Synergistic activity of quorum sensing inhibitor, pyrizine-2-carboxylic acid and antibiotics against multi-drug resistant V. cholerae

Abstract

Antibiotic resistance is one of the most pivotal health menaces in the human population worldwide. Use of conventional antibiotics at high concentration for prolonged periods facilitates a selective pressure on the bacterial strain to develop resistance to these antibiotics. The ascent of multi-drug resistance in Vibrio cholerae strains is a key issue for alternative drug development against cholera. A drug that could curb the virulence of V. cholerae is an elective choice for treatment. A few research studies have been carried out to discover such alternative medication that has an anti-virulent property rather than antibacterial activity. Combinatorial antibiotic treatments along with anti-biofilm compounds have diverse effects on bacterial growth i.e. it can be additive, synergistic or antagonistic. In the present investigation, combinatorial action of 2,3-pyrazine dicarboxylic acid derivatives (QSI^Vc targeted against the quorum regulator, LuxO) along with antibiotics was evaluated. The results indicated that QSI^Vc could inhibit biofilm at a very low concentration (IC₅₀ varying between 1 μM and 70 μM). The synergism of the compounds with the reported antibiotics (doxycycline, tetracycline, chloramphenicol and erythromycin) was evaluated by the checkerboard method. The fractional inhibitory concentration (FIC) values varied from 0.0 to 0.5 for each combination. The ΔE value (750–1900), according to the bliss independence model showed that the QSI^Vc synergistically enhanced the inhibitory effect of the reported antibiotics against the multidrug resistant clinical isolates of V. cholerae.

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Introduction

Antibiotics have dependably been viewed as one of the astounding revelations of the twentieth century. However, the ineffectual use of antibiotics results in the evolution of multi-drug resistant (MDR) strains.¹ MDR strains with enhanced virulence and transmissibility can also be referred to as “superbugs”.² Generally, resistance to multi-drugs can be either due to alteration in physiological states like biofilm formation or may be due to the genotypic resistance to the antimicrobial drugs.³ The adherence propensity of the microbes to both biotic and abiotic surfaces by forming a biofilm is considered to be one of the major reasons for attaining resistance.⁴ Biofilm can be defined as a densely packed community of surface associated microbes fenced in a self-secreted extracellular polymeric substance. Subsequently, antibiotic resistance also can be viewed as one of the virulence factors. As most of the V. cholerae strains emerge as superbug strains, treatment against disease outbreak due to MDR strains is of major health concern worldwide.⁵ The spread of MDR superbugs necessitates alternative therapeutic approaches. The general anti-virulent therapeutic approaches are to hinder specific mechanisms of bacteria that promote infection yet do not influence its growth.

Inhibition of quorum sensing (QS) pathway is one of the anti-virulent therapeutic approaches. QS is an illustrative term for cell to cell communication that occurs within bacteria to stay informed regarding their cell density.⁶ QS is mediated by the production and sensing of small molecules called auto-inducers to coordinate gene expression that involves the production of virulence factors (motility, biofilm production, toxin production etc.).⁷ In V. cholerae three parallel QS system exists which results in the production of cholera toxin (CT), toxin co-regulated pilus (TCP), biofilm and hemolysis.⁸ In this context, a drug that could inhibit the virulence property of V. cholerae by targeting the essential factor for infection (Biofilm, CT, TCP) is a novel method of treatment against cholera.

For instance, pyrazine, a symmetric aromatic heterocyclic compound that occurs almost ubiquitously in nature is a weaker base than pyridine, pyridazine and pyrimidine. Antibacterial activity of pyrazine and its derivatives against both environmental and clinical pathogens have been reported.^9,10 Pyrazine can be easily synthesized both biologically and chemically. Most importantly, pyrazine moiety and its related heterocyclic compounds are core parts of many clinically used drugs. It has been reported that 1,4-pyrazine derivative has good antimicrobial activity against a wide range of bacteria.¹¹ Various reports showing anti-mycobacterial property of pyrazine and related heterocyclic compound have been published.^10,12 Therefore, pyrazine and related heterocyclic compounds having good pharmacophore property could be considered for the development of new chemical compounds that may offer many possibilities in the drug development. Our earlier in silico study provided an insight to identify our lead compound, 3[4-morpholine-anilio] carbonyl-2-pyrazine carboxylic acid (QSI^Vc) that could possibly hinder the QS system of V. cholerae.¹³ Hence, in the present investigation, derivatives of 2,3-pyrazine dicarboxylic acid were chemically synthesized and investigated for the biofilm inhibition as well as for the synergistic activity with antibiotics.

