1. INTRODUCTION:

‏The discovery of antibiotics led to tremendous development in treating many bacterial diseases. However, this development did not continue due to the emergence of antibiotic-resistant bacteria.

Factors such as the overuse and inappropriate use of antibiotics may contribute directly to the emergence of antibiotic-resistant bacteria^1,². According to Threats 2020, over 2.8 million antibiotic-resistant infections have been reported in the United States each year, resulting in over 35,000 deaths³. The increased rate of infections is mainly due to carbapenem-resistant Acinetobacter, carbapenem-resistant Enterobacteriaceae (CRE), Candida auris, drug-resistant Neisseria gonorrhoeae, and drug-resistant Clostridioides difficile. To a lesser extent, Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a significant problem and one of the most frequently encountered antibiotic-resistant pathogens in U.S. hospitals⁴.

To overcome this challenge, an effort must develop a new, effective antibiotic therapy. There are several strategies to develop effective antibacterial agents and minimize antibiotic-resistant phenomena. The combination strategy is one of the most attractive strategies that could be relied upon to reduce the opportunity for bacteria to develop resistance⁵. Combining two effective antibacterial agents confers the ability to inhibit or kill bacteria by targeting multiple sites. Also, combining an antibiotic with an adjuvant may help lower the likelihood of bacteria developing resistance. A common adjuvant-antibiotic medication is the combination of amoxicillin and clavulanic acid, a beta-lactamase inhibitor. Clavulanic acid acts by inhibiting the production of the beta-lactamase enzyme by bacteria, allowing the antibiotic to impede the formation of bacterial cell walls. This combination has restored the effectiveness of beta-lactam antibiotics. Hence, they have been used as a drug of choice for the treatment of urinary and respiratory tracts infections and others⁶. This combination enables the ongoing use of beta-lactam antibiotics to combat diseases caused by beta lactamase-producing bacteria^7,⁸. It is also sparked the development of additional adjuvant-antibiotic regimens to overcome the antibiotic-resistant phenomenon.

Due to their broad biological activities, plants’ secondary metabolites have attracted scientists, and thousands of them have been discovered^9,¹⁰_. Eucalyptol, γ-terpinene, p-cymol and punicalagin are naturally occurring plant metabolites that have been screened and found to possess broad biological activities^11-17_. Eucalyptol, γ-terpinene and p-cymol belong to the terpene class, and are among the most common essential oil components of thyme and oregano species¹⁸. Punicalagin is tannin isolated from the Punica granatum plant¹⁹. Extracts rich in eucalyptol, γ-terpinene, p-cymol, or punicalagin have antibacterial activity^20-23_. Some of these compounds have been reported to induce the activity of conventional antibiotics^24,²⁵_. Therefore, this study was designed to evaluate the combination effect of eucalyptol, γ-terpinene, p-cymol and punicalagin with cefotaxime against three methicillin (oxacillin) resistant bacterial species including S. aureus, E. aerogenes and K. pneumoniae and two methicillin (oxacillin) susceptible bacterial species including S. epidermidis and E. coli. To the best of our knowledge, this is the first study about the combination effect of p-cymol and punicalagin with cefotaxime.

2. MATERIALS AND METHODS:

2.1 Antibiotics and tested compounds:

The antibiotics used in this study were purchased from Gulf Pharmaceutical industries (Ras Al Khaimah, UAE). The antibiotics discs were from Liofilchem (Italy). Eucalyptol, γ-terpinene, p-cymol and punicalagin were purchased from Sigma-Aldrich.

2.2. Bacterial strains:

Five bacterial isolates were collected from two different hospitals in Jordan, Karak Governorate Hospital and Al Bashir Hospital, from 9/2019 to 2/2020. These bacteria were S. aureus, S. epidermidis, E. coli, E. aerogenes and K. pneumoniae which were obtained from urine samples of patients who were diagnosed with urinary tract infections. All bacterial isolates were identified, and their antibiotic resistant profiles were determined using the Biomérieux VITEK® 2 system.

2.3 Confirmation of methicillin (oxacillin) resistant strains:

Oxacillin resistant strains were characterized using a cefoxitin-based method²⁶. The results were interpreted as previously described²⁷.

