Antistaphylococcal and Antibiotic Resistance Modulatory Activities of Thirteen Cameroonian Edible Plants against Resistant Phenotypes

Background In this study, 18 methanol extracts from Cameroonian edible plants were tested for their antibacterial activities against 26 strains of S. aureus; the role of efflux pumps in the resistance of tested bacteria and the antibiotic resistance-modulating activities against selected multidrug-resistant (MDR) phenotypes were also investigated. Methods Broth microdilution assay was used to evaluate the antibacterial activity, the role of efflux pumps, and the antibiotic resistance-modulating effects of plant extracts. Results Extracts from Dacryodes edulis seeds (DES) and Dacryodes edulis bark (DEB) were active against all 26 tested bacterial strains, within the minimal inhibitory concentration (MIC) range of 256–1024 µg/mL. MIC values varied from 64 to 1024 µg/mL against 96.2% of the 26 tested bacteria for Phaseolus vulgaris leaves (PVL), 92.3% for Azadirachta indica bark (AIB), Dacryodes edulis leaves (DEL), and Ricinodendron heudelotii leaves (RHL). The lowest MIC value of 64 µg/mL was obtained with the extract from Cucurbita maxima beans (CMB) against MRSA4 strain and from Uapaca guineensis bark (UGB) against MRSA9 strain. Bacterial efflux pump inhibitor (EPI), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), improved the activity of DES and UGB as well as that of extracts from Hibiscus esculentus leaves (HEL) and Uapaca guineensis leaves (UGL) against resistant S. aureus strains. Antibiotic-modulating effects against more than 70% of the S. aureus strains tested were obtained when RHL (at MIC/2) was combined with CIP, ERY, and KAN (88.89%), CHL (88.89%), TET (77.78%), and STR (88.89%). Conclusion The present study demonstrated that the 13 tested plants had antistaphylococcal effects and that DES, HEL, UGL, and UGB could be used in combination with EPI to combat resistance to Staphylococcus aureus. Also, it demonstrated that some studied extracts and mostly RHL could be used as antibiotic resistance modulators to fight against resistant strains of S. aureus.


Background
Bacterial infections caused by Staphylococcus aureus are globally responsible for 7-10% of deaths annually [1]. It is the most virulent species of the genus Staphylococcus and has emerged as one of the most important human pathogens in the last decades, being one of the main causes of hospital and community infections [2].
is bacterium causes a wide range of clinical infections, ranging from common infections such as skin and soft tissue infections to septicemia, pneumonia, and toxinosis [3,4]. e fight against S. aureus is hindered by the development of resistance of various strains to antibiotics [5][6][7][8][9]. e multidrug resistance (MDR) observed in Gram-positive bacteria is mostly attributed to overexpression of efflux pumps and antibiotics-degrading enzymes. is MDR of S. aureus propels the search of new antibacterials with more efficiency and low toxicity. Plant kingdom contains a variety of pharmacologically active secondary metabolites, and some of them have been reported for their antibacterial activities [10,11]. eir use to combat S. aureus antibiotic resistance is an attractive strategy. In regard to the loss of efficacy of several antibiotics and the scarcity of new antibacterial agents, it is also important to search for substances capable of restoring the activity of antibiotics. Antibacterial screenings of African plants have yielded promising results in the past [12][13][14][15]. e present study was set up to evaluate the antistaphylococcal potential of 13  . e study was extended on the role of efflux pumps in resistance to some plant extracts as well as the ability of extracts to potentiate the activity of selected antibiotics.  (Table 1). e dried and powdered material (100 g) of each plant was macerated in 300 mL of methanol at room temperature for 48 h and then filtered using Whatman filter paper No. 1. e filtrate obtained was concentrated using a rotary evaporator under reduced pressure to obtain the crude methanol extract, which was kept at 4°C until further use.

