Biosynthesis of Glycomonoterpenes to Attenuate

0 downloads 0 Views 8MB Size Report
XI. Function of mite body as geometrical isomerization and reduction of citral (the alarm pheromone). Carpoglyphus lactis. Applied Entomology and Zoology, 18, ...
Appl Biochem Biotechnol DOI 10.1007/s12010-016-2300-8

Biosynthesis of Glycomonoterpenes to Attenuate Quorum Sensing Associated Virulence in Bacteria Amrita Patil 1 & Kasturi Joshi-Navre 1 & Ruchira Mukherji 1 & Asmita Prabhune 1

Received: 25 May 2016 / Accepted: 20 October 2016 # Springer Science+Business Media New York 2016

Abstract The acquisition of multidrug resistance in bacteria has become a bigger threat of late, mainly due to the bacterial signaling phenomenon, quorum sensing (QS). QS, among a population of bacteria, initiates the formation of biofilms and offers myriad advantages to bacteria. Burgeoning antibiotic resistance in biofilm-producing bacteria has motivated efforts toward finding new alternatives to these traditional antimicrobials. In the present study, we report the increased solubility and additional quorum quenching as well as biofilm disruption activity of glyco-derivatives of monoterpenes (citral and citronellal). Glycomonoterpenes of citral and citronellal were synthesized via conjugation of the monoterpenes with glucose by the non-pathogenic yeast Candida bombicola (ATCC 22214). Structural elucidation of newly synthesized glycomonoterpenes showed that one synthesized using citronellal contains three major lactonic forms with molecular weight 492.43, 473.47, and 330.39 Da whereas the one produced using citral has an acidic form with molecular weight 389.33 and 346.23 Da. The glycomonoterpenes were able to individually inhibit QS, mediated through various medium-chain and long-chain N-acyl homoserine lactones (AHLs). These new compounds are interesting additions to the known range of quorum sensing inhibitors (QSIs) and could be further explored for potential clinical applications. Keywords Citral . Citronellal . Glycomonoterpenes . Quorum sensing inhibition

Introduction In recent years, an increasing number of bacterial strains have developed resistance to various commonly used antibiotics, due to genetic, social, and environmental factors, leading to the Electronic supplementary material The online version of this article (doi:10.1007/s12010-016-2300-8) contains supplementary material, which is available to authorized users.

* Asmita Prabhune [email protected]

1

Biochemistry Division, CSIR, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India

Appl Biochem Biotechnol

predominance of multiple drug-resistant (MDR) and extremely drug-resistant (XDR) bacteria [1]. The WHO’s 2014 report on global surveillance of antimicrobial resistance mentioned that the world is heading toward a post-antibiotic era in which infections which were easily curable for decades could once again cause havoc [2]. The biofilm forming ability of bacteria could be considered one of the major reasons behind the problem of antibiotic resistance. Biofilm is a unique multicellular structure where bacteria are embedded in self-secreted extracellular polymeric matrix or proteinaceous material, thus rescuing them from antimicrobial agents, environmental stresses, and the host immune system [3]. Biofilm-forming bacteria are more resistant to antibiotics as compared to their planktonic counterparts and are responsible for a range of problems related to human health and the shipping and aquaculture industries. As biofilms are recalcitrant in nature, they mainly result in chronic infections. Various human infections such as osteomyelitis, endocarditis, chronic and gastrointestinal ulcers, cystic fibrosis, periodontitis, and urinary tract infections have been shown to arise due to biofilms [4, 5] Bacteria can form biofilm on both biotic and abiotic surfaces, and hence, tooth surfaces, medical devices, and transplants are prone to biofilm formation [4, 5]. The formation and maintenance of biofilms depend on quorum sensing (QS)-dependent gene expression [6]. Quorum sensing is essentially the ability of bacteria to communicate with other bacteria. It involves small molecular weight easily diffusible signal molecules usually known as autoinducers which enable bacteria to sense their population density and to control the gene expression in response to the cell density [7]. QS is known to regulate a wide variety of bacterial functions such as bioluminescence, nitrogen fixation, expression of virulence factors, biofilm formation, and swarming motility which has been shown to contribute to bacterial pathogenesis [8]. Targeting QS pathways in bacteria could be an effective strategy to circumvent the problem of multiple drug resistance in bacteria. QS in bacteria can be impeded at several stages which ultimately might enable us to inhibit pathogenicity [6, 9]. Quorum sensing inhibitors (QSIs) are molecules that can incapacitate pathogenic bacteria by exclusively interrupting the QS mechanism without putting selective pressure on their growth [6, 9] and thus making them different from antibiotics. [6, 9–11]. There are three major bacterial quorum sensing mechanisms: (i) LuxI/LuxR of Gramnegative bacteria, dependent on N-acyl homoserine lactone (AHL) signals; (ii) the LuxS-based system present in both Gram-positive and Gram-negative bacteria; and (iii) an oligopeptidedependent two-component system exclusive to Gram-positive bacteria. Recently, essential oils have been reported as quorum sensing inhibitors [12–15], but the hydrophobic nature of these essential oils imposes major problem in exploring their real potential. Citral and citronellal are two major monoterpenes present in many essential oils which are easily available. Citral (3,7-dimethyl2,6octadienal or lemonal), is a major component in various citrus oils. Citronellal (3,7dimethyl-oct-6-en-1-al or rhodinal) is the major component of citronellal oil and gives the oil its distinctive lemon scent. Both citral and citronellal possess antimicrobial and insect-repellent activities [16–20]. These monoterpenes are highly hydrophobic in nature, hence their bioavailability is low. The rationale behind our work was to increase the solubility of the parent compounds (citral and citronellal) by converting them to glycomonoterpenes and then to assess their anti-QS activity in terms of inhibition of short- and long-chain AHL-dependent signaling. AHL, a signal molecule present in Gram-negative bacteria, has a homoserine lactone ring linked to either a short (C < 8) or a long (C ≥ 8) acyl chain through an amide bond. The short-chain AHL passively diffuses through bacterial membranes, and the long-chain AHL signals require active transportation mechanisms for their efflux [8].

