Research Article | Open Access
In Vitro Synergistic Effect of Psidium guineense (Swartz) in Combination with Antimicrobial Agents against Methicillin-Resistant Staphylococcus aureus Strains
The aim of this study was to evaluate the antimicrobial activity of aqueous extract of Psidium guineense Swartz (Araçá-do-campo) and five antimicrobials (ampicillin, amoxicillin/clavulanic acid, cefoxitin, ciprofloxacin, and meropenem) against twelve strains of Staphylococcus aureus with a resistant phenotype previously determined by the disk diffusion method. Four S. aureus strains showed resistance to all antimicrobial agents tested and were selected for the study of the interaction between aqueous extract of P. guineense and antimicrobial agents, by the checkerboard method. The criteria used to evaluate the synergistic activity were defined by the fractional inhibitory concentration index (FICI). All S. aureus strains were susceptible to P. guineense as determined by the microdilution method. The combination of the P. guineense extract with the antimicrobial agents resulted in an eight-fold reduction in the MIC of these agents, which showed a FICI ranging from 0.125 to 0.5, suggesting a synergistic interaction against methicillin-resistant Staphylococcus aureus (MRSA) strains. The combination of the aqueous extract of P. guineense with cefoxitin showed the lowest FICI values. This study demonstrated that the aqueous extract of P. guineense combined with beta lactamics antimicrobials, fluoroquinolones, and carbapenems, acts synergistically by inhibiting MRSA strains.
The widespread emergence of bacteria resistance to a large number of antimicrobial agents poses major health problems because of difficulties in treatment . Staphylococcus aureus is an important pathogen both in community acquired and nosocomial infections and a common etiological agent of infections of many different tissues and organs (e.g., furuncle, carbuncle, abscess, myocarditis, endocarditis, pneumonia, meningitis, bacterial arthritis, and osteomyelitis) . The multidrug resistant Staphylococcus aureus strains now pose serious problems to hospitalized patients and their care providers. The organism has successfully evolved numerous strategies for resisting, the action to practically all antimicrobial agents . Thus, it is extremely important to find new antimicrobial agents or new ways for the treatment of infectious diseases caused by multidrug resistant microorganisms .
The screening of plant extracts and phytochemicals for antimicrobial activity has shown that higher plants, especially their secondary metabolites, are a source to provide structurally diverse bioactive compounds with different pharmacological activities, including antimicrobials .
According to Hemaiswarya et al. , plants-derived antimicrobials are less potent, which enhances the need to adopt a synergistic interaction between its bioactive compounds to combat infections. Based on this knowledge, several studies have been published using crude plant extracts or phytochemicals combined with antimicrobial agents. These combinations can enhance the efficacy of the antimicrobial agents and are an alternative to treat infections caused by multidrug resistant microorganisms, which do not have any effective therapy available [4, 7, 8].
Few studies have been focused on the phytochemical profile and pharmacological activity of this plant; however, it is widely used in folk medicine in regions of South America to treat infections of the gastrointestinal and genitourinary tract . These authors associated the presence of mainly flavonoids and tannins in the fruits and leaves of P. guineense to be effective against Streptococcus mutans strains.
The aim of the present study was to evaluate the antimicrobial activity of the aqueous extract of P. guineense and to determine the synergistic potential of the combination of this extract with five antimicrobial agents against MRSA strains.
2. Materials and Methods
2.1. Plant Material and Extract Preparation
The plant materials used in this study consisted of Psidium guineense Swartz (leaves) that were collected in November/2009 from Moreno city, Pernambuco, Brazil, (08°07′07′′S-35°05′32′′W). This plant was identified by the biologist Olivia Cano, and the voucher specimen was deposited in the Herbarium Dárdano de Andrade Lima, Instituto Agronômico de Pernambuco and indexed under registration 83.564.
The leaves were air-dried for two weeks and then grinded into fine powder using an electric dry mill. A total of 50 g of the powder was soaked in 1000 mL of boiled distilled water and extracted exhaustively at room temperature for 48 hours. The extracts were filtered through Whatman No. 2 filter paper. The aqueous extract was lyophilized to obtain a dry powder extract.