Results and discussion

Synthesis and validation of 2,3-pyrazine dicarboxylic acid derivatives

Derivatives of 2,3-pyrazine dicarboxylic acid termed as QSI^Vc1–6 were synthesized in three steps involving the general synthetic route showed in Fig. 1. These compounds were synthesized starting from 4-fluronitrobenzene 2a or 3,4-difluronitrobenzene 2b and morpholine 1a, piperidine 1b, or pyrrolidine 1c. Initial aromatic nucleophilic substitution reaction was achieved in the presence of diisopropylethylamine in acetonitrile at room temperature to afford compounds 3a–3f.¹⁴ Subsequent reduction with Fe/NH₄Cl in methanol–water mixture yielded the amino compounds. Final amidation reaction with furo-[3,4-b]-pyrazine-5,7-dione 5 afforded the target compounds 6a–6f (QSI^Vc1–6) in good yield. The synthesized compounds 6a–f were characterized using ¹H, ¹³C NMR and FTIR spectroscopy. For instance, ¹H NMR spectrum of compound 6a has a broad singlet at 13.76 ppm and 10.62 ppm. These two signals can be attributed to the COOH and NH protons respectively. Two doublets at 6.95 ppm and 7.66 ppm are due to the para disubstituted aromatic ring hydrogens, and the singlet at 8.88 ppm can be assigned to the two hydrogens present in the pyrazine ring. The two triplets at 3.08 ppm and 3.75 ppm are due to the morpholine ring hydrogens. ¹³C NMR spectra also confirm the structure of the compounds. The NMR data of other compounds are given in the Experimental section.

Fig. 1 Synthetic scheme for as QSI^Vc1–6.

When piperidine was introduced in the place of morpholine, the antibiofilm property was decreased profoundly (QSI^Vc3 and QSI^Vc4). Antibiofilm property was regained when pyrrolidine moiety (QSI^Vc5 and QSI^Vc6) was added. Addition of fluoride to its benzene group did not alter the activity of the compound. This suggests that pyrrolidine moiety plays an important role in its activity.

Antibacterial property

Considering the need for an anti-adhesive compound, is one of the alternative approaches to overcome multi-drug resistance (MDR), a preliminary study was performed to test whether the compounds QSI^Vc1–6 had any antimicrobial activity. Results of the study (data not shown) showed that there was no growth inhibition against all three pathogens at tested concentrations (3.75–200 μM). There are many reports stating the anti-mycobacterial activity of pyrazine derivatives.^15,16 However, our results showed that when 2,3-pyrazine dicarboxylic acid were coupled with morpholine, piperidine and pyrrolidine moieties antimicrobial activity was significantly reduced or no growth inhibition was observed. There are many reports stating that quorum sensing inhibitors don't alter the cell population but inhibit the virulence factors production.¹⁷ Through our earlier studies we found that QSI^Vc1 was the lead compound that could be bind to the LuxO (response regulator protein) and would alter the expression of the virulence gene.¹³ Hence, this result suggests that QSI^Vc may disturb the QS signaling pathway without limiting the bacterial growth.

Anti-adhesive activity

As previously discussed, biofilm inhibition is one of the powerful strategies to control biofilm mediated bacterial infections. Bacteria have the tendency to adhere to any surface and can form biofilm. Physiological constituent and structure of biofilms reduce the efficiency of antimicrobial agent there by adding to their survival. Hence forth biofilm formation has been documented as a factor for bacterial survival.¹⁸ To our knowledge, there is no published data reporting the antibiofilm activity of 2,3-pyrazine dicarboxylic acid derivatives QSI^Vc1–6 and in the present study we have reported the antibiofilm activity of QSI^Vc1–6 as depicted in Table 1. Levels of antibiofilm activity differed among the 2,3-pyrazine dicarboxylic acid derivatives. The results demonstrated that the compound QSI^Vc1 and compound QSI^Vc2 showed moderate antibiofilm activity whereas QSI^Vc5 and QSI^Vc6 were the best among the all 6 compounds. On the other hand, the compound QSI^Vc3 and compound QSI^Vc4 did not show any antibiofilm activity.

