The Effect of Combinations of Antibiotics and Natural Products on the Antimicrobial Resistance of Staphylococcus aureus and Pseudomonas aeruginosa

All published articles of this journal are available on ScienceDirect.

RESEARCH ARTICLE

The Effect of Combinations of Antibiotics and Natural Products on the Antimicrobial Resistance of Staphylococcus aureus and Pseudomonas aeruginosa

The Open Infectious Diseases Journal 21 May 2024 RESEARCH ARTICLE DOI: 10.2174/0118742793303419240422094438

Abstract

Introduction/Background

The steadily increasing bacterial resistance to existing antimicrobial drugs is a significant issue, hence, it is imperative to look out for new approaches to bacterial therapy Occasionally, effective inhibitory action is not produced when antibiotics are used alone. To overcome this problem, a combination of drugs is often used. One approach to treat infectious diseases is the use of a combination of antibiotics together with plant extracts or phytochemicals. For patients with serious infections caused by pathogens resistant to drugs, combination therapy is beneficial and useful.

Materials and Methods

Seven antibiotics were obtained from a local pharmacy (gentamicin, ceftazidime, ciprofloxacin, doxycycline, amoxicillin, ceftriaxone, and azithromycin). Minimum inhibitory concentrations (MIC) were determined by broth micro-dilution method, and different antimicrobial combinations were studied on 20 Multidrug-resistant (MDR) clinical isolates (10 S. aureus and 10 P. aeruginosa). Moreover, the antibacterial activity of some volatile oils (limonene, rosemary, salvia, thymus, and black pepper), plant extracts (moringa seed, curcumin, and capsicum), and phytochemicals (thymol, and chitosan) was detected against S. aureus and P. aeruginosa isolates using broth micro-dilution method.

Results

According to our findings, ceftriaxone and ciprofloxacin or gentamicin together exhibited a substantial synergistic effect against S. aureus. Moreover, the combination of amoxicillin with ceftazidime was synergistic to reduce MIC by five to six times. Regarding MDR clinical isolates of P. aeruginosa, the combination of azithromycin with doxycycline exhibited a decrease of MIC of azithromycin by about five to sixfold. The combination of gentamicin with ceftriaxone was significant. For natural compounds, thymol, rosemary oil, curcumin, capsicum, and moringa seed extract exhibited the highest synergistic activity with the tested antibiotics against S. aureus and P. aeruginosa.

Conclusion

In conclusion, the lack of new antibiotics necessitates the improvement of existing ones. Our study shows that antibiotic combinations and antibiotic-natural plant combinations are very promising strategies for combating complex bacterial resistance.

Keywords: Antimicrobial resistance, Combination, Synergism, Plant extracts, Phytochemical structure-function relationship.

1. INTRODUCTION

There are many factors contributing to the emergence and dissemination of antibiotic resistance [1]. A significant factor to consider is the use of antibiotics by humans. Not surprisingly, the level of antibiotic-resistant infections strongly correlates with the level of antibiotic consumption [2-4]. Self-medication certainly lacks the attributes of a successful therapy, such as proper diagnosis, suitable antibiotic choice, correct usage, and treatment efficiency monitoring, thus contributing to the mounting resistance problem [5].

Combination antibiotic therapy is frequently used to treat severe infections. Potential achievements with combinations as compared with monotherapy include a broader antibacterial spectrum, synergistic effects, and reduced risk for emerging resistance during therapy. Combinations are increasingly employed to enhance the antibacterial effects of available drugs against MDR strains [6].

In the treatment of infections caused by MDR pathogens, including Staphylococcus aureus and Pseudomonas aeruginosa, combinations of antibiotics have often been used [7]. Combinations of antibiotics with drugs that block resistance mechanisms demonstrate an in vitro activity against resistant clinical isolates, which are more likely to result in successful therapeutic results. Therefore, evidence of in vitro synergism could be useful in selecting the most favorable combinations of antimicrobials for the therapy of serious bacterial infections [8].

Furthermore, because of their bactericidal qualities, plants produce a wide variety of secondary metabolites that play a significant role in shielding the plant against microbial pathogens and predators [9]. It is estimated that there are more than 3,000 essential oils (EOs) among these secondary metabolites [10, 11]. For these reasons, they are extensively utilized in the food and medical industries. The scientific research community is working to discover new applications and uses for alternative natural compounds because of the growing interest in these substances. These substances have been studied in vitro and have demonstrated promising actions against a variety of food-borne diseases and spoilage microbes [12].

Plants can be prospective sources of natural MDR inhibitors that can modulate the performance of antibiotics against resistant strains [13]. It is anticipated that the screening of plant components for antibiotic synergy may open up new avenues for MDR inhibitor isolation. Many substances that have been extracted from plants have also been shown to have the ability to lower the MICs of antibiotics against resistant organisms in vitro. For example, Polyphenols (epicatechin gallate and catechin gallate) have been reported to reverse β-lactam resistance in MRSA [14-16]. Moreover, diterpenes, triterpenes, alkyl gallates, flavones, and pyridines have also been reported to have resistance-modulating abilities to various antibiotics against S. aureus resistant strains [17, 18].

The present study aimed to combat such resistance by different combinations of antimicrobial agents and natural products against resistant isolates and assess the in vitro interaction of antimicrobial combinations.

2. MATERIALS AND METHODS

2.1. Bacterial Isolates

20 clinical isolates were obtained from the culture collection of the department. All specimens were originally obtained from septic surgical wounds, diabetic foot, diabetic ischemia, bed sore, abscess, respiratory catheters, and urine. Samples were collected aseptically from patients attending Zagazig University Hospital and transported to the Microbiology laboratory at Faculty of Pharmacy, Zagazig University, and were immediately processed.

