A SYNERGISTIC COMPOSITION OF AN ANTIBIOTIC WITH TRANS-CINNAMALDEHYDE AGAINST MULTI-DRUG-RESISTANT BACTERIA

Abstract

The invention relates to a synergistic composition of trans-cinnamaldehyde and tetracycline against extended spectrum beta-lactamase (ESBL), metallo-beta-lactamases (MBL) and biofilm producing bacterial strains.

Specification

Description:FIELD OF THE INVENTION:
The present invention relates to a synergistic composition of an antibiotic and a plant metabolite against the antibiotic resistance as well as biofilm formation in bacteria. Particularly, the invention provides a synergistic composition of tetracycline and trans-cinnamaldehyde against extended spectrum beta-lactamase (ESBL), metallo-beta-lactamases (MBL) and biofilm producing bacterial strains.

BACKGROUND OF THE INVENTION:
Incessant use of antibiotics has led to the increasing survival and propagation of antibiotic-resistant strains, especially in and around medical care facilities and hospitals. Antibiotic resistance and biofilm formation are two major factors, which augment and provide tenacity to bacteria for survival.

Escherichia coli and Klebsiella pneumoniae are known as extended spectrum beta-lactamase producing organisms and are isolated globally. The metallo-beta-lactamases render gram negative bacteria resistant to most ß lactams and have spread across numerous bacterial species with increasing diversity. Biofilm producing drug resistant strains are significantly resistant to a wide range of antibiotics as compared to non-biofilm forming strains. Some of the prior art discussing the synergistic effect of combining an antibiotic with plant metabolite have been listed below.

Bonincontro G et al., in the paper titled "Synergistic effect of plant compounds in combination with conventional antimicrobials against biofilm of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida spp." published in Pharmaceuticals, 2023, volume-16(11), 1531, 2023 summarises the current knowledge on the synergistic effects of plant metabolites in combination with conventional antimicrobials against biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. The synergism of conventional antimicrobials with plant compounds can modify and inhibit the mechanisms of acquired resistance, reduce undesirable effects, and obtain an appropriate therapeutic effect at lower doses.

Chauhan et al., in the paper titled "Synergistic effect of combined antibiotic and methanolic extracts of Withania somnifera and Catharanthus roseus against MDR Salmonella enterica Serovar Typhi" published in Advanced Gut & Microbiome Research, 8836886, 12 pages, 2023 illustrates the synergistic effect of bioactive constituents of medicinal plants in combination to synthetic drugs for enhancing the bacterial eradication mechanism. The synergistic activity was assessed with two nonantibiotics (ibuprofen and paracetamol) and 3 antibiotics (ceftazidime chloramphenicol, and trimethoprim) using methods of growth inhibitory indices (GIIs) and fractional inhibitory concentration index (FICI). The highly effective methanolic extracts were of W. somnifera and C. roseus, and the synergism was obtained in terms of GIIs and FICI values of 0.9 and 0.3 and 0.9 and 0.5, respectively, fulfilling the criteria for both extracts, respectively. The results for combinations of plant extracts and antibiotics ceftazidime, trimethoprim, and chloramphenicol and nonantibiotics (analgesic drugs) ibuprofen and paracetamol showed good synergistic activity against the MDR isolates of S. Typhi.

Haq, A et al., in the paper titled "Comprehensive investigation on the synergistic antibacterial activities of Jatropha curcas pressed cake and seed oil in combination with antibiotics", published in AMB Express, 9, 67, 2019 illustrates antibacterial potential of Jatropha curcas crude seed extracts, seed oil, commercially available antibiotics, and their combinations for their synergistic effect against clinical, MDR and ATCC bacterial strains by agar well diffusion assay. Methanolic extracts remained more active against Staphylococcus aureus (ATCC), with zone of inhibition (ZOI) of 21 mm, than clinical and methicillin-resistant S. aureus (MRSA) strains (ZOI range?~?15.0-17.0 mm). Moxifloxacin exhibited greater activity against Escherichia coli (ATCC) (ZOI?~?50.0 mm), followed by ofloxacin against Pseudomonas chlororaphis (47.3 mm), moxifloxacin against P. monteilii (47 mm), P. aeruginosa (46.3 mm) and MRSA2 (46 mm) and ofloxacin against S. aureus (ATCC) strains (45.7 mm).

Although many non-antibiotic compounds showing synergistic effects with antibiotics have been identified or discovered in vitro. However, the prior art doesn't talk about the combination's stability, efficacy, and safety. The combination's pharmacokinetics, pharmacodynamics, and bioavailability distributions. The toxicity and side effects of combination drug. There is an urgent need to provide a solution to these problems.