2.2. Phytochemical Screening
The preliminary phytochemical screening was carried out by various secondary metabolites present on aqueous extract of P. guineense using standard procedures [12–14]. The chromatographic analysis was made by TLC on Si gel (MERCK, Germany, 105553) developed by different solvent systems: (EtOAc-HCOOH–AcOH-H2O (100 : 11 : 11 : 26, v/v)) to investigate the presence of saponins, flavonoids, tannins, phenylpropanoids, alkaloids, condensed proanthocyanidins, leucoanthocyanidins, and iridoids and (n-BuOH–Me2CO-buffer phosphate pH = 5.0 (40 : 50 : 10 v/v)) to evaluate the presence of sugars.
2.3. Total Polyphenol Content (TPC)
The total polyphenol content (TPC) was determined colorimetrically by using Folin-Ciocalteu’s reagent and expressed as pyrogallol, according to the Brazilian Pharmacopoeia with modifications . Thus, samples of aqueous extract of P. guineense (1000 μg/mL) were prepared in distilled water. Concentrations (10 to 30 μg/mL) were measured. The standard pyrogallol (1000 μg/mL) was prepared in distilled water. 80 microliters of each solution was transferred to a 25 mL flask containing distilled water (10 mL) and Folin-Ciocalteu’s reagent (1 mL). The volume was completed with anhydrous sodium carbonate solution (10.75% w/v), resulting in final concentrations of 3.2 μg/mL of pyrogallol. A standard curve was determined with concentrations between 1.6 and 4.8 μg/mL (). Final results were given as pyrogallol equivalents. The samples were scanned in a UV/VIS spectrophotometer (Evolution 60S, Thermo Scientific), and measurements were obtained 15 min after addition of the sodium-carbonate solution and scanning ranging from 500 to 900 nm wavelength. Distilled water was used as a blank. All measurements were performed in triplicate.
2.4. Bacterial Strains and Inoculum Standardization
S. aureus strains () were isolated from clinical specimens and food. The standard strain used was S. aureus ATCC 25923. Strains were isolated in sheep blood agar and after identification they were stored in brain heart infusion (BHI) plus glycerol 20% v/v . The S. aureus strains used in this study showed a resistant phenotype, by the diffusion method, to several antimicrobial agents such as beta-lactams, aminoglycosides, macrolides, fluoroquinolones, tetracycline, chloramphenicol, and lincosamides. These strains were cultured onto Mueller Hinton Agar (MHA) (Acumedia Manufacturers, Baltimore, USA) and incubated at 37°C for 18 hours. Single colonies were selected and inoculated into Mueller-Hinton broth (Acumedia Manufacturers, Baltimore, USA) to turbidity comparable to that of 0.5 McFarland standard, which is equivalent to a bacterial count of approximately 108 CFU/mL.
After that, the bacterial suspension was diluted in saline (1 : 10) to obtain a final inoculum of 107 CFU/mL.
2.5. Antimicrobial Agents
The standard reference powders of ampicillin; amoxicillin/clavulanic acid; cefoxitin; ciprofloxacin; gentamicin; meropenem were provided by Eurofarma Laboratório LTDA, Brazil. Resistance was defined for each case: ampicillin (AMP, MIC ≥ 0.25 μg/mL); amoxicillin/clavulanic acid (AMC, MIC ≥ 8 μg/mL); cefoxitin (CFO, MIC ≥ 4 μg/mL); ciprofloxacin (CIP, MIC ≥ 4 μg/mL); gentamicin (GEN, MIC ≥ 8 μg/mL; meropenem (MER, MIC ≥ 16 μg/mL).
2.6. Antimicrobial Activity
The Minimal Inhibitory Concentration (MIC) test was performed by the microdilution broth method, following the recommendations established by Clinical Laboratory Standards Institute, , with some modifications. Serial two-fold dilutions of aqueous extract of P. guineense and antimicrobial agents were prepared in sterile 96-well microplates containing Mueller Hinton broth (MHB). Five microliters of bacterial suspension were inoculated in each well to give a final concentration of 104 CFU/mL. P. guineense extract and antimicrobial agents concentrations ranged from 7.25 to 1000 μg/mL and 3.12 to 400 μg/mL, respectively. The growth inhibition was demonstrated by optical density at 630 nm using a microplate reader (Thermo plate—TP Reader). Considering the total growth (100%) in the control well (MHB + bacteria), the percentage of growth reduction was attributed to the remaining wells. The MIC was reported as the lowest concentration of P. guineense extract or antimicrobial agents that inhibited the bacterial growth after 24 h of incubation at 37°C. In order to determine the Minimal Bactericidal Concentration (MBC), the contents of the well that showed higher or equal than 70% of growth inhibition were seeded into MHA. After 24 h of incubation at 37°C, the number of surviving S. aureus was determined. The MBC was defined as the lowest extract concentration at which 99.9% of the bacteria have been killed. All experiments were carried out in duplicate on two different days.