2.4. Antibacterial activity:

2.4.1. Disc diffusion method:

The disc diffusion method was used to evaluate the antibacterial activity of eucalyptol, γ-terpinene, P-cymol and punicalagin. Briefly, a bacterial culture containing 1 × 10⁸CFU/mL was prepared according to the 0.5 McFarland suspensions. Then, 100µl of the bacterial culture was transferred and spread on Mueller-Hinton agar plates using a sterile swab. Using sterile forceps, discs containing 1mg of eucalyptol, γ-terpinene, P-cymol or punicalagin were transferred to the surface of the inoculated agar. After 24 hours of incubation at 37°C, the inhibition zone was measured as mm in diameter. Each compound was tested in triplicates.

2.4.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC):

The Minimum Inhibitory Concentration (MIC) was determined using the microdilution method. This was performed using 96 well plates according to Wambaugh et al. (2018) with some modifications²⁸.

The MBC values were assigned by culturing the continent of the wells with concentrations equal to or greater than the MIC onto agar plates. MBC was defined as the lowest concentration of the compounds or antibiotics that kills the tested bacteria (no growth on the agar plate) after 24 hours at 37°C.

2.5. Synergistic effect:

2.5.1. Disc diffusion method and increase in folding area (IFA):

The combined effect of the compounds with antibiotics was determined using the disc diffusion method. Mueller-Hinton agar plates were inoculated with 100µl of the bacterial culture containing 1 × 108CFU/mL for this experiment. Disc containing antibiotics, tested compounds (1mg/disc), or a mixture of antibiotics and compounds were placed on the surface of inoculated agar. The inoculated plates were incubated at 37°C. After 24h, the inhibition zone was measured as mm in diameter.

2.5.2. Checkerboard Assays:

The checkerboard assay was used to determine the synergistic effect between cefotaxime and eucalyptol, γ-terpinene, p-cymol and punicalagin as previously described²⁸ with some modification.

2.6. Growth curve:

The effect of cefotaxime and p-cymol and punicalagin alone or in combination on the growth of S. aureus was evaluated at a concentration equal to ½ MIC. The test was performed using a 96-well plate and the incubation was done at 37°C. The growth of the treated and non-treated cultures was evaluated at fixed intervals (2h) using 96 well plate spectrophotometer at 600nm.

2.7. Statistical analysis:

A GraphPad Prism ANOVA was used to evaluate statistical differences between groups, followed by Dunnett's post hoc test. A p-value of less than 0.05 was considered statistically significant for all statistical analyses.

3. RESULTS:

3.1. Confirmation of methicillin (oxacillin) resistant strains:

Phenotypic confirmation of methicillin (oxacillin) resistant strains was made using a cefoxitin-based method. According to the CLSI (2020) breakpoints, S. aureus, E. aerogenes and k. pneumoniae were characterized as methicillin (oxacillin) resistant strains. However, S. epidermidis and E. coli were methicillin (oxacillin) susceptible strains. Similarly, the resistant pattern of these strains matches that of the Biomérieux Vitek^® 2 System.

3.2. Antibacterial activity of Eucalyptol, γ-Terpinene, p-Cymol and Punicalagin:

The antibacterial activity of eucalyptol, γ-terpinene, p-cymol and punicalagin was evaluated using the disc diffusion method (table 1) against five bacterial species. The result showed that eucalyptol, γ-terpinene and p-cymol exhibited weak or no antibacterial activity. This can be easily indicated by the low inhibition zone observed (0-8mm). Compared to other tested compounds, punicalagin showed significant antibacterial activity against all strains tested except S. epidermidis. The inhibition zones observed for punicalagin against E. aerogenes, K. pneumoniae, S. aureus and E. coli were 26, 23.5, 18 and 16.5mm, respectively.