Appl Biochem Biotechnol

Materials and Methods Bacterial Strains and Culture Conditions Synthesis of glycomonoterpenes (G-citral, synthesized from monoterpene citral, and G-citron, synthesized from monoterpene citronellal) was done using Candida bombicola ATCC 22214, as described below. Biosensor strains used for detection of QSI activity include Chromobacterium violaceum CV026 (a kind gift from Dr. Paul Williams, University of Nottingham), a mini-tn5 mutant of wild-type C. violaceum, and Agrobacteruim tumefaciens NTL4(pZLR4) (kindly given by Dr. Stephen Farrand, University of Illinois, USA). A. tumefaciens strain NTL4 is a non-pathogenic derivative of strain C58. Plasmid pZLR4 contains two key units, the traR gene and a traCDG::lacZ fusion.CV026 was grown in LuriaBertani (LB) medium supplemented with 100 μg/ml ampicillin and 30 μg/ml kanamycin at 28 °C. A. tumefaciens NTL4(pZLR4) was grown in nutrient broth with 30 μg/ml gentamicin at 28 °C. To study the antibiofilm activity of glycomonoterpenes, Pseudomonas aeruginosa NCIM 5029 and Vibrio cholerae MTCC 0139 were used. Protease inhibition and pyoverdine inhibition of glycomonoterpenes were studied against P. aeruginosa NCIM 5029 cultured in Kings’ B medium at 37 °C.

Synthesis of Glycomonoterpenes from Monoterpenes A pre-inoculum of C. bombicola ATCC 22214 in 10 ml malt extract, glucose, yeast extract and peptone (MGYP) broth (24 h at 28 °C and 180 rpm) was transferred to 90 ml MGYP broth and incubated for another 48 h. Cells were harvested by centrifugation at 5000 rpm for 10 min and then transferred to 100 ml production medium of 10% (w/v) glucose. Monoterpenes citral (purity 96%) and citronellal (purity 95%, HiMedia) were added (0.4% v/v) to the production medium as substrate. Flasks were incubated at 28 °C and 180 rpm for 7 days for the synthesis of respective glycomonoterpenes [12]. After harvesting the cell mass, supernatants from each flask were extracted thrice with an equal volume of ethyl acetate. The product was concentrated by rotary evaporation and further purged to remove any traces of solvent [12]. Synthesized glycomonoterpenes (G-citral and G-citron) were then characterized using different analytical techniques.

Oil Displacement Activity of Synthesized Glycomonoterpene Oil displacement is a qualitative preliminary test to confirm the surfactant property of a compound. The surfactant property of glycomonoterpenes was determined by adding 0.5 mg/ml of G-citral and G-citron in the Pongamia oil (Millettia pinnata)-water interphase. A clear zone formed after the addition of a glycomonoterpene to an oil-water interphase was measured [21].

High-Performance Liquid Chromatography Citral and citronellal were initially analyzed on a Hitachi Chromeline HPLC system using a Thermo Scientific C18 Reversed-Phase HPLC Column 250 × 4.6 mm. A 0–100% acetonitrile gradient in water over 60 min was used, followed by column re-equilibration for 10 min. A blank run was performed between each analysis. The flow rate was maintained at 1.0 ml/min,

Appl Biochem Biotechnol

column temperature was maintained at 40 °C, and compounds were detected at 220 nm. Synthesized glycomonoterpenes G-citral and G-citron were analyzed with same method.

FTIR Analysis of Synthesized Glycomonoterpenes FTIR spectroscopic analysis of the synthesized glycomonoterpenes and respective monoterpenes was done using Bruker ATR-FTIR Spectrophotometer over the spectral range of 400– 4000 cm−1. Spectral data obtained was plotted on a graph of transmittance versus wavenumber (cm−1) [22].

MALDI-TOF Analysis of G-Citral and G-Citron G-citral or G-citron in acetonitrile (2 mg/ml) was mixed with α-cyano-4-hydroxycinnamic acid (CHCA) matrix (10 mg/ml) in a 1:5 ratio and spotted on a matrix assisted laser desorption/ ionization (MALDI) plate. MALDI-MS study was done using the AB SCIEX TOF/TOF 5800 System [23].