Table 1 Table showing the biofilm inhibition concentration and eradication concentration of various strain for library member value in μM^a

Compound	Clinical isolate Vc4		Clinical isolate Vc3		MTCC 3905
	IC50 (μM)	EC50 (μM)	IC50 (μM)	EC50 (μM)	IC50 (μM)	EC50 (μM)
a V. cholerae strains used in the study; MTCC 3905-standard reference strain; Vc3-MDR strain (clinical isolate received from JSS Medical College, Mysore); Vc4-MDR strain (clinical isolate received from JSS Medical College, Mysore). IC50 – inhibitory concentration, EC50 eradication concentration.
6a (QSI^Vc1)	68.3	65	7.2	73.4	5.6	3.72
6b (QSI^Vc2)	68.3	65.4	16.4	—	3.9	5.3
6c (QSI^Vc3)	—	—	—	—	11.2	21.4
6d (QSI^Vc4)	—	—	—	—	3.4	—
6e (QSI^Vc5)	25	50.3	13.7	28.4	1.63	5.43
6f (QSI^Vc6)	30.5	55.5	8.3	22.4	3.02	3.75

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Antibiofilm activity mainly depends upon the type of functional groups and the concentration of the drug like compound.¹⁹ For compounds QSI^Vc1 and QSI^Vc2 biofilm inhibition was about 50% at the concentration of 68.3 μM whereas for compounds QSI^Vc5 and QSI^Vc6 a significant decrease in biofilm formation (73% and 71% respectively) was noted at the concentration of 25 and 30.5 μM respectively for the MDR Vc4. For MDR Vc3, the percentage of biofilm inhibition was 60% and 54% for QSI^Vc1 and QSI^Vc2 at the concentration of 7.2 μM and 16.4 μM respectively. Whereas, the percentage of inhibition for compounds QSI^Vc5 and QSI^Vc6 was 69% and 66% at the concentration of 13.7 μM and 8.3 μM. On the other hand, percentage of biofilm inhibition for compounds QSI^Vc1 and QSI^Vc2 was above 70% at the concentration of 5.6 and 3.9 μM respectively and for QSI^Vc5 and QSI^Vc6 at the concentration of 1.6 and 3.0 μM percentage of inhibition was above 80% for standard susceptible strain. In all the cases, there was no significant reduction of biofilm when the concentration of the synthesized compounds QSI^Vc1–6 was further increased. The overall results emphasized that not all compounds showed antibiofilm activity. Also, it is noteworthy to mention that ability for the compounds QSI^Vc1–6 to eradicate the preformed biofilm was very less when compared with its ability to inhibit biofilm formation. Ability of a compound to inhibit biofilm would be probably by altering the quorum-sensing pathway and the molecular basis of their target-specific action has to be further confirmed. Hence, inhibiting the biofilm formation by small ligand molecules would be an important strategy to resensitize the MDR strain to antibiotic compound and host immune system.²⁰

Antibiofilm compound has potential to restore the efficacy of the antibiotics that are inefficient in biofilm bacteria. Literature stating the antibiofilm activity of small molecule that includes from both the natural and synthetic source is very limited. In 2013, a report stated the antibiofilm activity of quinine analogs against V. cholerae.²¹ Also, polyphenol compounds extracted from coca and cranberries were found to exhibit the antibiofilm activity against S. mutans on primarily affecting the key enzymes, glucosyltransferase and fructosyltransferase activity on initial attachments.²² Examples depicted above prove that such therapeutic approach is an alternative mechanism to combat biofilm mediated infection.

Synergistic studies

One of the important consequences of phenotypic resistance is persistence of persister cells in biofilm population.³ Persister cells in biofilm causes various chronic infection as they limit the efficiency of the drug therapy. According to the report stated by NIH, about 60% of infections caused by microbes are biofilm mediated. Such common infections are UTI (Escherichia coli), catheter associated infection (Staphylococcus aureus), dental plaque (Porphyromonas gingivalis) that are hard to treat.²³ In case of V. cholerae, biofilm formation is one of the key factors for environmental survival and transmission of infection. A desirable strategy to be followed to tackle phenotypic resistant bacteria is to combine novel antibiofilm compounds with conventionally used antibiotics. These treatments would resuscitate the potency of the antibiotics, cooperating beneficially to combat an infectious threat. The antibiofilm agent will inhibit biofilm formation thus exposing the planktonic cells to sub inhibitory concentration of antibiotic and eliminate the bacterial population by easily penetrating and it is not possible in the case of biofilms as it acts as a diffusion barrier for antibiotics.^24,25 As mentioned earlier, we have hypothesized that our synthesized compounds might resensitize the MDR V. cholerae strains to antibiotics. Antibiotics would be effective in combination treatments exhibiting either a synergistic effect (cooperative effect by two or drugs) or an addictive effect (combined effect produced by two or more agents). On the other hand, the combination can also exhibit antagonistic behavior i.e., the effect of the combination treatment is less than the effect of the respective single-drug treatments.