Identification of S. aureus was confirmed by growth on MSA (oxoid), and P. aeruginosa was confirmed by using cetrimide agar (Sigma-Aldrich, USA). The antibiotic resistance pattern of S. aureus and P. aeruginosa was determined to be multidrug resistance bacteria (MDR).

2.2. Antibiotic Drugs and Natural Products

Seven Antimicrobials were evaluated in the current study, including gentamicin (EIPICO) and ceftazidime (Glaxo, Smithkline Pharmaceuticals). Ciprofloxacin (EIPICO), doxycycline (Nile Co. for Pharmaceuticals and Chemical Industries-A.R. E), amoxycillin (ADCO), ceftriaxone (SANDOZ), and azithromycin (Pfizer).

The volatile oils of the aerial parts of thyme (Thymus vulgaris), sage (Salvia officinalis), and rosemary (Rosmarinus officinalis); orange fruit`s rind (Citrus sinensis) and black pepper fruits (Piper nigrum) were obtained by hydrodistillation using Clevenger apparatus, according to Egyptian pharmacopeia [19]. The alcoholic extracts of morenga seeds (Morenga oleifera) and cayenne fruits (Capsicum minimum) were used, and the curcuminoid of turmeric rhizome (Curcuma longa) mixture was isolated from the alcoholic extract of Curcuma powder as described previously [20]. Thymol and VetoChitosan (hard gelatin capsule) were purchased from Sigma (Chemical Co, U.S.A).

2.3. Determination of Minimum Inhibitory Concentration (MIC) of Tested Antibiotics and Natural Compounds

For MIC determination, stock solutions of antibiotics were prepared by dissolving the drugs in DMSO (Sigma Aldrich). The stock solutions were serially diluted with Muller Hinton broth (Oxoid) in 96-well microtiter plates. Each antimicrobial agent received an addition of 100 μL bacterial suspension (equivalent to the volume of diluted antimicrobial solution). Plates were incubated at 37 °C for 16 hours, and the result was read. Inoculum density and preparation, incubation conditions, and determination of MIC endpoints followed the specifications given in Clinical and Laboratory Standard Institute (CLSI) guidelines [21, 22]. The amount of growth in each well compared with positive growth control and the MIC was recorded [23]. Furthermore, the tested natural compounds were serially diluted two-fold in Muller Hinton broth (MHB). The samples were incubated for 18 hours at 37°C. After incubation, the last tube without any visible growth of the bacteria was taken to represent the MIC. Control samples (positive and negative) were incubated under the same conditions [24].

2.4. Synergy Testing Methods

The assay of combinations was designed to include antimicrobials from different classes in 2-fold serial dilutions [25, 26]. The broth microdilution method is used to assess antibiotic combinations in vitro using static antibiotic concentrations (1/4, 1/8 MIC). The data produced by the broth microdilution assay were analyzed in terms of the fractional inhibitory concentration index (FIC). The FIC index was calculated according to the previous formula [27]. Antimicrobial combinations that resulted in a 4-fold reduction in the MIC compared with the MICs of agents alone were considered synergistic (FIC ≤ 0.5). FICs in the 0.5 to 1.0 range are considered non-synergistic or additive. FICs from 1 to 4 are defined as indifferent, while those of >4 are defined as antagonistic.

3. RESULTS

3.1. Antibiotic-antibiotic Combinations

Our results revealed that the combination of ceftriaxone with either gentamicin or ciprofloxacin for S. aureus was significantly synergistic (Table 1). The combination of azithromycin with doxycycline exhibited a decrease in MIC of doxycycline about five to six-fold. Moreover, the combination of amoxicillin with ceftazidime was synergistic and exhibited a decrease of MIC by 5-6 folds.

For MDR clinical isolates of P. aeruginosa, the combination of the antimicrobial agent gentamicin with ceftriaxone was significant as MIC of ceftriaxone decreased by fourfold. For P. aeruginosa, the combination of cefadroxil and gentamicin (β-lactam and amino- glycoside) was found to be the most effective (Table 2) as it showed more than 80% inhibition.

Table 1.
Antibiotic-antibiotic combinations against staphylococcus aureus.
Combination MIC of Drug Alone MIC in Combination FIC
CTX+ 1/4 MIC of gentamicin 2048 256 0.375 (S)
Doxycycline+1/4 MIC of azithromycin 128 4 0.28 (S)
CTX+1/4 MIC of ciprofloxacin 2048 64 0.28 (S)
Amoxicillin+1/4 MIC of ceftazidime 1024 32 0.28 (S)

Abbreviations: CTX: Ceftriaxone antibiotic, S: synergistic.

Table 2.
Antibiotic-antibiotic combinations against pseudomonas aeruginosa.
Combination MIC of Drug Alone MIC in Combination FIC
Ciprofloxacin+1/4 MIC of gentamicin 32 16 0.75 (A)
CTX+1/4 MIC of gentamicin 2048 256 0.375 (S)
Ceftazidime+1/4 MIC of gentamicin 2048 2048 1.25 (I)
Ceftazidime+1/4 MIC of ciprofloxacin 2048 2048 1.25 (I)

Abbreviations: S: synergistic, A: additive, I: indifferent.