C. violaceum CV026-Based Violacein Inhibition Assay CV026-based violacein inhibition assay is used to check inhibition mediated through shortchain AHL [24]. In the current study, violacein production was determined to study the potential of glycomonoterpenes to inhibit quorum sensing mediated by N-hexanoyl-Lhomoserine lactone (C6-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). CV026 (0.2% inoculum from overnight culture) was inoculated in 2 ml LB broth containing 100 μg/ml ampicillin and 30 μg/ml kanamycin, along with 0.1 μl of C6-HSL (25 mM stock) and the test sample (G-citral or G-citron). Six concentrations of test samples in water were used (0.05, 0.1, 0.25, 0.5, 0.75, and 1 mg/ml). After overnight incubation at 28 °C and 180 rpm, violacein pigment produced was extracted with dimethyl sulfoxide (DMSO) and measured spectrophotometrically at 580 nm. The effect of the glycomonoterpenes on C8-HSL-mediated quorum sensing was also studied using the same method [12]. Violacein inhibition (%) was calculated in comparison with the control using the following formula [25]: Violacein inhibition ð%Þ ¼

ðcontrol OD585nm −test OD585nm Þ  100 control OD585nm

ð1Þ

A. tumefaciens NTL4 (pZLR4)-Based Pigment Inhibition Assay A. tumefaciens NTL4 (pZLR4)-based pigment inhibition assay is used to check the inhibition of quorum sensing mediated through long-chain AHL [24]. In our study, we have executed this assay to check the inhibition of quorum sensing mediated through 3-oxo-C8-HSL (N-3-oxooctanoyl-L-homoserine lactone), C10-HSL (N-decanoyl-L-homoserine lactone), and C12-HSL (N-dodecanoyl-L-homoserine lactone). The reporter system in this strain is based on βgalactosidase activity and produces a blue pigment in the presence of externally provided Xgal and a signal molecule. A. tumefaciens NTL4 (pZLR4) was inoculated (10% inoculum from overnight culture) in 1 ml NB containing 30 μg/ml gentamicin, the respective signal molecule (0.25 μg/ml), X-gal (60 μg/ml), and a test compound. Five different concentrations of sample

Appl Biochem Biotechnol

were tested (0.1, 0.25, 0.5, 0.75, and 1 mg/ml). A tube without any test compound was used as control. Incubation was done overnight at 28 °C and 180 rpm. The blue pigment produced in each treatment due to substrate degradation was extracted with DMSO and quantified at 630 nm, to determine the extent of inhibition.

Antibiofilm Activity of Glycomonoterpenes The antibiofilm activity of the glycomonoterpenes was evaluated using V. cholerae and P. aeruginosa as test organisms. Initially, 10 μl of overnight grown cultures of V. cholerae and P. aeruginosa were subcultured to petri plates (30 mm diameter) containing 2 ml LB media and a sterile coverslip. G-citral or G-citron (1 mg) was then added to the plates. After 24 h at 37 °C, biofilms formed in control and treated plates were visualized by staining with crystal violet. The stained biofilm was then visualized under light microscope at ×400 magnification [23, 26, 27]. Antibiofilm activity of glycomonoterpenes was also evaluated quantitatively using V. cholerae, P. aeruginosa, and Cronobacter sakazakii. For quantitative evaluation of antibiofilm activity, initially biofilm was stained with crystal violet and then crystal violet was extracted using 30% acetic acid. O.D. was measured at 580 nm.

Inhibition of Pyoverdine Production by P. aeruginosa Quorum sensing in bacteria also governs virulence factor production. P. aeruginosa produce pyoverdine which is a virulence factor involved in chronic infection. For assessing the inhibition of pyoverdine production by P. aeruginosa due to action (in the presence) of glycomonoterpenes,10 μl overnight grown culture of P. aeruginosa was transferred to 2 ml of King’s medium B and incubated in the presence 1 mg Gcitral or G-citron at 35 °C. Pyoverdine which is produced after 12 h was measured by recording the fluorescence of the cell-free supernatant, with excitation at 405 nm and emission at 465 nm [28].

Statistical Analysis Experiments were performed in triplicates, and the data obtained from the experiments were given as mean values. To determine if two sets of data (control and treated) are significantly different from each other, Student’s t test was used [27].

Results Oil Displacement Activity The formation of a clear zone on addition of a test compound to an oil-water interphase is the measure of surfactant activity of a compound. G-citral and G-citron showed clear zones with diameter of 2.7 and 3.2 cm, respectively (Fig. 1), whereas parent monoterpenes citral and citronellal showed no oil displacement activity. As G-citron was showing better oil displacement activity, we measured the surface tension and critical micelle concentration (CMC) of the same. We found that G-citron reduced the surface tension of distilled water from

Appl Biochem Biotechnol

Fig. 1 Oil displacement activity of synthesized glycolipid: 1 mg/ml solution of a citronellal, b G-citron, c citral, and d G-citral displacing Pongamia oil (Millettia pinnata)

71.956 ± 0.5 mN/m to a minimum value of 34.359 ± 0.5 mN/m with a CMC value of 0.07 mg/ L. This clearly confirmed the conversion of monoterpenes to glycomonoterpenes having surfactant activity (method used for surface tension and CMC measurement is mentioned in the Supplementary Material).