OBJECTIVE OF THE INVENTION:
It is a main objective of the present invention is to provide a synergistic composition of an antibiotic with trans-Cinnamaldehyde against Multi-Drug-Resistant bacteria.
Another objective of the present invention is to provide a synergistic composition of an antibiotic with trans-Cinnamaldehyde that can act on extended spectrum beta-lactamase (ESBL) and metallo-beta-lactamases (MBL) producing bacterial strains.

Yet another objective of the present invention is to provide a synergistic composition of an antibiotic with trans-Cinnamaldehyde that can disrupt preexisting biofilm of bacterial strains

Yet another objective of the present invention is to provide a synergistic composition of an antibiotic with trans-Cinnamaldehyde that can inhibit biofilm synthesis of bacterial strains.

SUMMARY OF THE INVENTION:
In an embodiment, the present invention provides a synergistic composition against multi-drug-resistant bacteria comprising: an antibiotic and trans-cinnamaldehyde in a ratio of 2:1.

In another embodiment of the present invention, the antibiotic is tetracycline.

In another embodiment of the present invention, the composition is in the form of a solution.

In another embodiment of the present invention, the composition has antibacterial activity against gram negative bacteria selected from Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter diversus and Citrobacter amalonaticus.

In another embodiment of the present invention, the composition has antibacterial activity against gram positive bacteria selected from Staphylococcus epidermidis.

In another embodiment of the present invention, the composition has a fractional inhibitory concentration index for Staphylococcus epidermidis of 0.25.

In another embodiment of the present invention, wherein the composition has a fractional inhibitory concentration index for Escherichia coli of 0.5.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

Figure 1 illustrates scanning electron microscopy images of (a) Treated cells with the FIC value of 0.25 for the combination (b) Untreated cells.

Figure 2 illustrates MTT assay for the viability of vero cell line.

DETAILED DESCRIPTION OF THE INVENTION:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully interpreted and comprehended. However, any skilled person or artisan will appreciate the extent to which such embodiments could be generalized in practice.

It is further to be understood that all terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting in any manner the scope.

Unless defined otherwise, all technical and scientific expressions used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertains.

In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below which are known in the state of art.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Also, the term 'composition' does not limit the scope of the invention for multiple compositions that can be illustrated for best mode of the invention.

All modifications and substitutions that come within the meaning of the description and the range of their legal equivalents are to be embraced within their scope. A description using the transition "comprising" allows the inclusion of other elements to be within the scope of the invention.
As used herein, the term "Planktonic" refers to the floating or weakly swimming animal and plant life of a body of water. The planktonic bacteria is a free-living bacteria.

The combination of commercial antibiotics with plant metabolites could be a novel approach for targeting the issues associated with existing problems, like antibiotic resistance as well as biofilm formation. The approach behind using the combination is diversified targeting and will be addressed by different components of the formulations. The chances of developing resistance against plant compounds are negligible with less toxicity. To overcome the issues of antibiotic resistance and biofilm formation, the present invention has combined trans- cinnamaldehyde with tetracycline to curtail planktonic and biofilm growth of drug resistant clinical organisms.

In an aspect, the present invention provides a synergistic composition against multi-drug-resistant bacteria, comprising:
an antibiotic and trans-cinnamaldehyde in a ratio of 2:1.

In an embodiment of the present invention, the antibiotic is tetracycline.

In another embodiment of the present invention, the composition is in the form of a solution, suspension or tablet, preferably solution.

In another embodiment of the present invention, the solution has concentration of tetracycline and cinnamaldehyde as 15.6 and 31.25 µg/ml respectively.

In another embodiment of the present invention, the composition has antibacterial activity against gram negative bacteria selected from Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter diversus and Citrobacter amalonaticus.

In another embodiment of the present invention, the composition has antibacterial activity against gram positive bacteria Staphylococcus epidermidis.

In another embodiment of the present invention, the composition has a fractional inhibitory concentration index for Staphylococcus epidermidis of 0.25.

In another embodiment of the present invention, wherein the composition has the fractional inhibitory concentration index for Escherichia coli of 0.5.

In an embodiment of the present invention, the trans-cinnamaldehyde (125µg/ml) alone and tetracycline (125µg/ml) alone showed the inhibition of bacterial strain but in combination trans-cinnamaldehyde and tetracycline showed effective impact on planktonic as well as biofilm culture of ESBL and MBL producing clinical/bacterial strains.

EXAMPLES:
Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiments thereof. Those skilled in the art will appreciate that many modifications may be made in the invention without changing the essence of invention.