In synergistic study, the percentage growth inhibition of bacteria was calculated for all combination by comparing the OD of the treated with that of the control. Reduction in MIC value for culture that was treated with both the antibiofilm compounds and the antibiotic was observed when compared with the culture exposed to the antibiotics alone. As stated, ΔE and FIC are the two main nonparametric factors for analyzing the interaction between antibiofilm compounds and antibiotics. FIC index is commonly used for determination of the interaction between the two drugs. A vital observation grasped was that except QSI^Vc3 and QSI^Vc4 all other compounds (QSI^Vc1, QSI^Vc2, QSI^Vc5, and QSI^Vc6) interacted synergistically with all the antibiotics used in this study against MDR clinical isolates and standard reference strains. The FIC index ranged from 0.05 to 0.5. When using the ΔE method all the three microorganism taken in this study showed synergistic interaction, ranging from 764 to 1869 and the results were depicted in Table 2. Interaction between chloramphenicol and QSI^Vc1 showed a marginal synergy as the FIC value was 0.54 for the clinical isolate Vc3. Similarly, interaction between erythromycin and QSI^Vc1 was additive for clinical isolate Vc4 and the FIC index was 0.67. Results for the FIC model were interpreted as a single outcome even though the experiment was performed in triplicates for each combination. Values for FIC may vary sometimes between the experiments as FICs are determined from the effect of two drugs.²⁶ To overcome this, the ΔE model, an important method to analyze the interaction between the antibiotics and the test compounds used for this study. When ΔE was used for interpreting the interaction, the percentage of growth was directly derived from the experimental data and the results were in full agreement with the findings of FIC model. Moreover, the results of visual observation and OD determination methods for endpoint reading were also in good concordance, which confirm that the OD determination was suitable for interaction studies.

Table 2 Combined action of commercial antibiotics and QSI^Vc against V. cholerae^a

Nonparametric method
Strains		QSI^Vc1				QSI^Vc2
		D	T	C	E	D	T	C	E
a FIC model – FIC index of ≤0.5 was defined as synergy (SYN), FICI of >4.0 was defined as antagonism (ANT), and indifference (i.e. no interaction) as a FICI of >0.5 to 4. MTCC3905-standard strain; Vc3 and Vc4 – clinical isolates (MDR) – received from JSS Medical College, Mysore. D = doxycycline, T = tetracycline, C = chloramphenicol, E = erythromycin.
MTCC3905	FIC	—	—	0.1 (0.3–0.1)	0.3 (0.3–0.2)	—	—	0.2 (0.30–0.005)	0.1 (0.23–0.007)
	R	—	—	SYN	SYN	—	—	SYN	SYN
	ΔE	—	—	1479.2	919.2	—	—	1409.7	1541.5
	R	—	—	SYN	SYN	—	—	SYN	SYN
Vc3	FIC	0.2 (0.4–0.1)	0.1 (0.3–0.1)	0.5 (0.5–0.1)	0.5 (0.1–0.5)	0.3 (0.26–0.002)	0.2 (0.2–0.02)	0.1 (0.24–0.007)	0.1 (0.38–0.05)
	R	SYN	SYN	ADD	ADD	SYN	SYN	SYN	SYN
	ΔE	1106.1	1590.2	1301.3	1885.1	1157.0	1663.4	1368.7	1064.6
	R	SYN	SYN	SYN	SYN	SYN	SYN	SYN	SYN
Vc4	FIC	0.08 (0.3–0.01)	0.05 (0.2–0.01)	0.12 (0.3–0.2)	0.67 (0.2–0.6)	0.21 (0.19–0.01)	0.04 (0.1–0.007)	0.07 (0.18–0.005)	0.3 (0.26–0.17)
	R	SYN	SYN	SYN	ADD/ANT	SYN	SYN	SYN	SYN
	ΔE	1001.5	1759.0	897.7	1180.3	1051.4	1845.1	942.9	1337.8
	R	SYN	SYN	SYN	SYN	SYN	SYN	SYN	SYN

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Nonparametric method
Strains		QSI^Vc5				QSI^Vc6
		D	T	C	E	D	T	C	E
MTCC3905	FIC	—	—	0.3 (0.12–0.29)	0.06 (0.13–0.04)	—	—	0.1 (0.02–0.0.3)	0.2 (0.01–0.3)
	R	—	—	SYN	SYN	—	—	SYN	SYN
	ΔE	—	—	1754.4	1416.7	—	—	1919.8	1759.0
	R	—	—	SYN	SYN	—	—	SYN	SYN
Vc3	FIC	0.07 (0.02–0.25)	0.06 (0.1–0.04)	0.35 (0.13–0.4)	0.36 (0.13–0.34)	0.06 (0.1–0.06)	0.03 (0.04–0.03)	0.05 (0.003–0.11)	0.14 (0.28–0.03)
	R	SYN	SYN	SYN	SYN	SYN	SYN	SYN	SYN
	ΔE	805.4	1166.2	945.5	1868.8	921.9	1323.2	995.0	1195.8
	R	SYN	SYN	SYN	SYN	SYN	SYN	SYN	SYN
Vc4	FIC	0.2 (0.17–0.07)	0.05 (0.14–0.01)	0.06 (0.04–0.2)	0.12 (0.32–0.03)	0.07 (0.18–0.02)	0.04 (0.11–0.03)	0.04 (0.2–0.01)	0.05 (0.13–0.01)
	R	SYN	SYN	SYN	SYN	SYN	SYN	SYN	SYN
	ΔE	852.8	1483.2	763.8	1678.5	964.1	1675.8	877.7	1075.8
	R	SYN	SYN	SYN	SYN	SYN	SYN	SYN	SYN