HPLC Analysis HPLC analysis was carried out for both the monoterpenes (citral, citronellal) and the resultant glycomonoterpenes (G-citral and G-citron). The gradient elution method was used to analyze the difference in retention time of parent and newly synthesized glycomonoterpenes. Being hydrophobic in nature, citral showed a peak at 36.4 min and citronellal at 41.2 min when the acentonitrile/water ratio was high (Fig. 2). However, G-citral and G-citron eluted in the10–30 -min range when subjected to HPLC under identical conditions. This is also indicative of a polar nature of the compound and is in agreement with TLC results where multiple bands were seen in newly synthesized compounds (for TLC results, see Supplementary Material Fig. 1).

Appl Biochem Biotechnol

mAu

mAu

mAu

b

mAu

a

Time (in minutes)

Time (in minutes)

Time (in minutes)

mAU

mAu

mAU

d

mAu

c

Time (in minutes)

Fig. 2 HPLC analysis of monoterpenes citral and citronellal and their respective glycomonoterpenes G-citral and G-citron: HPLC chromatograms of compounds with a gradient elution method, which started with 0% acetonitrile and 100% water and ended with 100% acetonitrile and 0% water over 60 min, followed by column reequilibration for 10 min. The flow rate was 1.0 ml/min, and the absorption wavelength was set at 220 nm. a Citral chromatogram showing retention time 36.39 min. b G-citral spectra showing product elution earlier to 36.39 min. c Citronellal spectra showing retention time 41.21 min. d G-citron chromatogram where peaks earlier to 41.21 min represent a newly synthesized product, i.e., G-citron

These results are in accordance with previous reports [23] where glycomonoterpene alcohols were observed to elute at lower ratio of acetonitrile to water.

FTIR Analysis of Synthesized Glycomonoterpenes Analysis of the FTIR spectrum of G-citral and G-citron clearly showed incorporation of the parent moiety into the final product. The spectra clearly showed peaks corresponding to methyl (1379 cm−1) and methylene groups (1452 cm−1) from citral and citronellal. The 3063–3640 cm−1 region corresponding to the O–H stretch frequency in the glucose moiety was also observed in the G-citral and G-citron spectra, with the asymmetrical and symmetrical stretch modes of the methylene (CH2) groups of glucose and sophorose occurring at 2857 cm−1. Presence of a C=O stretch of saturated aliphatic cyclic six-membered ring of glucose (1720 cm−1) was also evident in the spectra. The carbonyl stretch C=O of saturated aliphatic aldehydes (citronellal) appears at 1729 cm−1, and the carbonyl stretch of the unsaturated aldehyde (citral) showed a shift to a lower wavenumber, 1674 cm−1 (Fig. 3). A similar FTIR spectrum has been observed recently for glyco-monoterpene alcohol with additional peak at 1060 cm for C–O stretching from primary alcohols, which was absent in the glycomonoterpenes in the present study.

MALDI-TOF Analysis of G-Citral and G-Citron In the case of G-citron, three major compounds having m/z 510.55, 438.55, and 348.42 were observed. Theoretical calculations suggest that m/z 510.55 [M1 + NH4]+ could correspond to the lactonic form of glycolipid possessing a sophorose head group without

Appl Biochem Biotechnol

Fig. 3 FTIR spectroscopic analysis of parent monoterpenes and synthesized glycomonoterpenes: a G-citron: peak at 1379 cm−1 corresponding to methyl groups indicated presence of citronellal in G-citron. Peak at 1452 cm−1 represents presence of methylene group. The 3063–3640 cm−1 region in spectra corresponds to the O–H stretch frequency in the glucose. The asymmetrical and symmetrical stretch modes of methylene (CH2) groups of glucose and sophorose occur at 2857 cm−1. Presence of C=O stretch of saturated aliphatic cyclic sixmembered ring of glucose (1720 cm−1) was also evident in the spectrum of G-citron. b Citronellal: the carbonyl stretch C=O of saturated aliphatic aldehydes (citronellal) appears at 1729 cm−1. c G-citral: peak at 1379 cm−1 corresponding to methyl groups indicated presence of citral in G-citral. Peak at 1452 cm−1 represents presence of methylene group in G-citral. The 3063–3640 cm−1 region in spectra corresponds to the O–H stretch frequency in the glucose. The asymmetrical and symmetrical stretch modes of methylene (CH2) groups of glucose and sophorose occur at 2857 cm−1 in G-citral spectrum. Presence of a C=O stretch of saturated aliphatic cyclic six-membered ring of glucose (1720 cm−1) was also evident in the spectrum of G-citral. d Citral: carbonyl stretch of the unsaturated aldehyde (citral) showed a shift to a lower wavenumber, 1674

acetylation where M1 = 492.43 (Fig. 4c), and the compound showing a peak at 438.55 [M2 + 2Na + NH4 + H]+ could correspond to the lactonic form of glycolipid with a monoacetylated glucose head group where M2 = 414.47 (Fig. 4c). On the other hand, the peak at m/z 348.42 [M3+ NH4 ] could be the lactonic form of glycolipid having a glucose head group without acetylation where M3 = 330.39 (Fig. 4d). Thus, all major compounds in G-citron were found to possess a lactonic form of glycolipid (Table 1). In the case of G-citral, two major compounds having m/z 454.4023 [M4 + 2Na + NH4 + H]+ and 370.4219 [M5 + Na + H]+were observed (Fig.4 a, b). The molecular weight of these compounds is well correlated with the acidic form of glycolipids having a monoacetylated glucose head group (M4 = 430.44) and a glucose head group without acetylation (M5 = 346.2296) (Fig. 4a, b, Table 1). The structures obtained in the present study are comparable with recent reports [20] that glycomonoterpene alcohols can be a mixture of compounds possessing either sophorose or glucose as head group.

