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

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

3. RESULTS

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.

REFERENCES