2.7. Determination of In Vitro Synergistic Activity
Combinations of P. guineense and antimicrobial agents were tested by the checkerboard method. The appropriate dilution of P. guineense extract and antimicrobial agents were performed into MHB. From these dilutions, one hundred microliters were added in 96-well microplates to obtain a final concentration equal to MIC or six dilutions lower than MIC to P. guineense and nine dilutions lower than MIC to antimicrobial agents. Each well received 5 μL of the bacterial suspensions (107 CFU/mL). Plates were incubated for 24 hours. Interpretation of the data was achieved by calculating the fractional inhibitory concentration index (FICI) as follows: (MIC of P. guineense in combination with antimicrobial agents/MIC of P. guineense) + (MIC of antimicrobial agents in combination with P. guineense/MIC of antimicrobial agents). The combination was considered to be synergistic when the FICI was ≤0.5, additive when it was 0.5 to ≤1, and antagonistic when ≥2 .
3.1. Phytochemical Profile
The phytochemical profile from the Psidium guineense extract showed the presence of tannins, flavonoids, condensed proanthocyanidins, leucoanthocyanidins, and sugar. On the other hand, the presence of alkaloids, phenylpropanoid, and saponins was not verified. The aqueous extract of P. guineense yielded 3.7% (w/w) over 50 g of plant material.
Concerning the total polyphenol content, the results of this study suggest that the aqueous extract of P. guineensis show an important amount of such secondary metabolites. Thus, the total polyphenol content was g% (1.51%) as pyrogallol equivalent.
3.2. Antimicrobial Activity
MIC and MBC values of P. guineense extract and of the ampicillin, amoxicillin/clavulanic acid, cefoxitin, ciprofloxacin, gentamicin, and meropenem against twelve S. aureus strains are shown in Table 1.
|MIC: minimal inhibitory concentration; MBC: minimal bactericidal concentration; ATCC: American Type Culture Collection; LFBM: Laboratório de Fisiologia e Bioquímica de Microorganismos; AMC: amoxicillin/clavulanic acid; AMP: ampicillin; AZI: azithromycin; CFO: cefoxitin; CIP: ciprofloxacin; GET: gentamicin; PEN: penicillin; MER: meropenem; AE: aqueous extract of P. guineense.|
1Resistance phenotype determined by the disk-diffusion method.
The aqueous extract of P. guineense showed a strong activity against all S. aureus strains with MIC values between 250 and 500 μg/mL. The S. aureus strains revealed a resistance profile against most antimicrobial agents tested, in particular to the beta-lactam antibiotics. The MIC values for ampicillin and cefoxitin ranged from 3.12 to 400 μg/mL which showed to be less effective against the S. aureus strains tested. For amoxicillin/clavulanic acid, the values ranged from 3.12 to 50 μg/mL. Among all S. aureus strains tested, seven showed to be resistant to ciprofloxacin. For meropenem, MIC values ranged from 3.12 to 25 μg/mL. All strains showed to be sensitive to gentamicin, except LFBM 26, LFBM 28, LFBM 33. Four strains of S. aureus (LFBM 01, LFBM 26, LFBM 28, LFBM 33) showed resistance to all antimicrobial agents tested. This resistance profile selected the microorganisms for the study of the interaction between P. guineense extract and antimicrobial agents. The values of MBC were higher than MIC in one dilution.