Appl Biochem Biotechnol

Fig. 4 MALDI-TOF analysis of G-citral and G-citron: a m/z of G-citral [M + 2Na + NH4 + H]+ where M = 430.44. b m/z of G-citral [M + Na + H]+ where M = 346.23. c m/z of G-citron [M + 2Na + NH4 +H]+ and [M + NH4]+ where M = 414.17 and 492.43, respectively. d m/z of G-citron [M + NH4] where M = 330.39

C. violaceum CV026-Based Violacein Inhibition Assay An earlier report [23] has shown that monoterpene alcohols, linalool and terpineol, acquire quorum quenching activity on biotransformation to their glyco form. The ability of glycomonoterpenes (G-citral and G-citron) and monoterpenes (citral and citronellal) to inhibit quorum sensing was studied using the C. violaceum CV026 reporter strain. QSI activity was Table. 1 Predicted structure and molecular formula of synthesized products (Supplementary Material Figs. 7 and 8) No.

1

2

Compound

Form

Structure of synthesized product

Elemental composition

Calculated mass (III)

454.4023 [M + 2Na + NH4 + 1− 1]+

Acidic product

Glucose head group + monoacetylation

C18112909

389.33

370.4219 [M + Na + 1 − 1]+

Acidic product

Glucose head group + no acetylation

C16H2608

346.2296

510.5551 [M ± NE14 ]−

Lactonic product

Sophorose head group (no acetylation)

C22H3 60 12

492.432

438.5512 [(M+ 2Na + NH4 + HI)]

Lactonic product

Glucose head group + monoacetylation

C1,14290

473.47

348.4244 [M + NH4]

Lactonic product

Glucose head group (no acetylation

C16H2607

330.39

G-citral m/z

G-citron

Appl Biochem Biotechnol

Fig. 5 Quantitative analysis of violacein inhibition in CV026 by G-citral and G-citron and effect of optimum concentration on growth of CV026: data are represented as percentage of violacein inhibition. Mean values of triplicate independent experiments and SE are shown a Violacein inhibition in the presence of C6 HSL as external signal molecule, significant at p < 0.005. b Violacein inhibition in the presence of C8 HSL as external signal molecule, significant at p < 0.005. c Effect of 0.5 mg/ml concentration of G-citron, G-citral, citronellal, and citral on growth of CV026

checked against C6 HSL and C8 HSL signal molecules. Citral and citronellal showed growth inhibition at 0.5 mg/ml (Fig. 5c). However, G-citron showed 100% pigment inhibition at the same concentration (Fig. 5a) when the C6 HSL signal molecule was added externally in the CV026 culture and at even lower concentration (0.25 mg/ml) when C8 HSL was used (Fig. 5b). G-citral completely inhibited pigment production at 0.5 mg/ml concentration, with both C6 HSL and C8 HSL (Fig. 5a, b). The reduction in pigment production was observed solely as a result of quorum sensing inhibition and not due to growth inhibition, as evidenced by the absence of any change in OD600nm (Fig. 5c) (Supplementary Material Fig. 2).

A. tumefaciens NTL4 (pZLR4)-Based Pigment Inhibition Assay A. tumefaciens NTL4 (pZLR4)-based assay was done to assess the inhibition of quorum sensing activity governed by long-chain signal molecules, viz., 3-oxo-C8-HSL, C10-HSL, and C12-HSL. The parent monoterpenes (1 mg/ml) showed growth inhibition whereas the same concentration of G-citral and G-citron did not affect bacterial growth (Fig. 6d). G-citral (0.75 mg/ml) showed complete inhibition of pigment production when 3-oxo-C8-HSL was added as external signal molecule. In the case of C10-HSL and C12-HSL, 0.75 mg/ml concentration of G-citral was enough to achieve more than 90% pigment inhibition, but to attain complete pigment inhibition, 1 mg/ml was required (Fig.6 b, c). Interestingly, we observed that 0.75 mg/ml concentration of G-citron could achieve more than 95% pigment inhibition with 3-oxo-C8-HSL as the signal, but a concentration of 1 mg/ ml was required for complete inhibition (Fig.6 a). However, 0.75 mg/ml concentration of G-citron was enough to achieve complete pigment inhibition when C10-HSL and C12-HSL signals were used (Fig.6 b, c) (Supplementary Material Fig. 3).