Table 3.
The effect of sub-inhibitory concentration (1/4MIC) of the essential oils (thymus and limonene, rosemary, salvia, and black pepper) on the MIC of different antimicrobials.
Antibiotic FIC in the Presence of 1/4MIC of
Thymus Limonene Salvia Black Pepper Rosemary
Doxycycline S 0.3125 (S) 0.3125 (S) 0.3125 (S) 0.3125 (S) 0.375 (S)
Ceftriaxone S 0.28125 (S) 0.5 (S) 0.375 (S) 0.28125 (S) 0.5 (S)
Amoxicillin S 0.2539 (S) 0.2656 (S) 0.2578 (S) 0.28125 (S) 0.2578 (S)
Azithromycin S 0.28125 (S) 0.28125 (S) 0.2656 (S) 0.28125 (S) 0.28125 (S)
Gentamicin P 0.5 (S) 0.75 (A) 0.5 (S) 0.75 (A) 0.5 (S)
Ceftazidime P 0.75 (A) 0.75 (A) 1.25 (I) 1.25 (I) 1.25 (I)
Ciprofloxacin P 0.3125 (S) 0.75 (A) 0.75 (A) 0.75 (A) 0.75 (A)

Abbreviations: FIC: fractional inhibitory concentration, S: synergistic, A: additive, I: indifferent, S:Staphylococcus aureus, P:Pseudomonas aeruginosa.

Table 4.
The effect of plant extracts (capsicum, moringa seed extract and curcumin) and phytochemicals (thymol and chitosan) on the MIC of tested antimicrobial agents.
Antibiotic FIC in the Presence of 1/4MIC of
Capsicum Extract) Moringa seed Extract) Curcumin Extract) Thymol Chitosan
Doxycycline S 0.2656 (S) 0.375 (S) 0.3125 (S) 0.2656 (S) 1.25 (I)
Ceftriaxone S 0.28125 (S) 0.375 (S) 0.2812 (S) 0.28125 (S) 1.25 (I)
Amoxicillin S 0.2578 (S) 0.2578 (S) 0.5 (S) 0.25195 (S) 1.25 (I)
Azithromycin S 0.5 (S) 0.28125 (S) 0.5 (S) 0.2656 (S) 1.25 (I)
Gentamicin P 0.3125 (S) 0.3125 (S) 0.5 (S) 0.28125 (S) 1.25 (I)
Ceftazidime P 1.25 (I) 1.25 (I) 0.75 (A) 0.5 (S) 1.25 (I)
Ciprofloxacin P 0.75 (A) 0.75 (A) 0.2812 (S) 0.3125 (S) 1.25 (I)

ABbreviations; FIC: fractional inhibitory concentration, S: synergistic, A: additive, I: indifferent, S:Staphylococcus aureus, P:Pseudomonas aeruginosa.

3.2. Antibiotics and Natural Plant Combinations

The tested essential oils, plant extracts, and phytochemicals showed varying degrees of antibacterial activity in combination with antibiotics (doxycycline, ceftriaxone, amoxicillin, and azithromycin) against S. aureus and (gentamicin, ceftazidime, and ciprofloxacin) against P. aeruginosa (Tables 3 & 4). As shown in Tables 3 and 4, all essential oils were synergistic with antibiotics used against MDR S. aureus strains. In addition, a few combinations were synergistic against P. aeruginosa, including the combination of thymus oil with gentamicin or ciprofloxacin and the combination of gentamicin with salvia oil and rosemary oil. Thymol was synergistic with all tested antibiotics, while chitosan showed indifferent effects.

4. DISCUSSION

The emergence of MDR pathogenic bacterial strains represents a significant global health threat. MDR P. aeruginosa and S. aureus strains, represent a large problem in therapy [5, 28, 29]. Hence, finding new therapeutic options is an urgent demand, and the use of different drug combinations may represent a solution to this resistance crisis [7, 8, 14, 30].

Our study and other previous studies reported that the combination of cefadroxil and amoxicillin (both are β-lactam drugs) was synergistic against S. aureus, inhibiting more than 80% of the isolates [31]. Synergism between β-lactam antibiotics against P. aeruginosa strains was also reported [32]. On the other hand, synergistic interactions of β-lactam/ aminoglycoside combinations have been reported in previous studies [33-35]. It is reported that the disruption of Gram-negative bacilli's cell walls by β-lactamases allows aminoglycosides to enter the periplasmic space [36, 37]. It was also observed that this combination was also synergistic against more than 75% of S. aureus isolates.

Our results showed that the efficacy of the combination of ceftazidime and gentamicin was non-significant. Dundar and Otkun detected synergy in ceftazidime tobramycin (67%) combination against resistant strains [38]. Moreover, the efficacy of the combination of ceftazidime with ciprofloxacin was not beneficial. Our results do not agree with the synergistic activity that has been reported for combinations of β-lactams and fluoroquinolones [39-42]. Reported results of in vitro synergy between β-lactams and fluoroquinolones against Gram-negative organisms ranged from 17-82% [39, 40, 43].

Other studies evaluated the ciprofloxacin /β-lactam combination against MDR P. aeruginosa isolates and demonstrated synergy with this combination [44]. An in vitro study found that the degree of synergy between β-lactam–aminoglycoside and β-lactam–fluoroquinolone combinations was shown to be the same in an in vitro investigation involving 12 clinical isolates of P. aeruginosa, with synergy percentages ranging from 58-79% [45].

There are many reports in the literature on quinolone and β-lactam interaction, with several rates observed [32, 46]. In a previous study, synergy with the imipenem/ ciprofloxacin combination was not demonstrated [38]. Moreover, the efficacy of combinations of ceftazidime/ ciprofloxacin and ceftazidime/gentamicin was not useful. In an in vitro study, synergy of ciprofloxacin with ceftazidime at rates of ≥50% was reported against ciprofloxacin-resistant P. aeruginosa isolates [47]. Pohlman and his colleagues evaluated the ciprofloxacin/ ceftazidime combination against different Gram-negative organisms [48]. They concluded that the synergy between ciprofloxacin and β-lactams was sporadic and was not consistent across drug concentrations or sampling times. Moreover, Tamma and coworkers reported that the synergistic potential of β-lactams and fluoroquinolones remains unclear [49].