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Confocal laser scanning microscopy (CLSM) analysis

Based on the results obtained from anti-adhesive assay, the inhibition of biofilm formation by Vc4 on glass slides in the presence and absence of QSI^Vc5 and QSI^Vc6 were qualitatively confirmed by CLSM analysis (Fig. 2). As evident from Fig. 2 there was a significant reduction in the biofilm formed on the surface of glass slide treated with QSI^Vc5 when compared to the untreated control. Distribution of biofilm formed was also depicted as 3 dimensional images, which showed a significant difference in biofilm inhibition between QSI^Vc5, treated (25 μM) and untreated culture after 24 h of incubation. Similar results were observed when the strains were treated with QSI^Vc6 (data not shown).

Fig. 2 Confocal laser scanning microscopy (CLSM) images showing the effect of QSI^Vc5 on bacterial biofilm.

From Fig. 3, it was clearly visible that there were no dead cells (red-stained cells) when the cultures were grown in the presence of QSI^Vc5 alone (25 μM) stating the absence of bactericidal activity. On the contrary, erythromycin when used alone, exhibited not much bactericidal activity at 15 μM (red-stained cells). However, QSI^Vc5 revealed its remarkable ability to act synergistically with erythromycin for both standard (MTCC 3905) and clinical isolates (Vc4) of V. cholerae like our in vitro results. Growth of MDR-Vc4 in the presence of 15 μM erythromycin and 25 μM QSI^Vc5 significantly reduced as biofilm formation has been inhibited by QSI^Vc5 thus increased the bactericidal activity of erythromycin. In case of MTTC 3905 biofilm growths was significantly reduced when the culture was treated with both the 15 μM erythromycin and 5.4 μM QSI^Vc5. These results clearly shows that the antimicrobial activity of erythromycin was enhanced when Vc4 and MTCC 3905 were treated with QSI^Vc5 along with erythromycin as compared to the samples treated with erythromycin alone. This could be possible because of the easy penetration of antibiotic into the planktonic cells rather than the biofilm cells. At this planktonic state, the antibiotics can easily penetrate and can kill bacteria thus reducing the infection.²⁴ Our experimental data is in coherence with the effective combinatorial therapy as it is more important than treating infections with higher concentration of conventional antibiotics.

Fig. 3 Live/dead staining images of bacterial isolates Vc4 and MTCC in the presence of 15 μM concentration of erythromycin and 25 and 5.43 μM of QSI^Vc5.

Experimental

Synthesis

Synthesis of compounds QSI^Vc1–6 was carried out using appropriate reactions and the general synthetic scheme is shown in Fig. 1.²⁷

General procedure for the synthesis of compounds 3. To a stirred solution of amine 1 (11 m mol) in 15 mL of acetonitrile was added diisopropylethylamine (11 mmol) at 0 °C. To this mixture fluronitorbenzenederivatives 2 (11 mmol) were added and stirring was continued at room temperature for overnight. The reaction mixture was poured into ice water and then separated yellow solid was filtered and dried under vacuum.

General procedure for the synthesis of compounds 4. Iron powder (30 mmol) and ammonium chloride (35 mmol) were added to a stirred solution compound 3 (5 mmol) in methanol : water (mixture) (2 : 1, 18 mL). The resulting mixture was refluxed for 5 h and ethyl acetate was added and filtered through a celite bed. The filtrate was washed with water (twice) and concentrated to afford a compound 4.

General procedure for the synthesis of compounds 6. To a solution of compound 5 (0.35 mmol) in THF (2 mL) was added compound 4 (0.35 mmol) at room temperature. The reaction was stirred for 30 min. During the course of the reaction solid product was precipitated which was filtrated and washed with petroleum ether.