Appl Biochem Biotechnol

Fig. 6 Quantitative analysis of pigment inhibition in A. tumefaciens NTL4(pZLR4) by G-citral and G-citron and effect of optimum concentration on growth of A. tumefaciens NTL4(pZLR4): data are represented as percentage of pigment inhibition. Mean values of triplicate independent experiments and SE are shown. a Pigment inhibition in the presence of 3-oxo-C8-HSL as external signal molecule, significant at p < 0.005. b Blue pigment inhibition in the presence of C12-HSL as external signal molecule, significant at p < 0.005. c Blue pigment inhibition in the presence of C10-HSL as external signal molecule, significant at p < 0.005. d Effect of 1 mg/ml concentration of G-citron, G-citral, citronellal, and citral on growth of A. tumefaciens NTL4(pZLR4)

Antibiofilm Activity of Glycomonoterpenes Using V. cholerae and P. aeruginosa as Test Organisms The formation of bacterial biofilms is largely governed by quorum sensing. Countering bacteria that possess the capacity to form biofilms is of prime significance in treating chronic infections. In this context, the antibiofilm activity of glycomonoterpenes (G-citral and G-citron) was assessed by using V. cholerae (MTCC 0139) and P. aeruginosa (NCIM 5029) as test organisms. Both G-citral and G-citron showed remarkable antibiofilm activity after 24 h at 1 mg/ml concentration (Figs. 7 and 8). Quantitative antibiofilm assay showed that 0.25 mg/ml G-citron and 0.5 mg/ml G-citral were sufficient to achieve more than 50% biofilm inhibition when tested against P. aeruginosa (Supplementary Material Fig. 4). As G-citron showed better antibiofilm activity, we also quantified antibiofilm activity of G-citron against V. cholerae and C. sakazakii (food pathogen) (Supplementary Material Fig. 5).

Inhibition of Pyoverdine Production by P. aeruginosa Pyoverdine is a siderophore produced by P. aeruginosa (NCIM 5029) that is linked to pathogenesis. G-citral and G-citron (1 mg/ml) could achieve 65% pyoverdine inhibition as compared to control (Fig. 9 and Supplementary Material Fig. 6).

Appl Biochem Biotechnol

Fig. 7 Inhibition of biofilm formation by P. aeruginosa (8 h static incubation at 37 °C). a Control biofilm in the absence of an inhibitor. b Biofilm formed in the presence of G-citral. c Biofilm formed in the presence of G-citron inhibition of biofilm formation by P. aeruginosa (24 h static incubation at 37 °C). d Control biofilm in the absence of an inhibitor. e Biofilm formed in the presence of G-citral. f Biofilm formed in the presence of G-citron (biofilms were stained with 0.1% crystal violet and were observed under light microscope ×400 magnification, and scale bar is 200,000 nm)

Fig. 8 Inhibition of biofilm formation by V. cholerae (8 h static incubation at 37 °C). a Control biofilm in the absence of an inhibitor. bBiofilm formed in the presence of G-citral. cBiofilm formed in the presence of G-citron. Inhibition of biofilm formation by V. cholerae (24 h static incubation at 37 °C). d Control biofilm in the absence of an inhibitor. eBiofilm formed in the presence of G-citral. f Biofilm formed in the presence of G-citron (biofilms were stained with 0.1% crystal violet and were observed under light microscope ×400 magnification, and scale bar is 200,000 nm)

Appl Biochem Biotechnol Fig. 9 Pyoverdine inhibition in P. aeruginosa by G-citral and Gcitron: fluorescence was taken at excitation wavelength 405 nm and emission wavelength of 465 nm and represented as percentage of pyoverdine inhibition. Mean values of triplicate independent experiments and SE are shown

Discussion A recent review on antimicrobial resistance (AMR) estimated that a rise in AMR at the current rate would account for 10 million deaths every year, thus probably costing the world up to 100 trillion USD by 2050 [29]. Antibiotic resistance can be mediated at the community level or at the cellular level. Quorum sensing (QS) is the major phenomenon through which bacteria acquire community level resistance. QS in bacteria plays a major role in biofilm formation which acts as a protective layer around bacteria. Due to this protective layer, antibiotics fail to penetrate, thus leading to antibiotic resistance. [8]. Targeting QS in bacteria does not impose a selective pressure on them, making it a promising approach to tackle the problem of multidrug resistance in bacteria [8]. Advance medical treatments such as organ transplants, joint replacements, cancer therapy, and treatment of chronic diseases such as diabetes, asthma, and rheumatoid arthritis are often combined with antibiotic doses to fight infections occurring during these treatments. Therefore, people undergoing the above treatments are at high risk, as infections caused by resistant microorganisms often fail to respond to the standard treatment of antibiotics [30]. Extensive research is therefore needed to achieve inhibition of bacterial quorum sensing and the phenotypes associated with it. In the present investigation, the QS inhibitory activity of glycomonoterpenes (Gcitral and G-citron) was assessed. It has been reported that plants produce anti-quorum sensing compounds. But it is important to find out their mode of action in order to establish whether they are a narrow or a broad spectrum. As QSIs from plant origin can specifically inhibit quorum sensing in bacteria; these QSIs can be useful in targeting only pathogenic bacteria without affecting normal microflora in the environment. Considering these features, the plant-derived quorum sensing inhibitors can be used as the next-generation drugs against infectious disease [8]. Recently, plant-derived essential oils have been reported as quorum sensing inhibitors [12–15], but biological activities of these essential oils become limited due to their hydrophobic nature. Citral and citronellal are major components of various easily available essential oils such as lemongrass and citronellal, respectively. There are two major problems in using citral and citronellal as quorum sensing inhibitors. First, these monoterpenes are highly hydrophobic, and second, they possess antibacterial activity [16–20]. Compounds having a hydrophobic nature are less bioavailable, and compounds possessing antibacterial activity cannot be used as