3.3. Determination of In Vitro Synergistic Activity
The MICs obtained by the combination of the aqueous extract of P. guineense with ampicillin, amoxicillin/clavulanic acid, cefoxitin, ciprofloxacin, and meropenem, against S. aureus strains (LFBM 01, LFBM 26, LFBM 28, LFBM 33) are listed in Table 2. The minimal inhibitory concentrations of P. guineense extract (250 μg/mL) enhanced the antistaphylococcal activity of all antimicrobial agents. The synergistic activity was detected by an eight-fold decrease in the MIC of the antimicrobial agents in the combination and determined by the FICI ≤0.5.
|MIC: minimal inhibitory concentration; FIC: fractional inhibitory concentration; AMC: amoxicillin/clavulanic acid; AMP: ampicillin; CFO: cefoxitin; CIP: ciprofloxacin; MER: meropenem; AE: aqueous extract of P. guineense; LFBM: Laboratório de Fisiologia e Bioquímica de Microorganismos.|
1%reduction of viable cells in wells containing the combination aqueous extract of P. guineense (1/2 MIC) and antimicrobials agents (1/512 MIC).
The combination of the aqueous extract of P. guineense and cefoxitin showed the lowest FICI whose values ranged from 0.125 to 0.5. The MIC of cefoxitin (individual MIC 100–400 μg/mL) was lowered to 1/512 (combined MIC ranged 0.19–0.78 μg/mL), when it was used in combination with the aqueous extract of P. guineense (MIC 250 μg/mL). For S. aureus LFBM 26 and LFBM 33 strains, this combination was also efficient on reducing the MIC of P. guineense extract to 1/8 × MIC (31.25 μg/mL) or 1/4 × MIC (62.25 μg/mL), respectively.
The inhibition of the growth of all the S. aureus strains by meropenem was enhanced by P. guineense extract (FICI 0.5). This combination was more effective against LFBM 28 (FICI 0.25) which individual MIC of P. guineense (250 μg/mL) lowered to 1/4 × MIC.
For S. aureus LFBM 33 strain, the MIC value of ciprofloxacin (individual MIC 50 μg/mL) was lowered to 0.78 μg/mL when combined with P. guineense extract (1/4 × MIC) enhancing the antistaphylococcal activity of this fluoroquinolone (FICI 0.25). All other combinations of the aqueous extract of P. guineense with antimicrobial agents demonstrated synergistic activity, enhancing its activity, with FICI 0.5.
Phytochemicals have great potential as antimicrobial compounds and have been proven to have great therapeutic potential . Some secondary metabolites have the ability to increase the susceptibility of the microorganism. When used in combination, these metabolites have the potential to either inhibit the modified targets or exhibit a synergy by blocking one or more of the targets in the metabolic pathway, acting as a modifier of multidrug resistance mechanisms .
The genus Psidium is native to America but is distributed worldwide. Their specimens are traditionally used in the prevention or treatment of a large number of diseases. Preparations of twigs or leaves of Psidium species are extensively used for the control of gastrointestinal and respiratory disorders as well as in the treatment of skin damage .
Ethnopharmacological evaluation demonstrated the antimicrobial activities of Psidium ssp. that explain its use for the treatment of infectious diseases of the digestive, respiratory, urinary tract as well as the skin and soft tissues. Within this genus, P. guajava is the most studied and used species . Thus, we are interested in P. guineense, an indigenous plant of Brazil, because it is characterized by its ample spectrum of uses including gastrointestinal disorders, infection of the genitourinary tract, treatment of colds, bronchitis, and ulcers .
According to Sartoratto et al. , strong activity is for MIC values between 50–500 μg/mL, moderate activity MIC values between 600–1500 μg/mL, and weak activity above 1500 μg/mL. Comparing with literature results, the aqueous extract of P. guineense (250–500 μg/mL) has a strong activity against MRSA strains tested.
The phytochemical analysis from the leaves of P. guineense indicated the presence of flavonoids, hydrolysable, and condensed tannins distributed throughout the leaf tissue . These results are in agreement with those obtained in the present study. Total polyphenol content g% determined in aqueous extract of Psidium guineense plays an important role on biological properties of this extract.
Neira González et al.  associated the presence of flavonoids, mainly avicularin, quercetin, and guaijaverin present in the ethanolic extract of P. guineense, with the activity against clinical isolated Streptococcus mutans strains.
Flavonoids have the ability to complex with proteins and bacterial cells forming irreversible complexes mainly with nucleophilic amino acids. This complex often leads to inactivation of the protein and loss of its function .