Moreover, the MIC and MBC values were determined to evaluate the bacteriostatic and bactericidal potential of eucalyptol, γ-terpinene, p-cymol and punicalagin. Generally, the zone of inhibition values was reflected in MIC values. The MIC values of eucalyptol, γ-terpinene and p-cymol were higher than those of punicalagin against all strains tested. As shown in table 2, the lowest MIC value observed was 0.08mg/mL for punicalagin against S. aureus. Punicalagin was also effective against E. aerogenes, E. coli, K. pneumoniae and S. epidermidis with MIC values of 0.16, 0.31, 0.63 and 1.25mg/mL, respectively. Ranked second is the p-cymol activity against S. aureus and E. coli with MIC values equal to 0.63 and 1.25mg/mL, respectively. The MIC values of eucalyptol and γ-terpinene ranged from 5 to ˃10mg/mL.

Interestingly, the MIC values of cefotaxime (table 2) were equal to the MIC values of cefoxitin. The lowest MIC value for cefotaxime was 7.5µg/mL against E. coli followed by S. aureus (60µg/mL). The MIC values for cefotaxime against K. pneumoniae, E. aerogenes and S. epidermidis were more than 120µg/mL.

On the other hand, the maximum bactericidal activity indicated was for punicalagin against S. aureus and E. aerogenes (0.16mg/mL, each), followed by K. pneumoniae (0.63mg/mL), E. coli (2.5mg/mL) and S. epidermidis (5 mg/mL). The MBC values of eucalyptol, γ-terpinene, p-cymol ranged from 5 to ˃10mg/mL. The exception to this was the MBC value of p-cymol against S. aureus (1.25mg/mL).

Table 1: Antibacterial activity using disc diffusion methods

Bacteria	Inhibition zone (mm±SD)
Bacteria	Eucalyptol	γ-Terpinene	p-Cymol	Punicalagin
*S. aureus*	0	0	0	18.0±0.0***
*S. epidermidis*	0	0	0	0
*E. coli*	0	0	8±0.0	16.5±0.5***
*E. aerogenes*	0	7±0.0	0	26.0±0.0***
*K. pneumoniae*	0	0	7±0.0	23.5±0.3***

Each disc contains 1 mg of the tested compounds

Table 2: MIC and MBC values of eucalyptol, γ-terpinene, p-cymol and punicalagin.

Bacteria	Cefotaxime µg/mL		Eucalyptol mg/mL		γ-Terpinene mg/mL		p-Cymol mg/mL		Punicalagin mg/mL
Bacteria	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
*S. aureus*	60	-	10	10	2.5	˃10	0.63	1.25	0.08	0.16
*S. epidermidis*	˃480	-	10	˃10	10	˃10	5	10	1.25	5
*E. coli*	7.5	-	5	10	10	˃10	1.25	5.0	0.31	2.5
*E. aerogenes*	240	-	10	˃10	5	˃10	5	5	0.16	0.16
*K. pneumoniae*	120	-	˃10	˃10	˃10	˃10	5	10	0.63	0.63

“-“ not determined

Table 3: IFA and Antibacterial activity of eucalyptol, γ-terpinene, P-cymol and punicalagin combined with β-lactam antibiotics against S. aureus.

Antibiotics (µg)	Antibiotics (mm)	+Eucalyptol (mm)	IFA	+ γ-Terpinene (mm)	IFA	+ p-Cymol (mm)	IFA	+Punicalagin (mm)	IFA
Oxacillin (1)	13.3	10.5	-0.38	10.5	-0.38	17.2	0.67	18.5	0.93
Ampicillin (10)	21.2	14.5	-0.53	15.3	-0.48	20.3	-0.08	21.2	0.00
Cefoxitin (30)	16.7	16.2	-0.06	19.7	0.39	21.3	0.63	25.7	1.37
Piperacillin (100)	16.3	15.7	-0.07	17	0.09	24.7	1.30	20.5	0.58
Cefotaxime (30)	15.3	15.3	-0.00	16.5	0.16	21.5	0.97	23.5	1.36
Cefixime (5)	13.7	14	0.04	18	0.73	20.3	1.20	20.2	1.17
Cefazolin (30)	35.0	26.5	-0.43	25.2	-0.48	28.3	-0.35	18.3	-0.73
Cefuroxime (30)	32.3	23.7	-0.46	24.3	-0.43	24	-0.45	31	-0.08
Imipenem (10)	35.5	7	-0.96	20.3	-0.67	18	-0.74	26	-0.46

Table 4: FIC index of eucalyptol, γ-terpinene, p-cymol and punicalagin combined with Cefotaxime against S. aureus.