3-(4-Morpholinophenylcarbamoyl) pyrazine-2-carboxylic acid 6a was obtained in 85% yield. Pale white color, IR (neat) 3248.5, 3056.6, 2918.7, 2866.7, 1683.6, 1642.1, 1521.6, 1404.9, 1315.2, 1085.7 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 3.08 (t, J = 4.8 Hz, 4H), 3.74 (t, J = 4.8 Hz, 4H), 6.95 (d, J = 9.0 Hz, 2H), 7.65 (d, J = 9.0 Hz, 2H), 8.87 (s, 2H), 10.60 (s, 1H); ¹³C NMR (75 MHz, DMSO-d₆) δ 48.7, 66.1, 115.2, 121.1, 130.5, 144.2, 145.2, 145.5, 146.6, 147.8, 161.6, 166.4.

3-(3-Fluoro-4-morpholinophenylcarbamoyl) pyrazine-2-carboxylic acid 6b was obtained in 87% yield. Pale white color, IR (neat) 3300.6, 3101.9, 2985.3, 2857.0, 1721.26, 1685.5, 1593.9, 1533.1, 1428.0, 1324.7, 1118.5 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 2.98 (t, J = 4.5 Hz, 4H), 3.74 (t, J = 4.5 Hz, 4H), 7.06 (t, J = 9.3 Hz, 1H), 7.51 (dd, J = 8.7, 1.5 Hz, 1H), 7.70 (dd J = 14.7, 2.1 Hz, 1H), 8.88–8.91 (m, 2H), 10.86 (s, 1H); ¹³C NMR (75 MHz, DMSO-d₆) δ 50.6, 66.1, 108.3 (d, J = 25.5 Hz), 116.2, 119.1 (d, J = 3.8 Hz), 133.3 (d, J = 10.5 Hz), 136.0 (d, J = 9.0 Hz), 144.4, 145.0, 145.8, 146.5, 154.3 (d, J = 241.5 Hz), 162.0, 166.3.

3-(4-(Piperidin-1-yl) phenyl carbamoyl) pyrazine-2-carboxylic acid 6c was obtained in 93% yield. Light bluish grey color, IR (neat) 3237.0, 3052.8, 2940.00, 1682.6, 1631.5, 1517.7, 1403.0, 1319.0, 1085.7 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 1.52–1.62 (m, 6H) 3.10 (t, J = 5.7 Hz, 4H), 6.93 (d, J = 9.0 Hz, 2H), 7.62 (d, J = 9.0 Hz, 2H), 8.87 (s, 2H), 10.56 (s, 1H); ¹³C NMR (75 MHz, DMSO) δ 23.8, 25.2, 49.9, 116.0, 121.1, 129.9, 144.2, 145.3, 145.5, 146.5, 148.5, 161.5, 166.4.

3-(3-Fluoro-4-(piperidin-1-yl) phenyl carbamoyl) pyrazine-2-carboxylic acid 6d was obtained in 86% yield. White color, IR (neat) 3241.75, 3056.62, 2944.8, 1688.4, 1627.6, 1522.5, 1401.0, 1274.7, 1083.8 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 1.52–1.65 (m, 6H), 2.94 (t, J = 5.4 Hz, 4H), 7.04 (t, J = 9.0, 1H), 7.48 (dd, J = 8.7, 1.5 Hz, 1H), 7.67 (dd, J = 15.0, 2.4 Hz, 1H), 8.85–8.90 (m, 2H), 10.83 (s, 1H); ¹³C NMR (75 MHz, DMSO) δ 23.7, 25.7, 51.6 (d, J = 3.0 Hz), 108.2 (d, J = 25.5 Hz), 116.1 (d, J = 2.3 Hz), 119.4 (d, J = 3.8 Hz), 132.9 (d, J = 10.5 Hz), 137.3 (d, J = 9.0 Hz), 144.4, 145.0, 145.7, 146.5, 154.3 (d, J = 241.5 Hz), 162.0, 166.3.

3-(4-(Pyrrolidin-1-yl) phenyl carbamoyl) pyrazine-2-carboxylic acid 6e was obtained in 81% yield. Maroon color, IR (neat) 3345.9, 2924.9, 2849.3, 1744.3, 1678.7, 1525.4, 1370.1, 1245.8, 1080.9 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 1.95 (t, J = 6.3, 4H) 3.22 (t, J = 6.3, 4H), 6.53 (d, J = 9.0 Hz, 2H), 7.60 (d, J = 9.0 Hz, 2H), 8.85 (s, 2H), 10.47 (s, 1H); ¹³C NMR (75 MHz, DMSO-d₆) δ 23.3, 45.8, 109.8, 120.0, 125.4, 142.5, 143.3, 143.5, 143.8, 145.1, 159.4, 165.0.