On the other hand, the combination of gentamicin/ciprofloxacin decreases MIC about 3-fold with two isolates and 1-fold with one isolate. Combinations of aminoglycosides with fluoroquinolones were shown to be synergistic [46, 50, 51] and support our results. Dundar and Otkun observed an additive effect with the ciprofloxacin-tobramycin combination [38]. Furthermore, none of the antimicrobial combinations tested in the current study demonstrated antagonism against any of the tested isolates. The variability of the results obtained in several studies may be due to differences in methodology, definitions of synergy, and choice of strains.

It has been demonstrated that plants produce not only intrinsic antimicrobial chemicals but also antimicrobial inhibitors, which can increase action of the antimicrobial compounds. For example, studies have described the synergistic and additive interactions of whole essential oils (EOs) or their constituents and antibiotics with different mechanisms of resistance [52, 53]. The results from broth microdilution assays showed a decrease in antibiotic MIC in the presence of plant EOs and phytochemicals [54, 55]. The antibacterial activity of orange EO (limonene) showed lower activity when compared to other tested compounds, and this was in agreement with previous results [56]. Combinations of limonene against P. aeruginosa were not useful with all tested antimicrobial agents. This was in agreement with the previous results that lemon oil and orange oil did not show inhibition against P. aeruginosa [56].

In our study, thymol exhibited a significant effect with almost all tested antimicrobial agents against Staphylococcus aureus and exhibited a good potentiating effect with gentamicin and ciprofloxacin against P. aeruginosa. Thymus had a synergistic effect with doxycycline (3-4-fold decrease in MIC) and amoxicillin (7-8-fold decrease in MIC) against S. aureus. For P. aeruginosa, about a 4-5-fold decrease in MIC of ciprofloxacin was observed. Previous studies showed that the EO of Thymus vulgaris and its major component, thymol, had bacteriostatic and bactericidal activities against Gram-negative strains [57]. Nonetheless, the activity of the EOs was superior to the compound alone. Such finding was explained by the fact that the high antimicrobial activity of some EOs generally results from the synergism of their major components [58].

In the current study, thymol showed the highest antimicrobial activity among the 10 tested plant materials, with MIC 2048 μg/mL for S. aureus and 16384 μg/mL for P. aeruginosa, followed by moringa seed, rosemary EO, and curcumin. Ivanovic reported significant antimicrobial activity of the extract and EO of thyme against Gram-negative strains, with MIC of 640 μg/mL, however our MIC ranged from 32768 μg/mL to 262144 μg/mL [59]. Such activity was attributed to the high concentration of thymol in the extract (39.7%) and in EO (48.49%). Previous studies also reported an antimicrobial activity of the EO of thyme [60, 61]. The study found three times stronger inhibition of pure thymol against different organism species than thyme oil, which is constituted mainly of p-cymene (36.5%), thymol (33%), and 11.3% of 1,8-cineole [57].

In this study, the combination of curcumin with doxycycline, amoxicillin, and ceftriaxone against S. aureus and with ciprofloxacin against P. aeruginosa exhibited significant synergistic effects, while other curcumin combinations were not synergistic. Other studies showed that the antibacterial activities of cefixime, cefotaxime, vancomycin, and tetracycline were increased in the presence of curcumin against the test strains. Curcumin significantly improved antibiotic efficacy against S. aureus when combined with cefixime, cefotaxime, vancomycin, and tetracycline, as curcumin inhibited the efflux pump system [62]. The result demonstrated that curcumin as a safe natural product could also serve as a valuable probe to study the structure-function relationships of antibiotic resistance reversal agents [63, 64]. Therefore, this compound or its future derivatives have a good potential for combination therapy against S. aureus [11, 65].

Rosemary was also used in combination with different antimicrobial agents against S. aureus. The combination was synergistic with doxycycline, azithromycin, CTX, gentamicin, and amoxicillin as MIC decreased about three folds with doxycycline and about 7-8 folds (1024μg/ml to 4μg /ml) with amoxicillin. Other rosemary–antimicrobial combinations were not useful either against S. aureus or P. aeruginosa.

El Hosseiny and El-Shenawy showed that the anti-pseudomonal activities of antibiotics were enhanced by a range of 12-33.3% in the presence of rosemary oil [66]. The antibacterial activity displayed by EO, alone and in association with antibiotics, is probably related to the major components (eucalyptol) identified in this oil. The MICs of the ethanolic extract of rosemary in our study were 1.1- 4.6 mg/mL against S. aureus isolates. Jarrar reported MICs in the range of 0.39–3.13 mg/mL [67]. They also demonstrated a synergistic effect between rosemary and cefuroxime against MRSA isolates. A significant modulatory effect on drug resistance was verified when rosemary was used in association with aminoglycosides against S. aureus MDR strains [68].

The EOs of rosemary and thymol had the highest antimicrobial activity in our study. Rosemary gave synergistic action combined with amoxicillin, doxycycline, ceftriaxone, azithromycin, and gentamicin. Thymol had a synergistic effect with all studied antimicrobials. The EOs of rosemary (Rosmarinus officinalis) and thyme (Thymus vulgaris) gave valuable synergistic effects with antimicrobial agents [12]. attributed synergism effects to phenolic and alcoholic compounds. Phenols and terpenes were the major antimicrobial compounds [68]. In general, Gram-positive bacteria are known to be more susceptible to EO than Gram-negative bacteria [69]. The weak activity against Gram-negative bacteria was attributed to the presence of their cell wall. P. aeruginosa appeared to be the most resistant to EOs and active phytochemicals [24].