Agents	MIC (µg/mL)		FIC index	Inference
	Alone	In combination
Cefotaxime	60	60	2.0	Indifferent
Eucalyptol	10000	10000
Cefotaxime	60	60	1.12	Indifferent
γ-Terpinene	2500	300
Cefotaxime	60	30	0.75	Additive
P-Cymol	630	160
Cefotaxime	60	7.5	0.38	Synergy
Punicalagin	80	20

3.4. Effect of ½ MIC of cefotaxime, p-cymol and punicalagin alone or in combination on the growth curve of S. aureus:

As shown in figure 1, punicalagin and p-cymol exhibited remarkable inhibition activity against S. aureus. A reduction in S. aureus growth was reported when treated with ½ MIC of punicalagin and p-cymol for the first 10 hours. After that, the growth of treated S. aureus gradually increased, restored at the end of the experiment. In the case of the combination of cefotaxime with punicalagin and p-cymol, the growth reduction was stable and there was no increase in the growth to the end of the experiment (24 h).

Fig. 1: S. aureus growth curve in the absent (Normal) and present of ½ MIC of cefotaxime, punicalagin, P-cymol, cefotaxime+ punicalagin and cefotaxime+P-cymol.

4. DISCUSSION:

Many researchers have concentrated on the identification of natural substances that improve bacterial drug susceptibility in order to combat the evolution of antibiotic-resistant strains. This therapeutic method has been found to be successful against bacteria resistant to antibiotics. In this study, we tested the antibacterial effects of four compounds; eucalyptol, γ-terpinene, p-cymol, and punicalagin against three methicillin (oxacillin) resistant strains and two methicillin (oxacillin) susceptible strains that have been isolated from urine samples of patients who have been diagnosed with urinary tract infection. The comination effect of these compounds on the antibacterial activity of methicillin (oxacillin) resistant S. aureus strain with nine β-lactam antibiotics was evaluated.

This study used a cefoxitin-based method to confirm the phenotypic confirmation of methicillin (oxacillin) resistant strains. The cefoxitin-based method is an alternative method to detect methicillin (oxacillin) resistant strains. The cefoxitin disc diffusion method is preferable over the oxacillin dilution method. The cefoxitin disc diffusion method is equivalent to the oxacillin dilution method, but it is easier to read and more accurate for identifying oxacillin-resistant strains ²⁷.

Methicillin (oxacillin) resistant clinically isolated strains were used in this study. These strains were resistant to beta-lactam antibiotics, including oxacillin, ampicillin, cefoxitin, piperacillin, cefotaxime and cefixime. In general, the mechanism of resistance is due to the ability of these bacteria to produce beta-lactamase enzyme, which in turn acts to break down the antibiotic's beta-lactam ring²⁹.

In this study, the antibacterial activity of eucalyptol, γ-terpinene, p-cymol and punicalagin was evaluated using disc diffusion and microdilution methods. Punicalagin was the most effective compound tested, followed by p-cymol. Eucalyptol and γ-terpinene showed weak or no antibacterial activity, as recorded by the higher MIC values. In particular, p-cymol exhibited bacteriostatic and bactericidal activity against S. aureus and E. coli (0.63 to 5mg/mL). Miladi reported similar results, for the inhibitory effect of p-cymol against seven orally isolated S. aureus strains (MIC values ranged from 32-512μg/mL and MBC values ranged from 128-512 μg/mL)²⁰. In another study, p-cymol was effective against Bacteroides fragilis and Clostridium perfringens with an inhibition zone of 15 and 20mm, respectively³⁰. Other studies have found that p-cymol has no antimicrobial activity against Shigella sonnei, Shigella flexneri, Escherichia coli, or Vibrio cholera^15,31,³²^.

