti-QS activity by agar well diffusion method. The TLC band which demonstrated anti-QS potential was labelled as F-b and was subjected to GC-MS analysis.

GC-MS Analysis: The F-b was dissolved in methanol and then subjected to GC-MS analysis at Sophisticated Analytical Instruments facility, IIT Powai, Mumbai. The instruments used for GC and MS were Agilent 7890 and Joel Accu TOF GCV respectively. The carrier gas was helium and a flow rate of 1ml/min was used. The length and internal diameter of HP5 column were 30 mm and 0.32 mm and the film were 0.25 mm. 5 ml of the sample was injected in split mode with injector split ratio 1:10. The temperature used was 80 °C to 200 °C at a rate of 8°/min and hold for 1 min. The temperature was raised to 275 °C at a rate of 8°/min and hold for 3 min. The temperature was then raised to 280 °C at the rate of 5°/min and hold for 5 min. The complete elution time was 35 min. Identification of compounds was done by comparing spectra of unknown compounds with known compounds via library search with HPCHEM software.

QS Inhibition in S. marcescens:

Prodigiosin Assay: 1% inoculum was added to a nutrient broth containing different concentrations of F-b (252-1020 μg/ml) and incubated overnight. After incubation, the cells were pelleted by centrifugation. The supernatant was discarded, and the pellet was re-suspended in 1 ml of 4% 1 M HCl for extracting prodigiosin from the bacterial cells. The mixture was again centrifuged to remove cell debris. The supernatant was collected, and its absorbance was read at 534 nm. ⁶ The relative prodigiosin concentration per cell was calculated ¹⁸ as:

Relative Prodigiosin concentration per cell =   A₅₃₄ ml^-1  /OD ₆₀₀ unit

Swarm Assay: 5 ml of an overnight culture of S. marcescens was inoculated on dried 0.5% LB agar plates containing different concentrations of F-b (252-1020 μg/ml). The plates were incubated for 24 h and observed for swarming pattern changes against the control plate ¹⁹.

Biofilm Formation Assay: Effect of F-b on biofilm formation by S. marcescens was quantified using crystal violet. 1% inoculum was added to LB broth and 100 ml of this freshly inoculated broth was transferred to the wells of the microtiter plate. Different concentrations of F-b (252-1020 μg/ml) were then added to the wells and incubated overnight. After the completion of incubation, the planktonic cells were removed by washing with sterile water twice and biofilm was stained with 0.4% crystal violet. After two minutes, excess stain was removed by using sterile water. The cells bound to crystal violet were solubilized in 1 ml of ethanol, and its absorbance was read using spectrophotometer at 570nm. ²⁰

Air-Liquid Interface Assay: This assay was performed for microscopic analysis of bacterial cells forming biofilm in the presence of F-b. Briefly, a sterile 24-well microtiter plate was adjusted at an angle of 30 - 50° and inoculated with 250 μl broth containing 0.1% inoculum of S. marcescens. Different concentrations of F-b (252-1020 μg/ml) were then added to the wells and incubated overnight in the angled position. After incubation, spent medium was removed, followed by washing twice gently with sterile medium. 200 μl of medium was then added to the wells and plate was laid on the flat surface of an inverted microscope to visualize the cells ²¹ using suitable controls.

QS Inhibition in P. mirabilis:

Swarm Assay: 5 μl of an overnight culture of P. mirabilis was inoculated on dried 1.5% LB agar plates containing different concentrations of F-b (133, 333, 533 and 733 μg/ml). The plates were incubated for 24 h and observed for changes in swarming pattern against a suitable control.

Urease assay: Effect of F-b (133-733 μg/ml) on urease production was analyzed by determining the amount of un-hydrolyzed urea in urea-LB broth cultures (filter sterilized urea was added to LB broth with a final concentration of 0.5 mg/ml). Urea-LB broth culture containing bioactive principle was inoculated with 2% overnight culture of P. mirabilis and incubated for 48 h at 37 °C. The amount of urea remaining was estimated to ascertain the urease activity using suitable control ⁹.