3-(3-Fluoro-4-(pyrrolidin-1-yl) phenyl carbamoyl) pyrazine-2-carboxylic acid 6f was obtained in 82% yield. Light yellow color, IR (neat) 3276.5, 3102.9, 2949.6, 1685.5, 1535.1, 1427.1, 1150.3, 1082.8 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 1.89 (t, J = 6.3, 4H), 3.30 (t, J = 6.3 Hz, 4H), 6.74 (t, J = 9.6 Hz, 1H), 7.42 (dd, J = 9.0, 1.8 Hz, 1H), 7.64 (dd, J = 15.9, 2.1 Hz, 1H), 8.83–8.89 (m, 2H), 10.72 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆) δ 24.6, 49.5 (d, J = 4.5 Hz), 108.5 (d, J = 25.5 Hz), 115.3 (d, J = 6.0 Hz), 116.5 (d, J = 2.3 Hz), 128.8 (d, J = 9.8 Hz), 133.8 (d, J = 9.8 Hz), 144.2, 144.8, 145.6, 146.7, 150.5 (d, J = 237.0 Hz), 161.5, 166.4.

Bacterial strains and growth conditions

Two multi-drug resistant clinical isolates of Vibrio cholerae (Vc3 and Vc4) and one standard reference strain (MTCC 3905) were obtained from JSSC medical college Mysore and MTCC Chandigarh respectively. Bacteria were maintained in Luria Bertani (LB) media at 37 °C. All the strains were confirmed as Vibrio cholerae by conventional biochemical methods described previously.²⁸

Inoculum preparation

Inoculum size was standardized according to the national committee for clinical laboratory standards guidelines²⁹ (NCCLS 1993). In brief, the cultures were revived and re-suspended in fresh LB media to obtain a turbidity of 0.5 McFarland units that contained 5 × 10⁵ CFU mL⁻¹ in all the experiments.

Antimicrobial activity

Antimicrobial susceptibility test for the reported antibiotics and anti-biofilm compounds QSI^Vc1–6 was carried out by broth microdilution method in accordance with the clinical and laboratory standard institute (NCCL 2007) guidelines.³⁰ Briefly, 2-fold serial dilutions of the antimicrobial agent were prepared in the LB broth to obtain the required concentration. 100 μL of the each of the compound dilutions and 90 μL of blank media with 10 μL of prepared inoculum were added in each well. The positive control (media alone) and the negative control (media with inoculum alone; without drug treatment) was maintained. Plates were incubated at room temperature for 24 h. The growth/inhibition of the cells was observed at 595 nm using 96-well microtiter plate reader (BioRadi-Mark). MIC is defined as the lowest concentration of the drug that inhibits the visible growth of the microbes after overnight incubation in comparison with the control (without drug treatment). Antibiotics used in this study were erythromycin (250–3.75 μM), doxycycline (250–3.75 μM), tetracycline (250–3.75 μM), and chloramphenicol (250–3.75 μM). The dilution range for the test compounds was as follows: QSL^Vc1 (75–1.1 μM), QSL^Vc2 (75–1.1 μM), QSL^Vc3 (75–1.1 μM), QSL^Vc4 (75–1.1 μM), QSL^Vc5 (75–1.1 μM), QSL^Vc6 (75–1.1 μM).

Antiadhesive and biofilm dispersion assay

The standard microtiter plate method was carried out to quantify biofilm as described by O'Toole et al. (1999).³¹ In brief, desired concentrations of the synthesized compound were added to the plate and ∼5 × 10⁵ CFU mL⁻¹ of bacteria was inoculated in each well. The plates were incubated at 37 °C for 24 h. Afterwards, plates were washed to remove the unattached and loosely bound cells. Attached cells were then fixed with methanol (100%) and then stained with crystal violet dye (0.2%) for 20 min. Excess stain was removed by washing and the bound dye was eluted with 96% ethanol. The eluted dye was measured at OD 595 nm. Biofilm dispersion assay was performed by the addition of 100 μL of the prepared inoculum with the 100 μL of the blank medium alone into a 96-well microtiter plate. Plate was incubated at 37 °C for 24 h. Planktonic cells were removed by repeated wash with PBS. Preformed biofilm was treated with varying concentrations of the QSI^Vc and incubated for 5 h. Plate was then processed by the addition of crystal violet followed by washing and eluted the bound dye with 96% of ethanol. Optical density was read at 595 nm in ELISA plate reader (BioRadi-Mark).