Example 1. Microtiter plate assay for determination of minimum inhibitory concentration.
The different concentrations of trans-cinnamaldehyde and tetracycline alone as well as their combinations were incubated with 1x106 cfu/ml of the indicator organisms S. epidermidis and E. coli in each well of the 96 well plate. Bacterial culture without drug and media alone served as control. The plates were incubated at 37°C for 16-18 hours and then examined for the growth inhibition with respect to bacterial growth control. The concentration at which the antimicrobial agent was able to inhibit >90% growth of indicator micro-organism was considered as minimum inhibitory concentration. The assay was carried out in triplicates and repeated thrice. The % inhibition was calculated by following formula:

% ????h?????????????? = [1 - {???? ?????????????????? ??????????/???? ???? ?????????????????? ??????????}]100.

The antimicrobial activity of combination (Tran-cinnamaldehyde and tetracycline) against ESBL and MBL producing bacterial strains have been tabulated in tables 1 and 2. Tran-cinnamaldehyde and tetracycline showed effective impact on planktonic as well as biofilm culture of ESBL and MBL producing clinical strains. The microtiter plate assay was used for determination of MIC of compounds alone as well as FIC of their combinations. The Tran-cinnamaldehyde and tetracycline showed FIC 0.25 and 0.5 against Gram positive and Gram-negative indicator microorganisms respectively, which falls under synergistic category. Synergistic combinations enhance the effectiveness of the combined compounds. The combination of Tran-cinnamaldehyde and tetracycline at their respective FIC values showed remarkable antibiofilm activity.

Table 1: Antimicrobial activity of combination (Tran-cinnamaldehyde and tetracycline) against ESBL producing bacterial strains.

S. no. Isolate no. Isolate name Combination's Zone of inhibition (ZOI)
(mm) MIC of ceftazidime (µg/ml) MIC of
Cefotaxime
(µg/ml) MIC of
Ceftriaxone
(µg/ml)
1 250 E. coli 23 8 =256 =256
2 253 E. coli 29.7 8 =256 =256
3 254 E. coli 27 8 =256 =256
4 263 E. coli 26.3 =256 =256 =256
5 264 E. coli 21.2 4 16 0.5
6 210 K. pneumoniae 29.5 16 =256 =256
7 275 K. pneumoniae 23.7 2 8 =256
8 296 K. pneumoniae 22.3 16 =256 0.5
9 674 K. pneumoniae 17.5 =256 =256 =256
10 9b P. mirabilis 22 64 =256 =256
11 110 P. mirabilis 26.4 =256 =256 =256
12 150 P. mirabilis 24 =256 =256 =256
13 191 P. mirabilis 24.6 32 =256 =256
14 15 C. diversus 23.2 32 =256 =256
15 30 C. diversus 21.5 64 =256 =256
16 59 C. amalonatus 25.7 128 =256 =256
17 123 C. diversus 22 =256 =256 =256


Table 2: Antimicrobial activity of combination (Tran-cinnamaldehyde and tetracycline) against MBL producing bacterial strains.

S. no. Isolate
no. Isolate
name Combination's ZOI
mm MIC of Meropenem
(µg/ml)
1 95 K. pneumoniae 29 8
2 101 K.pneumoniae 31 16
3 85 P. aeruginosa 28 16
4 214 P. aeruginosa 18 16
5 216 P. aeruginosa 28 0.064
6 463 E. coli 23 16
7 467 E. coli 25 16
8 526 E. coli 29 8
9 621 E. coli 21 2.5
S. no. Isolate
no. Isolate
name Combination's ZOI
mm MIC of Meropenem
(µg/ml)
1 95 K. pneumoniae 29 8
2 101 K.pneumoniae 31 16
3 85 P. aeruginosa 28 16
4 214 P. aeruginosa 18 16
5 216 P. aeruginosa 28 0.064
6 463 E. coli 23 16
7 467 E. coli 25 16
8 526 E. coli 29 8
9 621 E. coli 21 2.5

Example 2. Determination of Fractional Inhibitory Concentration Index (FIC index).
In combinatorial studies, the interaction among different compounds was analyzed by comparing the inhibitory concentration alone and in combination. Fractional inhibitory concentration (FIC), a measure of the interaction, which could be synergistic / additive / indifferent / antagonistic, depends on the calculated value of FIC index (FICI). The checkerboard assay on 96 well plate was performed to calculate FIC. Indicator strain was treated with the different dilutions of the compound alone as well as with the combination of two compounds. The stock solutions of different antimicrobial compounds were prepared by keeping one component as constant and varying the concentration of another antimicrobial compound. The dilutions of plant compounds alone without any bacterial cells were also analyzed to take into account color effect. The FIC was calculated by using the formula given.