INT Colorimetric Assay for MIC and Minimal Bactericidal Concentration (MBC) Determinations.
e MIC and minimal bactericidal concentration (MBC) determinations on various strains of S. aureus were performed using the rapid INT colorimetric assay [56] with some modifications as previously described [14,38]. e samples were dissolved in DMSO/MHB. e final concentration of DMSO was lower than 2.5%. e twofold dilutions of the samples were made in a 96-well microplate, and the tested bacterial concentration was 1.5 × 10 6 colony-forming unit (CFU)/mL. e microplates were incubated at 37°C for 18 h. All assays were performed in triplicate and repeated thrice. Wells containing MHB, 100 µL of inoculum, and DMSO to a final concentration of 2.5% served as negative control. e MIC of each sample was detected after 18 h incubation at 37°C, following addition (40 µL) of 0.2 mg/mL of INT and incubation at 37°C for 30 minutes as the lowest sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth [56]. e MBC was determined by adding 50 µL aliquots of the preparations, which did not show any growth after incubation during MIC assays, to 150 µL of MHB. ese preparations were further incubated at 37°C for 48 h. e MBC was regarded as the lowest concentration of samples, which did not produce a color change after addition of INT as mentioned above [57,58]. Chl r , Cyp r , Erm r , Flx r , Im/Cs r , Kan r , Met r , Ofxa r , Tet r , Vm r , Amp r , Dox r , Aug r , Gen r , and Nis r resistance to chloramphenicol, ciprofloxacin, erythromycin, flomoxef, imipenem/cilastatin sodium, kanamycin, methicillin, ofloxacin, tetracycline, vancomycin, ampicillin, doxycycline, augmentin, gentamicin, and nisin, respectively, SA: Staphylococcus aureus. pumps in the resistance of selected bacterial strains to some of the active plant extracts, Dacryodes edulis seeds (DES), Hibiscus esculentus leaves (HEL), Uapaca guineensis leaves (UGL), Uapaca guineensis bark (UGB), and CIP (reference drug) were tested in the absence or presence of EPI (CCCP (0.5 µg/mL) or CPZ (25 µg/mL)). MICs of samples alone or in combination with EPI were determined as above, and the increase in activity was determined as the ratio of MIC of sample alone versus sample in combination with EPI. All assays were performed in triplicate and repeated thrice.

Antibiotic Activity Modulation Assays.
To evaluate the antibiotic resistance modulation activity of the most active extracts: Azadirachta indica bark (AIB), Dacryodes edulis seeds (DES), Dacryodes edulis bark (DEB), Dacryodes edulis leaves (DEL), Phaseolus vulgaris leaves (PVL), Ricinodendron heudelotii leaves (RHL), and Uapaca guineensis bark (UGB), a preliminary assay was performed in order to assess the MICs of antibiotics in the absence and presence of these extracts using the broth microdilution method as previously described [14,38,56]

Phytochemical Composition of Plant
Extracts. e major classes of phytochemicals were screened in the 18 studied plant extracts (Table 3). It appears that all extracts contained polyphenols and tannins. Other classes of phytochemicals were selectively present. Only the extract of the beans of eobroma cacao contained all the investigated classes of secondary metabolites.

Antibacterial Activity.
e antibacterial activities of the 18 tested extracts and ciprofloxacin against 26 strains of S. aureus are summarized in Table 4  International Journal of Microbiology  (IGB). e lowest MIC value of 64 µg/mL was obtained with CMB against MRSA4 strain and UGB against MRSA9 strain.  Tables 5 and 6. It appears that CCCP significantly improved the activity of the 4 tested extracts against the majority of S. aureus strains (Table 5). e increase of activity in the presence of CCCP ranged from 2-fold to >128-fold. e highest increase of activity (>128-fold) was obtained when DES, UGB, and UGL were tested in the presence of CCCP on at least one S. aureus strain. In contrast, in the presence of CPZ, no improvement in the activity of the four extracts was observed (Table 6). is is clear indication that CCCP was the appropriate EPI of the studied S. aureus strains.

Phytochemical Composition of Extracts.
Polyphenols and tannins were detected in all extracts. e role of several molecules belonging to polyphenols as antibacterials has been demonstrated [10,11,38,61]. Tannins also belong to a class of polyphenols, and its presence in all extracts could in part explain the fact that all the tested extracts were active in at least one strain of the tested bacteria [10]. However, it should be made clear that the presence of a class of secondary metabolite with reported antibacterial effect is not a guarantee of the good activity of a plant. e antibacterial effect depends on the structure and the amount of a particular phytochemical in the plant or possible interactions with other compounds. is could explain why the extract from the beans of eobroma cacao that contained all the investigated classes of secondary metabolites (Table 3) was not the most active sample (Table 4).