Biofilm Formation Assay: Effect of the bioactive compound on biofilm formation by P. mirabilis was quantified using crystal violet. 1% inoculum was added to LB broth and 100 μl of freshly inoculated broth was transferred to the wells of a microtiter plate. Different concentrations of F-b (133-733 μg/ml) were then added to the wells and incubated overnight. After the completion of incubation, the planktonic cells were removed by washing twice with sterile water and biofilm was stained with 0.4% crystal violet. After two minutes, excess stain was removed with the help of sterile water. The cells bound to crystal violet were solubilized in 1 ml of ethanol and its absorbance was read using spectrophotometer at 570nm. ²⁰

Air-Liquid Interface Assay: Briefly, a sterile 24-well microtiter plate was adjusted at an angle of 30 – 50º and inoculated with 250 μl broth containing 0.1% inoculum of P. mirabilis. Different concentrations of F-b (133-733 μg/ml) were then added to the wells and incubated overnight in the angled position. The spent medium was removed after incubation, followed by washing gently twice with sterile medium. 200 μl of medium was then added to the wells and plate was laid on the flat surface of an inverted microscope to visualize the cells ²¹ using suitable controls.

Bacterial Growth Assay: The bacterial growth was monitored in the presence of F-b using growth curve assay described by Hall et al., ²² with few modifications. 0.1% of overnight cultures of P. mirabilis and S. marcescens were inoculated into sterile broth tubes containing different concentrations of F-b. 100 μl of this freshly inoculated broth was transferred to the different wells microtiter plate. The plates were covered, and immediately the absorbance of the plate was read at 600 nm using plate reader. The plates were now transferred to shaker incubator, and OD₆₀₀ was read at 2, 4, 6, 8 and 24 h. The readings were recorded and compared with control.

Statistical Analysis: All the experiments were accomplished in triplicates. The results are calculated and expressed as mean ± standard deviation. The statistical significance was evaluated using Student’s t-test (p<0.05) and ANOVA using Microsoft Excel (version 16.4.1).

RESULTS AND DISCUSSION: QS in S. marcescens and P. mirabilis is a population density-dependent phenomenon that regulates expression of virulence factors. It directs the production of antibiotics and exoenzymes, swarming, adhesion to the surfaces ²³ and biofilm formation ²⁴. Thus, disruption of QS might prove to be a contemporary method of controlling virulence and pathogenesis in S. marcescens and P. mirabilis.

Preliminary Phytochemical Analysis: Preliminary phytochemical analysis revealed the presence of various phytoconstituents as shown in Table 2. Presence of flavonoids was detected in petroleum ether extract (PX), ethyl acetate extract (EX), acetone extract (AX) and methanolic extract (MX). PX, chloroform extract (CX), EX and AX also demonstrated the presence of alkaloids. The occurrence of phenols and saponins was observed in all extracts. The presence of tannins, flavonoids, alkaloids, triterpenoids, and saponins has been previously reported in plants belonging to the Euphorbia genus ^{12, 25}. Several reports suggest that phytochemicals can restrain QS ²⁶. Various phenolics, tannins, stilbenes, flavonoids, diarylheptanoids, triterpenoids, sulfur-containing phytochemicals, coumarins, quinones, and alkaloids have been described to possess QS inhibitory potential ^{27, 28}. Thus, phytoconstituents detected in extracts of E. trigona Mill. might be responsible for its QS inhibitory properties.

TABLE 2: PRELIMINARY PHYTOCHEMICAL ANALYSIS OF EUPHORBIA TRIGONA EXTRACTS

+: Phytochemical Present; -: Phytochemical absent

Analysis of Anti-QS Activity of Crude Extracts Against S. marcescens, Isolation of Bioactive Principle and Preparative TLC of Bioactive Principle: Quorum quenching potential of crude extracts of PX, CX, EX, AX, and MX was evaluated using S. marcescens as indicator strain ^{19, 29}. The work presented here reports the novel use of S. marcescens as indicator strain to identify QS inhibitor(s) from terrestrial plant E. trigona Mill. by agar well diffusion method. Synthesis of Prodigiosin pigment in S. marcescens is regulated by QS ⁷. PX, CX, and AX demonstrated QS inhibitory potential, as no prodigiosin synthesis was observed around the wells in the plates, while EX showed no such effect as shown in Fig. 1. MX (as shown in Fig. 1) was found to enhance synthesis of pigment prodigiosin, in turn demonstrating QS augmenting potential. The activity of crude extracts is graphically represented in Fig. 2. Several extracts of plants like K. Africana ³⁰, T. chebula ³¹, P. nigrum, P. betle, G. gnemon ³², C. verum ³³ and many others have exhibited anti- QS potential.