Appl Biochem Biotechnol

quorum sensing inhibitors as they impose selective pressure on bacteria. Therefore, to increase bioavailability and to achieve QSI activity, these monoterpenes were converted into glycomonoterpenes. Conversion of citral and citronellal to their respective glycol derivative was confirmed by doing an oil displacement activity, HPLC FTIR analyses of parent and converted compounds, respectively. Earlier work in our group has shown that glycomonoterpene alcohols can be synthesized using non-pathogenic yeast C. bombicola in the presence of oleic acid as an inducer and a newly synthesized product showed increased solubility and quorum sensing inhibitory activity [23]. In this study, we showed that C. bombicola was able to convert monoterpenes (citral and citronellal) to glycomonoterpenes (G-citral and G-citron) even in the absence of an inducer, and the converted product showed QSI activity. G-citral and G-citron significantly inhibited quorum sensing mediated through five different signal molecules—C6-HSL, C8-HSL, 3oxo-C8-HSL, C10-HSL, and C12HSL—without affecting bacterial growth (Figs.5 and 6). To the best of our knowledge, this is the first report in which the quorum sensing inhibitory activity of a compound has been checked against five different signal molecules. As G-citral and G-citron can inhibit quorum sensing mediated through five different signal molecules, it can be used to effectively combat bacterial infections. G-citral and Gcitron both inhibited biofilm formation by P. aeruginosa, V. cholerae, and C. sakazakii without affecting the growth of the bacteria, showing their efficacy as QSIs and specificity for quorum sensing systems (Figs 7 and 8). Quorum sensing also leads to production of virulence factors. Pyoverdine is a fluorescent, water-soluble siderophore produced by P. aeruginosa which competes directly with mammalian transferrin for iron and acts as a crucial element for virulence to the host cell. Interestingly, both G-citral and G-citron significantly inhibited pyoverdine production in P. aeruginosa (Fig. 9). Overall, G-citron was observed to be more effective than G-citral. These results can be attributed to a high lactonic percentage present in G-citron (Table 1). In conclusion, this is the first report on the application of glycomonoterpenes as a quorum sensing inhibitor showing complete inhibition of pigment production in both CV026 and Ag NTL4 (pZLR4) without affecting bacterial growth. We are still working on the purification of different forms of G-citral and G-citron to reveal the mode of action of these compounds in quorum sensing inhibition. Once the mechanism of action is known, it can be used to avoid implant-associated infections or to avoid biofilm formation on medical devices. Acknowledgments Amrita Patil thanks UGC for the Ph.D. fellowship and AcSIR for Ph.D. registration. Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest.

References 1. Bhardwaj, A. K., Vinothkumar, K., & Rajpara, N. (2013). Bacterial quorum sensing inhibitors: attractive alternatives for control of infectious pathogens showing multiple drug resistance. Recent Patents on AntiInfective Drug Discovery, 8, 68–83. 2. WHO media centre: antimicrobial resistance fact sheets http://www.who.int/mediacentre/factsheets/fs194/en/ Accessed Jan2016.