In our study, the salvia plant was combined with different antimicrobials against S. aureus and P. aeruginosa. The combination was synergistic with amoxicillin (about 7-fold reduction in MIC), doxycycline (about 4-fold reduction in MIC), ceftriaxone (about 3-fold decrease in MIC), and azithromycin (about 6-fold reduction in MIC). In the presence of subinhibitory concentration (1/4 MIC to 1/32 MIC) of sage, the MIC values of antibiotics were found to be decreased by 2- to 10-fold. The essential oils of S. officinalis exhibited the lowest antibacterial activity in the disc diffusion method and did not affect P. aeruginosa [69].

In our study, moringa seed extract gave antibacterial activity against Gram-positive (MIC 4-8.1mg/ ml) and negative bacteria (MIC 16.3-32.7 mg/ml). Alikwe and Omotosho revealed that high antibacterial activity was observed in Moringa oleifera seed extract [70], which is similar to the findings of others [71-73]. They found out that the ethanol extracts of the seed extracts of M. oleifera were active against all bacteria tested [70].

In this study, moringa seed extract had a great potentiating effect with doxycycline, amoxicillin, ceftriaxone, and azithromycin against S. Aureus, with no noticeable effect on P. aeruginosa except with gentamicin, where MIC decreased about 3-4 folds. Other studies revealed that moringa ethanol extract efficiently inhibited the growth of Gram positive and negative strains [74, 75]. Moreover, other studies [75-77] showed the ability of substances in moringa seeds to inhibit mainly Bacillus subtilis, Mycobacterium phlei, Serratia marcescens, E. coli, Pseudomonas aeruginosa, Shigella and Streptococcus sp.. The observation of both Gram-negative and Gram-positive effects in the same plant extract may be explained by the presence of a wide spectrum of bactericidal substances [77].

Capsicum extract exhibited a synergistic effect with amoxicillin (7 folds decrease in MIC), ceftriaxone (2-6 folds), and doxycycline (6-7 folds) but the other combinations with azithromycin against S. aureus and ciprofloxacin and ceftazidime against P. aeruginosa were not synergistic. The MIC of capsicum extract in our study ranged from 103125μg/mL to 412500 μg/mL. Other studies reported lower MIC ranged from 256 to 1024μg/mL [76]. Capsicum extract in other studies displayed a large spectrum of activity (73%) against the tested bacteria strains [78].

Piperine, a major plant alkaloid isolated from the family Piperaceae, including black pepper (Piper nigrum) and long pepper (P. longum), has been reported to increase the accumulation of antibiotics by S. aureus [79]. Moreover, our results showed that black pepper exhibited a synergistic effect with doxycycline, ceftriaxone, amoxicillin, and azithromycin against S. aureus but no synergistic effects against P. aeruginosa.

For chitosan, our study showed no antimicrobial activity nor any synergistic effect with any of the antibiotics studied. Studies showed that COS exhibited synergistic effects with azithromycin against P. aeruginosa infection in both wild-type and resistant strains [80]. It was also implied that macrolides have a more positive interaction with selected oligosaccharides than the other antibiotics (oxytetracycline, cefotaxime, and ampicillin). This is in agreement with our results except for azithromycin [81]. The addition of ADO and COS drastically decreased the MICs of AZM by 2.8 and 5-fold, respectively; however, there was no synergistic effect in the rest of the treatments [82, 83, 84].

CONCLUSION

A good approach for the development of successful combinations would be to aim for broad and potent activities against a wide range of pathogens, including S. aureus and P. aeruginosa. Our results revealed that the combination of ceftriaxone with either gentamicin or ciprofloxacin for S. aureus was significantly synergistic. Moreover, the combination of amoxicillin with ceftazidime was synergistic and exhibited a decrease of MIC by 5-6 folds. Regarding MDR clinical isolates of P. aeruginosa, a combination of azithromycin with doxycycline exhibited a decrease of MIC of azithromycin by about 5-6 folds. The combination of gentamicin with ceftriaxone was significant. For natural compounds, thymol, rosemary oil, curcumin, capsicum, and moringa seed extract exhibited the highest synergistic activity with the tested antibiotics against S. aureus and P. aeruginosa.

In conclusion, the lack of new antibiotics necessitates the improvement of existing ones. Our study shows that antibiotic combinations and antibiotic-natural plant combinations are very promising strategies for combating the complex bacterial resistance. However, future efforts should be made to conduct in vivo studies with the extracts on animal models to confirm the present in vitro findings that are not only affected.

LIST OF ABBREVIATIONS

MDR = Multidrug-resistant
MIC = Minimum Inhibitory Concentrations
EOs = Essential Oils
CLSI = Clinical and Laboratory Standard Institute
MHB = Muller Hinton Broth
FIC = Fractional Inhibitory Concentration Index

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

Not applicable.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The data introduced in this study was a part of the results of the Master thesis of the first author, The datasets used /or analysed in the current study are available from the corresponding author [A-E] upon reasonable request.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