Synergistic studies

Checkerboard assay was used for evaluate the interaction between conventional antibiotics with synthesized compounds³² Antibiotics of varying concentration from 1/32 × MIC to 4 × MIC was combined with pure synthesized compound were prepared in Muller Hinton broth (MHB) with standard inoculum size of 5 × 10⁵ CFU mL⁻¹. Two models were used to analyse the data obtained by the spectrometric method i.e. FIC index and ΔE. The FIC index is the sum of FIC of each drug. FIC value below 0.5 indicates synergy, and values between 0.5 and 1 is said to be additive. Values between 1 and 2 is said to be indifferent and it is said to be antagonistic when the value is above 2.

The bliss independence model or BI theory can be defined by the equation I_i = (I_A + I_B) − (I_A × I_B), where I_i is the predicted percentage of inhibition of the theoretical combination of drug A and B; I_A and I_B are the experimental percentages of inhibition of each drug acting alone. Since I is equal to 1 − E, where E is the percentage of growth, and by substitution into the former equation, and thus a resultant equation is derived as E_i = E_A × E_B, where E_i is the predicted percentage of growth of the theoretical combination of drug A and drug B, respectively, and E_A and E_B are the experimental percentages of growth of each drug action alone respectively. The interaction is described by the difference ΔE between the predicted and the measured percentages of growth at various concentrations (ΔE = E_predicted − E_measured) whereas, the E_A and E_B are obtained directly from the experimental data by the nonparametric approach described by Prichard et al. The nature of interactions were studied using the microtiter plate assay with a twofold dilution of either drug would result in a ΔE for each drug combination. In each of the three independent experiments, the observed percentages of growth obtained from the experimental data were subtracted from the predicted percentages, and then the average difference of the three experiments was calculated. When the average difference as well as its 95% confidence interval among the three replicates was positive, statistically found to be significant and synergy was claimed; when the difference as well as its 95% confidence interval was negative, a significant antagonism was claimed and in other cases, BI was concluded. To summarize the interaction surface, the sums of the percentages of all statistically significant synergistic ([summ] SYN) and antagonistic ([summ] ANT) interactions were calculated. Interactions with <100% statistically significant interactions were considered weak, interactions with 100% to 200% statistically significant interactions were considered moderate, and interactions with >200% statistically significant interactions were considered strong, as described previously.²⁶ In addition, numbers of statistically significant synergistic and antagonistic combinations among all tested drug combinations were calculated for each strain.

Confocal laser scanning microscopy imaging

Biofilm was developed onto glass slides under various conditions of treatment using drug and antibiotic. After the biofilms were grown for 24 h, the glass slides were removed carefully and the biofilms were rinsed delicately with 1× PBS to remove loosely attached cells. The attached cells were stained with 300 μL of Live/Dead BacLight dye for 15 min, and then the excess dye was removed by washing with 1× PBS. The slides were then dried in dark condition for 2 min under ambient temperature. Confocal imaging was performed using 488 multi argon ion laser in a confocal laser scanning microscope (Carl Zeiss) to obtain the live/dead imaging.³³

Statistical analysis

Graph pad prism software (version 6.01) was used for statistical analysis. The minimum level of significance was set at P ≤ 0.05. All assays were conducted in triplicate and statistical analysis was done.

Conclusions

Our data were the first report to show pyrizine-2-carboxylic derivatives (QSI^Vc1, QSI^Vc2, QSI^Vc6 and QSI^Vc5) has antibiofilm activity against V. cholerae. Results of antibiofilm assay and confocal microscopy imaging strongly indicates that QSI^Vc5 and QSI^Vc6 would be better lead compounds due to their potent ability to reduce the biofilm formation in clinical isolates as well as standard MTCC culture. Development of selective pressure towards resistance would be less for these compounds since it inhibits the biofilm formation rather than inhibiting the growth of V. cholerae. Moreover, in combinatorial assay QSI^Vc5, QSI^Vc6, QSI^Vc1 and QSI^Vc2 showed potent synergistic antimicrobial effects when combined with conventional antibiotics. In this context, we propose that pyrizine-2-carboxylic derivatives (QSI^Vc1, QSI^Vc2, QSI^Vc6 and QSI^Vc5) would be a potent lead compound for developing novel drugs that could be used alone or in combination with antibiotics to tackle the infections. This study provides an insight into alternative approach of controlling bacterial infections. However, an insight into the molecular mechanism of the quorum quencher effect of (QSI^Vc) has to be established.

Acknowledgements

We sincerely thank SASTRA University and its management for providing us with the infrastructure needed to accomplish our research work. We also acknowledge the DST-PURSE sponsored CLSM facility (No. SR/FT/LS-113/2009) at Bharathidasan University, Trichy, and Tamil Nadu for confocal imaging facility.

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