Appl Biochem Biotechnol 3. Mukherji, R., Patil, A., & Prabhune, A. (2015). Role of extracellular proteases in biofilm disruption of gram positive bacteria with special emphasis on Staphylococcus aureus biofilms. Enz Eng, 4, 126. doi:10.4172/2329-6674.1000126. 4. Heilmann, C., & Götz, F. (2010). Cell–cell communication and biofilm formation in gram-positive bacteria. In R. Krämer & K. Jung (Eds.), Bacterial signaling (pp. 7–17). Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA. 5. Chen, M., Yu, Q., & Sun, H. (2013). Novel strategies for the prevention and treatment of biofilm related infections. International Journal of Molecular Sciences, 14, 18488–18501. 6. Kalia, V. C., & Purohit, H. J. (2011). Quenching the quorum sensing system: potential antibacterial drug targets. Critical Reviews in Microbiology, 37, 121–140. doi:10.3109/1040841X.2010.532479. 7. Williams, P. (2007). Quorum sensing, communication and cross-kingdom signalling in the bacterial world. Microbiology, 153, 3923–3938. 8. Kalia VC (2015). Quorum sensing vs quorum quenching: a battle with no end in sight. Springer ISBN 97881-322-1981-1 DOI 10.1007/978-81-322-1982-8. 9. Kalia, V. C. (2013). Quorum sensing inhibitors: an overview. Biotechnology Advances, 31, 224–245. doi:10.1016/j.biotechadv.2012.10.004. 10. Romero, M., Acuna, L., & Otero, A. (2012). Patents on quorum quenching: interfering with bacterial communication as a strategy to fight infections. Recent Patents on Biotechnology, 6, 2– 12. doi:10.2174/187220812799789208. 11. LaSarre, B., & Federle, M. J. (2013). Exploiting quorum sensing to confuse bacterial pathogens. Microbiology and Molecular Biology Reviews, 77, 73–111. doi:10.1128/MMBR.00046-12. 12. Mukherji, R., & Prabhune, A. (2014). Novel glycolipids synthesized using plant essential oils and their application in quorum sensing inhibition and as antibiofilm agents. Scientific World Journal, 2014, 1–7. 13. Kerekes, E.-B., Deák, É., Takó, M., Tserennadmid, R., Petkovits, T., Vágvölgyi, C., & Krisch, J. (2013). Anti-biofilm forming and antiquorum sensing activity of selected essential oils and their main components on feed related micro-organisms. Journal of Applied Microbiology, 115, 933–942. doi:10.1111/jam.12289. 14. Jesus Olivero-Verbel, Elena E. Stashenko, Irene Wagner-Döbler, and Brigitte Kunze(2011) Anti-quorum sensing activity of essential oils from Colombian plants Natural Product Research Vol. 26, Sensors 2012, 12 (4), 4016-4030 doi: 10.1080/14786419.2011.557376. 15. Ahmad, A., Viljoen, A. M., & Chenia, H. Y. (2015). The impact of plant volatiles on bacterial quorum sensing. Letters in Applied Microbiology, 60, 8–19. doi:10.1111/lam.12343. 16. Onawunmi, G. O. (1989). Evaluation of the antimicrobial activity of citral. Lett. Appl. Microbial., 9(3), 105– 108. doi:10.1111/j.1472765X.1989.tb00301.x. 17. Kuwahara, Y., Suzuki, H., Matsumoto, K., & Wada, Y. (1983). Pheromone study on acarid mites. XI. Function of mite body as geometrical isomerization and reduction of citral (the alarm pheromone) Carpoglyphus lactis. Applied Entomology and Zoology, 18, 30–39. 18. Robacker, D. C., & Hendry, L. B. (1977). Neral and geranial: components of the sex pheromone of the parasitic wasp, Itoplectis conquisitor. Journal of Chemical Ecology, 3(5), 563–577. doi:10.1007/BF00989077. 19. Kim, J. K., Kang, C. S., Lee, J. K., Kim, Y. R., Han, H. Y., & Yun, H. K. (2005). Evaluation of repellency effect of two natural aroma mosquito repellent compounds, citronella and citronellal. Entomological Research, 35(2), 117–120. doi:10.1111/j.17485967.2005.tb00146.x. 20. Kazuhiko Nakahara, Najeeb S. Alzoreky, Tadashi Yoshihashi, Huong T. T. Nguyen and Gassinee Trakoontivakorn (2003).Chemical composition and antifungal activity of essential oil from Cymbopogon nardus (citronella grass). JARQ 37 (4). 21. Rodrigues, L. R., Teixeira, J. A., van der Mei Henny, C., & Oliveira, R. (2006). Physiochemical and functional characterization of a biosurfactant produced by lactococcus lactis 53. Colloids and surfaces. B. Biointerfaces, 49(1), 79–86. 22. Dubey, P., Selvaraj, K., & Prabhune, A. (2014). Physico-chemical, analytical and antimicrobial studies of novel sophorolipids synthesized using cetyl alcohol. World journal of pharmacy and pharmaceutical sciences, 3(3), 993–1010. 23. Mukherji, R., & Prabhune, A. (2015). A new class of bacterial quorum sensing antagonists: glycomonoterpenols synthesized using linalool and alpha terpineol. World Journal of Microbiology and Biotechnology, 31, 841–849. doi:10.1007/s11274-015-1822-5. 24. Lade, H., Paul, D., & Kweon, J. H. (2014). Review Article N-Acyl homoserine lactone-mediated quorum sensing with special reference to use of quorum quenching bacteria in membrane biofouling control. BioMed Research International, 2014(162584), 25. 25. Hafizah Y. Chenia (2013). Anti-Quorum Sensing Potential of Crude Kigelia africana Fruit Extracts Sensors. 13, 2802-2817; doi:10.3390/s130302802.

Appl Biochem Biotechnol 26. Gowrishankar, S., Mosioma, N. D., & Pandian, S. K. (2012). Coral-associated bacteria as a promising antibiofilm agent against methicillin-resistant and -susceptible Staphylococcus aureus biofilms. Evidencebased Complementary and Alternative Medicine, 2012(862374), 16. doi:10.1155/2012/862374. 27. Packiavathy, I. A. S. V., Priya, S., Pandian, S. K., & Ravi, A. V. (2014). Inhibition of biofilm development of uropathogens by curcumin—an anti-quorum sensing agent from Curcuma longa. Food Chemistry, 148, 453–460. 28. Alasil, S. M., Omar, R., Ismail, S., & Yusof, M. Y. (2015). Inhibition of quorum sensing-controlled virulence factors and biofilm formation in Pseudomonas aeruginosa by culture extract from novel bacterial species of Paenibacillus using a rat model of chronic lung infection. International Journal of Bacteriology, 2015(671562), 16. doi:10.1155/2015/671562. 29. The guardian-antibiotics: http://www.theguardian.com/society/2014/dec/11/drug-resistant-infections-deathssoar-10m-by-2050-report Accessed May 20, 2016. 30. Frieden, T. (2013). Antibiotic resistance threats. Cdc 22–50. doi:CS239559-B http://www.cdc. gov/drugresistance/threat-report-2013/ Accessed May 18, 2016.