Tannins are not crystallizable substances and when they are in the aqueous system, form colloidal solutions. The antimicrobial activity of tannins can be summarized as follows: (i) binding with proteins and adhesins, inhibiting enzymes, (ii) complexation with the cell wall and metal ions, and (iii) disruption of the plasmatic membrane .
Synergism of natural products and antimicrobial agents is a thrust area of phytomedicinal research, developing novel perspective of phytopharmaceuticals. The synergism of plant-derived compounds and antimicrobial agents has been evaluated previously against pathogenic microorganisms. The approach is not exclusive for extract combinations, since effective combinations between single natural products, essential oils or extracts with chemosynthetics or antibiotics have been described [6, 29].
In addition to achieving these synergistic effects, the combinations of two or more compounds are essential for the following reasons: (1) to prevent or suppress the emergence of resistant strains, (2) to decrease dose-related toxicity, as a result dosage, and (3) to attain a broad spectrum of activity .
In this study, a growth inhibitory effect of P. guineense extract on MRSA strains was observed for its combinations with beta-lactams, carbapenems, and fluoroquinolones.
The combination of P. guineense extract and beta-lactam may help to reduce the amount of antimicrobial agents used and deliver a medicine with similar or greater potency as antimicrobial. More importantly, since phytochemicals are structurally different from antimicrobial agents and often have different modes of action, they may provide new means of studying the mechanisms of bacterial control at a molecular level. With the increase prevalence of multidrug resistant S. aureus, synergism testing using various combinations of phytochemicals with antimicrobial agents could be a powerful tool in helping to select appropriate antimicrobial therapy [6, 29].
The indiscriminate use of antimicrobial agents in the treatment of bacterial infections has led to the emergence and spread of resistant strains, and it resulted in a great loss of clinical efficacy of previously effective first-line antimicrobials which results in shifting of antimicrobial treatment regimen to second-line or third-line antimicrobial agents that are often more expensive with many side effects . In fact, studies have showed that crude extracts of plants possess the ability to enhance the activity of antimicrobial agents [4, 7, 8].
There is a wide list of phytochemicals which act as inhibitors, and a few of them are glycosylated flavones suppressing topoisomerase IV activity, myricetin inhibiting DnaB helicase, allicin inhibiting RNA synthesis. Corilagin, a polyphenol from Arctostaphylos uva-ursi is found to markedly reduce the MIC of beta-lactam agents against MRSA. According to Hemaiswarya et al. , there are two possibilities regarding the mechanism of action of corilagin, namely, inhibition of penicillin binding protein (PBP2a) activity or inhibiting its production.
The polyphenol epigallocatechin gallate (EGCg) from green tea is believed to be synergic with beta-lactam agents since both attacked the same target site, namely, peptidoglycan. EGCg inhibits the penicillinase produced by S. aureus thereby restoring the activity of penicillin. The combination of EGCg with ampicillin/sulbactam reduced the MIC90 to 4 μg/mL from its initial value of 16 μg/mL .
Hatano et al.  investigated the synergistic activity of two proanthocyanidins isolated from the fruits of the Zizyphus genus. Although the MICs of these polyphones were 512–1024 μg/moL, both reduced the MIC for oxacillin to 1/2–1/16 of those in the absence of the polyphenols.
It is interesting to note that most plant secondary metabolites have weak antimicrobial activity, several orders of magnitudes less than that of common antimicrobial agents produced by bacteria and fungi. In spite of the fact that plant-derived antimicrobials are less potent, plants fight infections successfully. Hence, is becomes apparent that plants adopts a synergistic mechanism between their compounds .
According to Rosales-Reyes et al. , the use of the crude extract in our study was intentional and based on the belief that it would be the closest representation to that of traditional preparations.
Therefore, it is speculated that the efficacy of a crude extract may be due to the interplay between the different active constituents that may be present in the extract leading to better activity and/or decrease in potential toxicity of some individual constituents .
An antibacterial effect of P. guineense extract and a synergistic effect in combination with antimicrobial agents were reproducibly observed and might be an interesting alternative therapy for infectious diseases caused by MRSA strains. In addition, more studies, including toxicity tested in vivo, need to be conducted on this plant before therapeutic treatments are implemented.
The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for granting the fellowship as well as the financial support and infrastructure provided by the Universidade Federal de Pernambuco (UFPE) which made this study possible.
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Copyright © 2012 Tiago Gomes Fernandes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.