FIG. 1: AGAR WELL DIFFUSION ASSAY AGAINST S. MARCESCENS TO DETECT TO QUORUM SENSING INHIBITORY POTENTIAL OF CRUDE EXTRACTS

FIG. 2: EFFECT OF CRUDE EXTRACTS ON PIGMENT INHIBITION IN S. MARCESCNES. The error bars shown indicate that the experiments are carried out in triplicates and p<0.001 as compared with control

Crude PX, CX, and AX demonstrating QS inhibitory potential were subjected to column chromatography. Different fractions of column chromatography of each extract were collected, concentrated and examined for QS inhibitory property using S. marcescens as an indicator organism. PX yielded 12 fractions (named as PXF1 – PXF12) while CX and AX both yielded 13 fractions each (named as CXF1 – CXF13 and AXF1 – AXF13 respectively) when subjected to column chromatography. The effect of these fractions of column chromatography of PX, CX, and AX are shown in Fig. 3.

Those fractions that inhibited prodigiosin synthesis in S. marcescens were subjected to TLC. R_f values of bands obtained on TLC plates were calculated, and each of the bands was scraped, extracted and then checked for its anti - QS activity against S. marcescens using agar well diffusion assay. Fraction PXF5, CXF11, and AXF12 demonstrated the presence of common bands on TLC and also showed QS inhibitory potential. Various bands obtained of PXF5, CXF11 and AXF12 on TLC, their R_f values are shown in Fig. 4.

Only a single band each from PX, CX, and AX (as indicated by the blue rectangle), with common R_f value showed inhibition of prodigiosin synthesis in S. marcescens and hence this band was selected and named as F-b for further assays.

GC-MS Analysis: GC-MS analysis of F-b in methanol yielded a total 12 peaks, of which 4 were major peaks and the rest of them were minor peaks. Comparison of mass spectra of peaks with the database led to the identification of various compounds present in F-b. Remarkably, compounds detected in F-b also displayed bio-activities. The details of compounds identified, nature of the compound, molecular weight, molecular formula, retention time, peak area (%), NIST match factor are as indicated in Table 3. Reported biological activities/functions of these compounds are shown in Table 4.

TABLE 3: COMPOUNDS IDENTIFIED FROM F-B BY GC-MS ANALYSIS

TABLE 4: BIOLOGICAL ACTIVITY/FUNCTIONS OF COMPOUNDS IDENTIFIED BY GC-MS ANALYSIS OF F-B

GC-MS analysis of F-b revealed the presence of various fatty acids and their derivatives. 9,12-Octadecadienoyl chloride (Z, Z)-, was found to be the highest component, 54.82%, in F-b followed by Hexadecanoic acid, 2 - hydroxyl - (1 - hydroxymethyl) ethyl ester 9.12%, oleic acid 8.42% and 7 - Methyl - Z - tetradecane - 1 - ol acetate 7.82%. Different peaks obtained by GC-MS analysis of F-b can be seen in Fig. 5. The mass spectra of different compounds identified are shown in Fig. 6.

FIG. 5: GC-MS CHROMATOGRAM OF F-B DISSOLVED IN METHANOL

FIG. 6: MASS SPECTRA OF COMPONENTS IDENTIFIED IN F-B DISSOLVED IN METHANOL

Various components of F-b have been reported to possess antibacterial properties as well as QS inhibitory properties. 9, 12-Octadecadienoyl chloride (Z, Z)- [derivative of linoleic acid], present in the highest concentration, might be responsible for QS inhibition of virulence factors seen in S. marcescens and P. mirabilis. Linoleic acid has been reported to possess antimicrobial activity in addition to bacteriostatic functions ⁴⁷. Linoleic acid and oleic acid have also been earlier reported as QS inhibitors against Vibrio harveyi and Escherichia coli ⁴⁸. Inhibitory action of fatty acids is a result of inhibition of FabI, in turn inhibiting fatty acid synthesis in bacteria ⁴⁹. Work presented in this research paper reports fatty acids and their derivatives as QS inhibitors isolated from E. trigona Mill. against S. marcescens and P. mirabilis responsible for causing nosocomial infections. As per our knowledge, it is one of the primary reports of fatty acids and their derivatives as QS inhibitors from the terrestrial succulent plant. Hence, the identified factors might be useful to develop drugs for effective infection control and elimination of pathogens.