Antibacterial Potential of Extracts.
Resistance of bacteria to antibiotics propels the search of new agents to fight against MDR phenotypes. In the present study, clinical strains of S. aureus used were previously reported as resistant to at least one commonly used antibiotic [8,9] (Table 2). Several locally isolated strains of S. aureus [54] were used herein, to better adapt the study to our environment. According to established criteria, MIC values in the range of 100-1000 µg/mL are indication that plant extracts bear antimicrobial activities [62]. Also, the antibacterial activity of botanicals is considered significant if MIC values are below 100 µg/mL, moderate if 100 ≤ MICs ≤ 625 µg/mL, and weak if MICs > 625 µg/mL [37,60]. On these bases, it can be deduced that all the tested plant extracts had antistaphylococcal activities, except Irvingia gabonensis beans (IGB), with MICs above 1000 µg/mL against all tested strains of S. aureus (Table 4).
is activity was significant for CMB against MRSA4 strain and UGB against MRSA9 strain (MIC: 64 µg/mL). Most of the recorded MIC values ranged from 512 to 1024 µg/mL, indicating that extracts rather exhibited moderate to low antistaphylococcal effects. However, this activity could be considered important because the clinical strains of S. aureus used were resistant phenotypes while extracts were from edible plants. In effect, it was suggested that if botanicals are food plants, as they are allegedly nontoxic or less toxic than other medicinal plants, their antibacterial activity is significant in a range of 100 ≤ MIC ≤ 512 µg/mL and moderately active in a range of 512 < MIC ≤ 2048 µg/mL [63].

Role of Efflux Pumps in Susceptibility of S. aureus Strains to
the Extracts. Bacterial efflux systems are associated with major human health concerns as they are involved in the resistance of pathogenic bacteria such as S. aureus [64][65][66]. Previously, inhibition of efflux pumps by natural products has been found to improve the activity of antibiotics against S. aureus. For example, inhibition of the TetK efflux pump was reported with the essential oil of Chenopodium ambrosioides and its constituent α-terpinene against S. aureus IS-58 strain [66]. In the present study, two well-known EPIs (CCCP and CPZ) were used to assess the implication of efflux pumps in the resistance of the studied S. aureus strains to plant extracts. CCCP is an inhibitor of the proton-motive force of ATPbinding cassette (ABC) transporters of several Gram-negative and Gram-positive bacteria, including S. aureus [5][6][7]. CPZ is capable of reversing or reducing the antibiotic resistance of bacteria including S. aureus [67], due to its indirect effects on ATPase activity that is dependent upon Ca 2+ [68]. In the present study, it was found that CCCP contrary to CPZ improved the activity of the four extracts (DES, HEL, UGL, and UGB) (Tables 5 and 6). is indicates that ABC transporters are involved in the resistance of the studied strains of S. aureus and that combination of extracts such as HEL, UGL, and UGB with an inhibitor of ABC transporters could improve the antistaphylococcal fight.

Antibiotic Modulation Effects of Extracts.
e antibiotic resistance-modulating effects of several botanicals and phytochemicals against resistant bacteria have been documented [12-15, 59, 69]. It has been suggested that extracts capable of potentiating the activity of antibiotics on more than 70% of bacteria could be potential efflux pump inhibitors [70]. In this study, antibiotic modulation activity of extracts at their MIC/2 on more than 70% tested strains of S. aureus was obtained with the association of DEB and DEL and with 1/8 (12.5%) tested antibiotics (Tables S2 and S3; Supplementary Materials), UGB with 2/8 (25%) antibiotics (Table S6; Supplementary Materials), 8 International Journal of Microbiology DES with 4/8 (50%) antibiotics (Table S4; Supplementary Materials), and RHL with 6/8 (75%) antibiotics (Table S5; Supplementary Materials). Hence, the tested extracts and mostly RHL may act as efflux pump inhibitors [70]. e use of CCCP indicated that ABC transporters were the efflux pumps involved in the resistance of the tested bacteria, suggesting that the above extract could be the inhibitors of such pumps. e potential of the R. heudelotii leaf extract (RHL) to reverse antibiotic resistance in Gram-negative MDR bacteria was previously reported [18]. e present study therefore provides more information about the ability of this plant to modulate the activity of antibiotics against resistant strains of S. aureus.

Conclusion
In conclusion, the present work provides informative data about the antistaphylococcal potential of 13 Cameroonian food plants. It also indicates that some extracts such as DES, HEL, UGL, and UGB could be used in combination with EPI to combat resistance of Staphylococcus aureus to antibiotics. Finally, this study also demonstrates that some studied extracts and mostly RHL could be used as antibiotic resistance modulators, providing a new weapon against the resistance of S. aureus to antibiotics.