The lowest MIC observed for γ-terpinene was 2.5 and 5 mg/mL against S. aureus and E. aerogenes, respectively, while the MBC values were ˃10mg/mL against all tested strains. These results agreed with Eftekhar et al. (2009), who found the MIC values of γ-terpinene against S. aureus and E. coli were ˃15 and 7.5mg/mL, respectively²¹. A study conducted by Li, Li et al., (2014) showed that γ-terpinene has bacteriostatic and bactericidal activity against S. aureus ATCC 25923, S. enteritidis CMCC (B) 50041 and E. coli ATCC 25922 with equal MIC and MBC values of 0.781, 3.125 and 1.562µL/mL, respectively³³.

Eucalyptol in this study exhibited weak antibacterial activity as indicated by the highest MIC (5-10mg/mL) and MBC (10- ˃10mg/mL) values. The results of this study concerning the activity of eucalyptol were similar to those reported by Lima, Silva et al. (2021) who found that the MBC of eucalyptol (1, 8 Cineole) was 28.6 mg/mL against S. aureus, E. faecalis and S. pyogenes ³⁴. Similarly, eucalyptol was bacteriostatic against S. aureus, MRSA, E. coli and P. aeruginosa at concentrations ranging from 16 to ˃256mg/mL whereas it was bactericidal at concentrations ranging from 128- 512mg/mL. Several studies reported the antibacterial activity of 1, 8 Cineole against 28 MRSA strains. They found that most of the tested strains were susceptible to 1, 8 cineole at MIC values of 0.049mg/mL and an MBC value of ˂50mg/mL^22,³⁵_.

This study revealed a remarkable bacteriostatic and bactericidal activity of punicalagin against all tested strains with MIC values ranging from 0.08 to 1.25 mg/mL and MBC values ranging from 0.16 - 5mg/mL. Gosset-Erard et al. (2021) reported that the MIC values of punicalagin against S. aureus ATCC 6538 and E. coli ATCC 25922 were 0.60 and 1.2µg/mL, respectively²³ . Xu Y et al. (2017) found that punicalagin possessed antibacterial activity against S. aureus as indicated by the inhibition zone observed at 1mg (11.1mm) and an MIC value of 0.25mg/mL³⁶.

Here, the combination of beta-lactam antibiotics (Oxacillin, Ampicillin, Cefoxitin, Piperacillin, Cefotaxime, and Cefixime) and punicalagin and p-cymol showed better antibacterial activity against S. aureus. The synergistic activity between cefotaxime and punicalagin has been evaluated here using the checkerboard assay. This synergistic potential can be confirmed from the results of the growth curves since there was no significant increase in the growth of S. aureus to the end of the experiments when treated with the combination formula.

In this study, an additive effect was observed for the combination of p-cymol with cefotaxime. However, the combination of p-cymol with cefotaxime led to a significant reduction in the MIC of cefotaxime from 60 to 7.5µg/mL against S. aureus. p-Cymol containing essential oils combined with cefotaxime exhibited synergistic potential against beta-lactamase-producing E. coli²⁵. In addition, significant improvements in carvacrol, thymol, γ-Terpinene were reported when tested in combination with p-cymol^20,³⁷.

5. CONCLUSION:

Although it was tested using one strain of methicillin (oxacillin) resistant S. aureus, punicalagin-induced reversal activity on methicillin (oxacillin) resistant S. aureus in this study. Punicalagin improved S. aureus susceptibility to cefotaxime, suggesting that using these two medicines together can reverse beta-lactam resistance in methicillin (oxacillin) resistant S. aureus. Further works are required to generalized this result and then it may be useful for treating diseases caused by methicillin (oxacillin) resistant strains.

6. DECLARATION OF COMPETING INTEREST:

All authors declare no conflict of interest concerning the manuscript.

7. ABBREVIATIONS:

E. coli, Escherichia coli, E. aerogenes, Enterobacter aerogenes, FICI, fractional inhibitory concentration index; IFA, Increase in Folding Area; K. pneumoniae, Klebsiella pneumoniae, MBC, Minimum Bactericidal Concentration; MIC, Minimum Inhibitory Concentration, MRSA, Methicillin-resistant Staphylococcus aureus, S. aureus, Staphylococcus aureus; S. epidermidis, Staphylococcus epidermidis.

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Received on 03.02.2022 Modified on 25.05.2022

Research J. Pharm. and Tech 2022; 15(9):3905-3911.

DOI: 10.52711/0974-360X.2022.00654