Prodigiosin Assay: QS regulates the synthesis of secondary metabolite-prodigiosin in S. marcescens ⁷. When the effect of F-b was studied on prodigiosin synthesis in S. marcescens, it was found that prodigiosin synthesis decreased in a dose-dependent manner as shown in Fig. 7. The highest inhibition was seen at a concentration of 1020 μg/ml demonstrating the least prodigiosin synthesis of 24.66%.

The concentrations of 252 μg/ml, 508 μg/ml and 764 μg/ml resulted in 52.05%, 45.21% and 35.62% of prodigiosin synthesis by bacteria. Similar results were also observed in QS inhibition studies of plants like Cinnamomum verum ³³ and Anethum graveolens ²⁰ which resulted in 38.3% and 29% of prodigiosin synthesis in the bacterium, indicating effectivity of F-b in inhibiting prodigiosin synthesis. Thus, F-b was effective in inhibiting prodigiosin synthesis, in turn, influencing QS in S. marcescens.

FIG. 7: EFFECT OF F-B ON PRODIGIOSIN SYNTHESIS IN S. MARCESCNES. All the experiments were performed in triplicates and *p<0.001.

Urease Assay: Urease is a nickel metalloenzyme produced by P. mirabilis. Urea is hydrolyzed by enzyme urease to yield ammonia and carbon dioxide, subsequently increasing local pH of urine. This increased alkaline pH ensues precipitation of calcium and magnesium ions, consequently forming apatite (calcium phosphate) and struvite (magnesium ammonium phosphate), i.e., urinary stones, respectively ⁸. Stone formation and alkaline pH facilitates the bacteria to adhere to cell surfaces of host and form a biofilm. Besides, stone formation benefits the bacteria as it protects the host immune system. It also triggers ammonia toxicity, the direct damage of tissues and blocking of ureters ⁵⁰.

FIG. 8: EFFECT OF F-B ON UREASE ACTIVITY OF P. MIRABILIS. Error bars indicate that the experiment was performed in triplicate and *p<0.01 concerning control

Study of the effect of F-b on the urease activity of P. mirabilis yielded in steadily decreased urease activity with increasing concentration of F-b as seen in Fig. 8. Urease activity was found to be 89.25%, 86.46%, 86.01% and 84.69% at concentrations of 133, 333, 533 and 733µg/ml respectively. There was a decrease of urease activity at a concentration of 133µg/ml followed by a gradual reduction in enzyme activity at increased concentrations of F-b.  Inhibition of urease activity has also been revealed by the study of the effect of allicin from garlic ⁵¹, and antibacterial agents like fluoroquinolones ⁵². Thus, inhibition of urease activity would prevent struvite and apatite formation and accordingly benefit in the elimination of pathogens by the host immune system.

Swarm Assay: Swarming in bacteria is a multicellular behavior characterized by movement over surfaces using rotating flagella ⁵³. Swarming motility assists bacteria in better proliferation by mechanisms like the production of antimicrobial surfactants, prevention of engulfment by macrophages, toxin production ⁵⁴, biofilm formation and invading eukaryotic cell ⁵⁵.

Work reported here establishes concentration-dependent inhibition of swarming in S. marcescens. Swarm diameter was reduced to 5.00 cm, i.e. 61.54% at a concentration of 1020 µg/ml concerning control. Effect of F-b on swarming is graphically represented in Fig. 9. Swarm diameter was found to be 7.70 cm, 6.68 cm and 6.00 cm signifying 94.77%, 82.15% and 73.85% swarming at concentrations of 252 μg/ml, 508 μg/ml and 764 μg/ml, thus indicating a consistent dose-dependent reduction. Studies conducted using Plectranathus ambonicus ⁵⁶, Capparis spinosa ⁵⁷ and petroselinic acid ⁵⁸ have similarly demonstrated swarm inhibition in S. marcescens.