REFERENCES

1
Aminov RI. A brief history of the antibiotic era: Lessons learned and challenges for the future. Front Microbiol 2010; 1: 134.
2
Chopra I, O’Neill AJ, Miller K. The role of mutators in the emergence of antibiotic-resistant bacteria. Drug Resist Updat 2003; 6(3): 137-45.
3
Goossens H, Ferech M, Vander Stichele R, Elseviers M. Outpatient antibiotic use in Europe and association with resistance: A cross-national database study. Lancet 2005; 365(9459): 579-87.
4
Grigoryan L, Burgerhof JGM, Degener JE, et al. Attitudes, beliefs and knowledge concerning antibiotic use and self‐medication: A comparative European study. Pharmacoepidemiol Drug Saf 2007; 16(11): 1234-43.
5
Usha PTA, Jose S, Nisha A. Antimicrobial drug resistance a global concern. Vet World 2010; 3: 138-9.
6
Kamatou GPP, van Zyl RL, van Vuuren SF, et al. Chemical composition, leaf trichome types and biological activities of the essential oils of four related Salvia species indigenous to Southern Africa. J Essent Oil Res 2006; 18(sup1): 72-9.
7
Rybak MJ, McGrath BJ. Combination antimicrobial therapy for bacterial infections. Guidelines for the clinician. Drugs 1996; 52(3): 390-405.
8
Abdel-Karim SAAM, El-Ganiny AMA, El-Sayed MA, Abbas HAA. Promising FDA-approved drugs with efflux pump inhibitory activities against clinical isolates of Staphylococcus aureus. PLoS One 2022; 17(7): e0272417.
9
Chanda S, Dudhatra S, Kaneria M. Antioxidative and antibacterial effects of seeds and fruit rind of nutraceutical plants belonging to the Fabaceae family. Food Funct 2010; 1(3): 308-15.
10
Van de Braak SAAJ, Leijten GCJJ. Essential oils and oleoresins. A Survey in the Netherlands and other Major Markets in the European Union. Centre for the Promotion of Imports from Developing Countries 1999; 2: 116.
11
Lv F, Liang H, Yuan Q, Li C. in vitro antimicrobial effects and mechanism of action of selected plant essential oil combinations against four food-related microorganisms. Food Res Int 2011; 44(9): 3057-64.
12
Bassolé IHN, Juliani HR. Essential oils in combination and their antimicrobial properties. Molecules 2012; 17(4): 3989-4006.
13
Tegos G, Stermitz FR, Lomovskaya O, Lewis K. Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials. Antimicrob Agents Chemother 2002; 46(10): 3133-41.
14
Betoni JEC, Mantovani RP, Barbosa LN, Di Stasi LC, Fernandes Junior A. Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases. Mem Inst Oswaldo Cruz 2006; 101(4): 387-90.
15
Sibanda T, Okoh AI. in vitro evaluation of the interactions between acetone extracts of Garcinia kola seeds and some antibiotics. Afr J Biotechnol 2008; 7(11)
16
Aiyegoro OA, Afolayan AJ, Okoh A. Synergistic interaction of Helichrysum pedunculatum leaf extracts with antibiotics against wound infection associated bacteria. Biol Res 2009; 42(3): 327-38.
17
Marquez B, Neuville L, Moreau NJ, et al. Multidrug resistance reversal agent from Jatropha elliptica. Phytochemistry 2005; 66(15): 1804-11.
18
Shibata H, Kondo K, Katsuyama R, et al. Alkyl gallates, intensifiers of β-lactam susceptibility in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2005; 49(2): 549-55.
19
Egyptian Pharmacopoeia. The English Text of Arab Republic of Egypt. Ministry of Health and Population 4th. 2005; 1295: 31-3.
20
Revathy S, Elumalai S, Benny M, Antony B. Isolation, purification and identification of curcuminoids from turmeric (Curcuma longa L.) by column chromatography. J Experim Sci 2011; 2(7): 21-5.
21
Clinical and Laboratory Standards Institute, CLSI.. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals 5th. 2020.
22
Clinical and Laboratory Standards Institute, CLSI.. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically 10th. 2015.
23
Kahlmeter G, Brown DFJ, Goldstein FW, et al. European committee on antimicrobial susceptibility testing (EUCAST (technical notes on antimicrobial susceptibility testing. Clin Microbiol Infect 2006; 12(6): 501-3.
24
Bosnić T, Softić D, Grujić-Vasić J. Antimicrobial activity of some essential oils and major constituents of essential oils. Acta Med Acad 2006; 35(1): 9-14.
25
Doern CD. When does 2 plus 2 equal 5? A review of antimicrobial synergy testing. J Clin Microbiol 2014; 52(12): 4124-8.
26
Tängdén T. Combination antibiotic therapy for multidrug-resistant Gram-negative bacteria. Ups J Med Sci 2014; 119(2): 149-53.
27
Saiman L. Clinical utility of synergy testing for multidrug-resistant Pseudomonas aeruginosa isolated from patients with cystic fibrosis: ‘The motion for’. Paediatr Respir Rev 2007; 8(3): 249-55.
28
Baiomy AA, Serry FE, Kadry AA, et al. Genome analysis of Pseudomonas aeruginosa strains from chronically infected patients with high levels of persister formation. Pathogens 2023; 12(3): 426.
29
El-Baz AM, Yahya G, Mansour B, et al. The Link between occurrence of class I integron and acquired aminoglycoside resistance in clinical MRSA isolates. Antibiotics 2021; 10(5): 488.
30
Kadry AA, El-Antrawy MA, El-Ganiny AM. Impact of short chain fatty acids (SCFAs) on antimicrobial activity of new β-lactam/β-lactamase inhibitor combinations and on virulence of Escherichia coli isolates. J Antibiot 2023; 76(4): 225-35.