The FICI was determined using the following formula:
???????? = ???????? + ????????

In the formula, MICA+B represents the MIC of A in combination with B and MIC B+A is vice versa. If the FICI value is less than or equal to = 0.5 then it is considered as a synergistic combination, and value >0.5-1 is for additive combination and in between 1-4 is for antagonistic activity.

The antibacterial activity of Trans-cinnamaldehyde alone as well as in combination with tetracycline against planktonic cultures of S. epidermidis and E. coli has been tabulated in table 3 and the results show that the combination of trans-cinnamaldehyde and tetracycline showed synergistic impact on antibacterial activity with FIC value 0.25. The effect of combination (trans-cinnamaldehyde and tetracycline) with FIC 0.25 against bacterial strain can be seen in the SEM image for the treated cells Figure 1(a) in comparison to untreated cells Figure 1(b).

Table 3. Determination of antibacterial activity of Trans-cinnamaldehyde alone as well as in combination with tetracycline against planktonic cultures of S. epidermidis and E. coli.
Combi Combination nation MIC of compound alone/ MIC of combination (µg/ml) FIC FICI Result
S. epidermidis E. coli S. epidermidis E. coli S. epidermidis E. coli S. epidermidis E. coli
Trans-cinnamaldehyde /(Trans-cinnamaldehyde +
Tetracycline) 125/15.6

125 /15.6
125/31.25
125 / 31.25 FIC A = 0.125
FIC B = 0.125 FIC A = 0.25FIC B =
0.25 FICI = 0.25 FICI = 0.50 Synergy Synergy

Example 3: MTT assay.
The toxicity studies were performed using the MTT assay. The results show that cells treated with combination of tetracycline and trans-cinnamaldehyde has viability of about 99% against multidrug resistant bacterial strain. The viability shown by combination is much higher than viability for cells treated with tetracycline or trans cinnamaldehyde alone (Figure 2).

Antibiofilm activity
Antibiofilm activity of antimicrobial substance was tested against preformed biofilm and also for the inhibition of biofilm formation of indicator organism.

Example 4. Inhibition of biofilm formation.
Antimicrobial agent was incubated with 1x106 cfu/ml of indicator microorganisms in the 96 well plate and incubated at 37°C for 48 hours to allow cell attachment and biofilm development. The plate was washed two times with 20 mM phosphate buffer saline (PBS) pH 7.4 and stained with 0.2% crystal violet for 5-10 minutes. The plate was again washed with PBS and allowed to dry. The remaining stain was dissolved in 100% ethyl alcohol and the reading was taken at 595 nm. The % reduction in biofilm was evaluated as

% inhibition = ((OD of untreated biofilm - OD of treated biofilm))/((OD of untreated biofilm) ) x100
The concentration at which compound inhibited =90% inhibition of bacterial biofilm, was considered as minimum biofilm inhibitory concentration (MBIC).

Example 5. Disruption of the established biofilm.
The plate with 1x106 cfu/ml of indicator microorganism in each well was incubated at 37°C for 48 hours to allow cell attachment and biofilm development. The well containing established biofilm was treated with different concentrations of the compound and incubated at 37°C for 24 hours. The plate was properly washed with PBS after incubation to remove unbound cells. The remaining procedure was followed as previously described.

The combination of trans-cinnamaldehyde with tetracycline shows antimicrobial activity against drug resistant bacterial strains. In addition, to this the combination of trans-cinnamaldehyde with tetracycline not only disrupts the preexisting biofilm of bacterial strains but also inhibits the formation of biofilms in bacterial strains.
, Claims:1. A synergistic composition against multi-drug-resistant bacteria, comprising:
an antibiotic and trans-cinnamaldehyde in a ratio of 2:1.

2. The composition as claimed in claim 1, wherein the antibiotic is tetracycline.

3. The composition as claimed in claim 1, wherein the composition is in the form of a solution.

4. The composition as claimed in claim 1, wherein the composition has antibacterial activity against gram negative bacteria selected from Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter diversus and Citrobacter amalonaticus.

5. The composition as claimed in claim 1, wherein the composition has antibacterial activity against gram positive bacteria Staphylococcus epidermidis.

6. The composition as claimed in claim 1, wherein the composition has a fractional inhibitory concentration index for Staphylococcus epidermidis of 0.25.

7. The composition as claimed in claim 1, wherein the composition has the fractional inhibitory concentration index for Escherichia coli of 0.5.