FIG. 9: GRAPHICAL REPRESENTATION OF EFFECT OF F-B ON SWARMING IN S. MARCESCENS. Experiments were performed in triplicates and p<0.001.

Swarming in P. mirabilis is unique with a distinctive bull’s eye pattern ⁸. The current study analyses change in swarming pattern of P. mirabilis in the presence of F-b. The changes in swarm pattern of P. mirabilis are represented in Fig. 10. As the concentration of F-b increased, the no. of swarm rings formed by bacterium decreased accompanied by a reduction in swarm diameter. The decline in swarm diameter was gradual, but the no. of swarm rings reduced to 50% at the highest concentration of 733 μg/ml. Swarm inhibition and changes in swarm diameter have also been previously documented in work conducted using thioridazine ⁵⁹, bacteriocins from probiotic bacteria ⁶⁰, lauric acid, myristic acid and palmitic acid ⁶¹.  F-b was more effective in inhibiting swarming of S. marcescens. While F-b inhibited swarming in P. mirabilis, it decreased the ability to form more swarm rings, a representative attribute of QS and virulence. Thus, additional insight into the process at the molecular level might facilitate a better understanding of inhibition observed.

FIG. 10: EFFECT OF F-B ON SWARMING IN P. MIRABILIS. A: GRAPHICAL REPRESENTATION OF EFFECT OF F-B ON SWARMING IN P. MIRABILIS. EXPERIMENTS WERE PERFORMED IN TRIPLICATES AND *P<0.005. B: CONTROL SWARM PLATE OF P. MIRABILIS. C: SWARM INHIBITION PLATE OF P. MIRABILIS CONTAINING 733 μg/ml OF F-B

Biofilm Formation: Pathogenic bacteria adhere to surfaces and propagate to form a biofilm. Formation of biofilm results in altered gene expression as well as modified phenotype. Development of biofilm provides an advantage of the decreased vulnerability to antibiotics ³³ and increased ability to survive against host defenses. Consequently, biofilm formation on medical devices such as catheters and implants result in long-term or persisting infections. Hence, inhibiting biofilm formation would help in the control of bacterial pathogenesis.

S. marcescens forms a biofilm of undifferentiated cells arranged as micro-colonies, regulated by QS ⁶². Biofilm formation reduced with increasing concentration of F-b. Least biofilm formation was observed to be 61.61% at a concentration of 1020 μg/ml while, the concentrations of 252 μg/ml, 508 μg/ml and 764 μg/ml resulted in the formation of 88.89%, 86.11% and 79.17% biofilm concerning control as shown in Fig. 11.

The results observed were coherent with the QS inhibition studies of plants like Vitex trifolia ⁶³, Cinnamomum verum ³³ and Anethum graveolens ²⁰, thus indicating that F-b was able to reduce the biofilm formation ability of S. marcescens.

FIG. 11: EFFECT OF F-B ON BIOFILM FORMATION

(A) GRAPHICAL REPRESENTATION OF BIOFILM FORMATION IN S. MARCESCENS IN PRESENCE OF F-B. ERROR BARS INDICATE THAT EXPERIMENTS HAVE BEEN PERFORMED IN TRIPLICATE (#P<0.02, $P<0.007, ^P<0.01 AND & P<0.005). (B) GRAPHICAL REPRESENTATION OF BIOFILM FORMATION IN P. MIRABILIS IN PRESENCE OF F-B ERROR BARS INDICATE THAT EXPERIMENTS HAVE BEEN PERFORMED IN TRIPLICATE (*P<0.001)

P. mirabilis forms crystalline biofilms, which is mushroom-shaped. These crystalline biofilms instigate various problems like kidney and bladder stones, further worsening the infection ⁶⁴. Biofilm formation is also regulated by QS; hence, the effect of F-b was analyzed on biofilm formation in P. mirabilis. 733 μg/ml diminished the biofilm formation to 52.17%, while 133, 333 and 533 μg/ml reduced biofilm formation to 69.57%, 56.52%, and 56.52% respectively as depicted in the Fig. 11. F-b actively inhibited biofilm formation in P. mirabilis demonstrating the potential of fatty acid and their derivatives to interfere with QS. Investigations made by Liaw et al., on the effect of fatty acids- stearic acid and palmitic acid also demonstrate their QS inhibitory potential ⁶¹. Likewise work described by Widmer et al., also establishes the QS inhibitory nature of linoleic acid mediated by AI - 2 inhibition ⁴⁸. Thus, the presence of fatty acids and their derivatives in F-b corroborates its QS inhibitory potential.