31
Ahmed Z, Saeed Khan S, Khan M. in vitro trials of some antimicrobial combinations against Staphylococcus aureus and Pseudomonas aeruginosa. Saudi J Biol Sci 2013; 20(1): 79-83.
32
Siriyong T, Murray RM, Bidgood LE, et al. Dual β-lactam combination therapy for multi-drug resistant Pseudomonas aeruginosa infection: enhanced efficacy in vivo and comparison with monotherapies of penicillin-binding protein inhibition. Sci Rep 2019; 9(1): 9098.
33
Burgess DS, Hastings RW. Activity of piperacillin/tazobactam in combination with amikacin, ciprofloxacin, and trovafloxacin against Pseudomonas aeruginosa by time-kill. Diagn Microbiol Infect Dis 2000; 38(1): 37-41.
34
CLSI- Clinical and Laboratory Standard Institute. Document: M100-S16 CLSINCCLS 2006; 2-8.
35
American Thoracic Society; Infectious Diseases Society of America. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005; 171(4): 388-416.
36
Poole K. Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2005; 49(2): 479-87.
37
Bryant RE, Fox K, Oh G, Morthland VH. β-lactam enhancement of aminoglycoside activity under conditions of reduced pH and oxygen tension that may exist in infected tissues. J Infect Dis 1992; 165(4): 676-82.
38
Dundar D, Otkun M. In-vitro efficacy of synergistic antibiotic combinations in multidrug resistant Pseudomonas aeruginosa strains. Yonsei Med J 2010; 51(1): 111-6.
39
Milatovic D, Wallrauch C. in vitro activity of trovafloxacin in combination with ceftazidime, meropenem, and amikacin. Eur J Clin Microbiol Infect Dis 1996; 15(8): 688-93.
40
Gimeno C, Borja J, Navarro D, Valdés L, García-Barbal J, García-de-Lomas J. in vitro interaction between ofloxacin and cefotaxime against gram-positive and gram-negative bacteria involved in serious infections. Chemotherapy 1998; 44(2): 94-8.
41
Gradelski E, Kolek B, Bonner DP, Valera L, Minassian B, Fung-Tomc J. Activity of gatifloxacin and ciprofloxacin in combination with other antimicrobial agents. Int J Antimicrob Agents 2001; 17(2): 103-7.
42
Pankuch GA, Lin G, Seifert H, Appelbaum PC. Activity of meropenem with and without ciprofloxacin and colistin against Pseudomonas aeruginosa and Acinetobacter baumannii. Antimicrob Agents Chemother 2008; 52(1): 333-6.
43
Jenkins SG, Lewis JW. Synergistic interaction between ofloxacin and cefotaxime against common clinical pathogens. Infection 1995; 23(3): 154-62.
44
Giamarellou H. Prescribing guidelines for severe Pseudomonas infections. J Antimicrob Chemother 2002; 49(2): 229-33.
45
Burgess DS, Nathisuwan S. Cefepime, piperacillin/tazobactam, gentamicin, ciprofloxacin, and levofloxacin alone and in combination against Pseudomonas aeruginosa. Diagn Microbiol Infect Dis 2002; 44(1): 35-41.
46
Drago L, De Vecchi E, Nicola L, Colombo A, Guerra A, Gismondo MR. Activity of levofloxacin and ciprofloxacin in combination with cefepime, ceftazidime, imipenem, piperacillin-tazobactam and amikacin against different Pseudomonas aeruginosa phenotypes and Acinetobacter spp. Chemotherapy 2004; 50(4): 202-10.
47
Bustamante CI, Wharton RC, Wade JC. in vitro activity of ciprofloxacin in combination with ceftazidime, aztreonam, and azlocillin against multiresistant isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1990; 34(9): 1814-5.
48
Pohlman JK, Knapp CC, Ludwig MD, Washington JA. Timed killing kinetic studies of the interaction between ciprofloxacin and β-lactams against gram-negative bacilli. Diagn Microbiol Infect Dis 1996; 26(1): 29-33.
49
Tamma PD, Cosgrove SE, Maragakis LL. Combination therapy for treatment of infections with gram-negative bacteria. Clin Microbiol Rev 2012; 25(3): 450-70.
50
Gerçeker AA, Gürler B. In-vitro activities of various antibiotics, alone and in combination with amikacin against Pseudomonas aeruginosa. J Antimicrob Chemother 1995; 36(4): 707-11.
51
Mayer I, Nagy E. Investigation of the synergic effects of aminoglycoside- fluoroquinolone and third-generation cephalosporin combinations against clinical isolates of Pseudomonas spp. J Antimicrob Chemother 1999; 43(5): 651-7.
52
Kon KV, Rai MK. Plant essential oils and their constituents in coping with multidrug-resistant bacteria. Expert Rev Anti Infect Ther 2012; 10(7): 775-90.
53
Langeveld WT, Veldhuizen EJA, Burt SA. Synergy between essential oil components and antibiotics: A review. Crit Rev Microbiol 2014; 40(1): 76-94.
54
Mikulášová M, Chovanová R, Vaverková Š. Synergism between antibiotics and plant extracts or essential oils with efflux pump inhibitory activity in coping with multidrug-resistant staphylococci. Phytochem Rev 2016; 15(4): 651-62.
55
Abdel-Halim MS, Askoura M, Mansour B, Yahya G, El-Ganiny AM. in vitro activity of celastrol in combination with thymol against carbapenem-resistant Klebsiella pneumoniae isolates. J Antibiot 2022; 75(12): 679-90.
56
Soković M, Glamočlija J, Marin PD, Brkić D, Griensven LJLD. Antibacterial effects of the essential oils of commonly consumed medicinal herbs using an in vitro model. Molecules 2010; 15(11): 7532-46.
57
Santurio DF, Kunz de Jesus FP, Zanette RA, Schlemmer KB, Fraton A, Martins Fries LL. Antimicrobial activity of the essential oil of thyme and of thymol against Escherichia coli strains. Acta Sci Vet 2014; 42(1): 1-4.
58
Höferl M, Buchbauer G, Jirovetz L, et al. Correlation of antimicrobial activities of various essential oils and their main aromatic volatile constituents. J Essent Oil Res 2009; 21(5): 459-63.
59
Ivanovic J, Misic D, Zizovic I, Ristic M. in vitro control of multiplication of some food-associated bacteria by thyme, rosemary and sage isolates. Food Control 2012; 25(1): 110-6.
60
Burt SA, Reinders RD. Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7. Lett Appl Microbiol 2003; 36(3): 162-7.
61
Alves-Silva JM, Dias dos Santos SM, Pintado ME, Pérez-Álvarez JA, Fernández-López J, Viuda-Martos M. Chemical composition and in vitro antimicrobial, antifungal and antioxidant properties of essential oils obtained from some herbs widely used in Portugal. Food Control 2013; 32(2): 371-8.
62
Shahverdi AR, Moghaddam KM, Iranshahi M, Yazdi MC. The combination effect of curcumin with different antibiotics against Staphylococcus aureus. Int J Green Pharm 2009; 3(2): 141-3.
63
Menichetti F. Current and emerging serious Gram-positive infections. Clin Microbiol Infect 2005; 11(s3)(Suppl. 3): 22-8.
64
Ejim L, Farha MA, Falconer SB, et al. Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy. Nat Chem Biol 2011; 7(6): 348-50.
65
Stavri M, Piddock LJV, Gibbons S. Bacterial efflux pump inhibitors from natural sources. J Antimicrob Chemother 2007; 59(6): 1247-60.
66
El Hosseiny L, El-Shenawy M. in vitro evaluation of the combination between some antibiotics and essential oils of clove and rosemary against Pseudomonas aeruginosa. Antimicrob Resist Infect Control 2015; 4(S1): P169.
67
Jarrar N, Abu-Hijleh A, Adwan K. Antibacterial activity of Rosmarinus officinalis L. alone and in combination with cefuroxime against methicillin–resistant Staphylococcus aureus. Asian Pac J Trop Med 2010; 3(2): 121-3.
68
Issabeagloo E, Kermanizadeh P, Taghizadieh M, Forughi R. Antimicrobial effects of rosemary (Rosmarinus officinalis L. (essential oils against Staphylococcus species. Afr J Microbiol Res 2012; 6(23): 5039-42.
69
Stefanović OD, Stanojević DD, Comić LR. Synergistic antibacterial activity of Salvia officinalis and Cichorium intybus extracts and antibiotics. Acta Pol Pharm 2012; 69(3): 457-63.
70
Alikwe PCN, Omotosho MS. Evaluation of the proximate, chemical and phytochemical composition of <i>Moringa oleifera</i> leaf meal as potential food/feedstuff for man and non ruminant livestock. Agrosearch 2013; 13(1): 17-28.
71
Jamil B, Hasan R, Zafar A, et al. Dengue virus serotype 3, Karachi, Pakistan. Emerg Infect Dis 2007; 13(1): 182-3.
72
Thilza IB, Sanni S, Isah Z, Sanni F, Talle M, Joseph M. in vitro antimicrobial activity of water extract of Moringa oleifera leaf stalk on bacteria normally implicated in eye diseases. Academia Arena 2010; 2(6): 80-2.
73
Saadabi AM, Zaid IA. An in vitro antimicrobial activity of Moringa oleifera L. seed extracts against different groups of microorganisms. Aust J Basic Appl Sci 2011; 5(5): 129-34.
74
Batool S, Saba S, Iqbal A, Naveed A, Zia A. Evaluating the antibacterial activity of Moringa oleifera leaves extracts against pathogenic bacterial isolates. Bio/Scient Rev 2023; 5(4): 25-37.
75
Viera GHF, Mourão JA, Ângelo ÂM, Costa RA, Vieira RHSF. Antibacterial effect (in vitro) of Moringa oleifera and Annona muricata against Gram positive and Gram negative bacteria. Rev Inst Med Trop São Paulo 2010; 52(3): 129-32.
76
Areeba A, Malika A. Water treatment and purification using Moringa Oleifera seed extract. Int J Trend Scient Res Develop 2020; 4(4): 2456-6470.
77
Kostova I, Dinchev D. Saponins in Tribulus terrestris chemistry and bioactivity. Phytochem Rev 2005; 4(2-3): 111-37.
78
Touani FK, Seukep AJ, Djeussi DE, Fankam AG, Noumedem JAK, Kuete V. Antibiotic-potentiation activities of four Cameroonian dietary plants against multidrug-resistant Gram-negative bacteria expressing efflux pumps. BMC Complement Altern Med 2014; 14(1): 258.
79
Inouye S, Uchida K, Yamaguchi H, Miyara T, Gomi S, Amano M. Volatile aroma constituents of three Labiatae herbs growing wild in the Karakoram-Himalaya district and their antifungal activity by vapor contact. J Essent Oil Res 2001; 13(1): 68-72.
80
He X, Hwang H, Aker WG, et al. Synergistic combination of marine oligosaccharides and azithromycin against Pseudomonas aeruginosa. Microbiol Res 2014; 169(9-10): 759-67.
81
He X, Hwang HM. Nanotechnology in food science: Functionality, applicability, and safety assessment. Yao Wu Shi Pin Fen Xi 2016; 24(4): 671-81.
82
Isik A, Poyanli A, Tekant Y, et al. Incomplete or inappropriate endoscopic and radiologic interventions as leading causes of cholangitis. PolJ Surg 2021; 93(6): 47-52.
83
Işık A, Fırat D. Letter to the editor concerning “Most cited 100 articles from Turkey on abdominal wall hernias: A bibliometric study”. Turk J Surg 2021; 37(2): 193-4.
84
Eladl A, Elganiny A, Abdullatif H. Evaluation of the potency of some antibiotic formulations in the Egyptian market. Zag J Pharm Sci 2017; 26(1): 48-54. ISSN 2356-9786