Growth Curve Assay: Growth curve analysis was performed to assess whether changes seen in QS-regulated properties of S. marcescens and P. mirabilis was a consequence of QS inhibition by F-b and not its antibacterial activity. Analysis indicated no significant change in cell densities at 24 h when compared to control, as shown in Fig. 12 and Fig. 13, indicating that changes in various virulence factors observed ensued from QS inhibition by F-b.

Air-Liquid Interface Assay: Observation of biofilm of S. marcescent and P. mirabilis under the inverted microscope in the presence of F-b (1020 μg/ml and 733 μg/ml) revealed diminished biofilm when compared to control Fig. 14. This observation further ascertains the QS inhibitory capacity of F-b.

FIG. 14: AIR LIQUID INTERFACE ASSAY OF BIOFILM

A. CONTROL BIOFILM OF MARCESCENS

B. MARCESCENS BIOFILM IN PRESENCE OF 1020 μg/ml OF F-B

C. CONTROL BIOFILM OF MIRABILIS

D. MIRABILIS BIOFILM IN PRESENCE OF 733 μg/ml OF F-B

As 9,12-Octadecadienoyl chloride (Z, Z)- [chloride derivative of linoleic acid] formed the primary component of F-b, hence, linoleic acid was used to assess QS inhibition, by analyzing swarm pattern changes in S. marcescens and P. mirabilis.

FIG. 15: EFFECT OF LINOLEIC ACID ON SWARMING IN S. MARCESCENS. A: GRAPHICAL REPRESENTATION OF EFFECT OF LINOLEIC ACID ON SWARMING OF S. MARCESCENS. ERROR BARS INDICATE THAT THE EXPERIMENT WAS PERFORMED IN TRIPLICATES AND P<0.001. B: CONTROL - SWARM PLATE OF S. MARCESCENS. C: SWARM PLATE OF S. MARCESCENS CONTAINING 3500 μg/ml OF LINOLEIC ACID

Concentrations of 1400, 2100, 2800 and 3500 μg/ml of linoleic acid were used to analyze its swarm inhibitory potential. The effect of linoleic acid on swarm motility of S. marcescens and P. mirabilis is represented in Fig. 15 and 16. It was more effective in inhibiting swarm motility of S. marcescens than P. mirabilis. Linoleic acid reduced swarming to 53.06% at 1400 μg/ml and 28.03% at 3500 μg/ml in S. marcescens. The pattern of anti-swarm ability of linoleic acid was comparable to that of F-b, however, F-b was effective in bringing about QS inhibition as evident from the concentrations used for investigation, implying higher QS inhibitory ability of fatty acid derivatives.

FIG. 16: EFFECT OF LINOLEIC ACID ON SWARMING OF P. MIRABILIS. Error bars indicate that the experiment was performed in triplicates and p<0.05

CONCLUSION: The work presented here depicts QS inhibitory effects of fatty acid derivatives on S. marcescens and P. mirabilis isolated from terrestrial plant E. trigona Mill. While the exact mechanism of QS inhibition by fatty acids or their derivatives is not known, this study paves the way for developing drugs, containing fatty acid derivatives and promulgating the importance of lipids. Further, analysis of the interaction of fatty acid derivatives with different receptors and its effect on various genes of bacteria might furnish a better understanding of impeding bacterial pathogenesis and contribute to better treatment of infections.

ACKNOWLEDGEMENT: We would like to acknowledge CSIR for funding the study in the form of Fellowship [File no. 08/611(0001)2013-EMR-I]. Further, we would also like to express our gratitude to IIT-POWAI SAIF for helping us with GC-MS analysis.

CONFLICT OF INTEREST: No Conflict of Interest.

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    Marathe K, Nashikkar N, Bundale S and Upadhyay A: Analysis of quorum quenching potential of Euphorbia trigona Mill. Int J Pharm Sci & Res 2019; 10(3): 1372-86. doi: 10.13040/IJPSR.0975-8232.10(3).1372-86.

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