Our aim was to determine the antimicrobial activity of three selected plants (
Disease causing bacteria have always been considered a major cause of morbidity and mortality in humans. The appearance of resistant microorganisms paved the way to the occurrence of infections that are only treated by a limited number of antimicrobial agents. The emergence of resistant Gram negative bacteria presents a major challenge for the antimicrobial therapy of infectious diseases and increases the incidence of mortality and morbidity. Bacterial resistance to antimicrobial agents is a medical problem with public health, socioeconomic, and even political implications [
For centuries, plants have been used as remedies and treatments of diseases. The Middle Eastern Mediterranean region is rich in plant species; there are about 2,600 species of which many are considered to have medicinal effects. However, there is relatively limited research on medicinal plants in this region [
Many studies have investigated the antimicrobial activity of different plant species in various geographical regions in search for new antibiotics. The use of plant derivatives as antimicrobials has not been extensively addressed until recently since most antibiotics were derived from bacterial or fungal origin. With the increase in resistance and the realization that the effective life span of any antibiotic is limited, new sources especially plant sources are currently being heavily investigated. Thousands of phytochemicals with antimicrobial activity have already been identified but they should be subjected to animal and human studies to study their toxicity and their effectiveness in whole organism systems. Several phytochemicals are already being studied in humans [
The aim of this study was to determine the antimicrobial activity of selected indigenous Lebanese plants (
Twenty strains of
The herbal sample consisted of three different Lebanese plants: leaves of
The Antimicrobial Susceptibility Testing was performed as recommended by the Clinical and Laboratory Standards Institute (CLSI) [
Fresh plants were dried in the shade at room temperature and ground in a coffee bean grinder. The dried plant material was weighed and then soaked in 80% ethanol for 7 days with continuous shaking in a shaker at room temperature. At day seven, the plant material was filtered and the filtrate collected. This was repeated and the filtrates were combined and concentrated in a rotary evaporator to obtain the crude extract (fraction 1).
The crude extract of each plant was further partitioned by extraction with different solvents in a 1 : 1 (v/v) ratio in order to subfractionate the plant components according to their polarity: petroleum ether (fraction 2), dichloromethane (fraction 3), and ethyl acetate (fraction 4). Extractions were repeated three times and fractions were combined. The remaining aqueous layer was collected as fraction number 5. Fractions 1 and 5 were dried using a freeze dryer, but fractions 2, 3, and 4 were dried under the hood to dryness due to the inconvenience of introducing vapor solvent into the freeze dryer. Controls were prepared for each fraction by drying the same amount of solvent and following the same subfractionation method without plant extract (solvent control).
The plant powders were weighed and dissolved in sterile distilled water. The solutions were filtered through 0.22
The Microdilution Broth Method was used for the determination of the MIC of plant extracts as recommended by the Clinical and Laboratory Standards Institute [
All experiments were performed three independent times in duplicate form. The MIC90 is defined as the Minimum Inhibitory Concentration required to inhibit the growth of 90% of organisms; it was calculated as the percentile below which 90% of the individual MICs values fall. In view of the relatively small population of tested bacteria, it was not advantageous to calculate MIC50.
As shown in Table ESBL positive, Quinolone resistant, with no overproduction of AmpC, ESBL positive, Quinolone susceptible, with no overproduction of AmpC, ESBL positive, Quinolone resistant, with overproduction of AmpC, ESBL positive, Quinolone susceptible, with overproduction of AmpC,
Phenotypic profiles of susceptibility of
Number of strains | AM | AMC | PIP | TZP | CF | CXM | FOX | CTX | CRO | CAZ | CEF | IMP | GN | AN | SXT | OF | CIP | TGC | Resistance profile | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
5 | R | R | R | S | R | R | S | R | R | R | R | S | S | S | S | S | S | S | ESBL+ QS | |
5 | R | R | R | S | R | R | R | R | R | R | R | S | R | S | R | R | R | S | ESBL+ QR AmpC + | |
10 | R | R | R | S | R | R | S | R | R | R | R | S | S | S | R | R | R | S | ESBL+ QR | |
5 | R | R | R | S | R | R | S | R | R | R | R | S | R | S | R | S | S | S | ESBL+ QS | |
5 | R | R | R | R | R | R | S | R | R | R | R | S | R | S | R | R | R | I | ESBL+ QR |
AM: ampicillin, AMC: amoxicillin/clavulanic acid, PIP: piperacillin, TZP: piperacillin/tazobactam, CF: cephalotin, CXM: cefuroxime, FOX: cefoxitin, CTX: cefotaxime, CRO: ceftriaxone, CAZ: ceftazidime, CEF: cefepime, IMP: imipenem, GN: gentamicin, AN: amikacin, SXT: trimethoprim/sulfamethoxazole, OF: ofloxacin, CIP: ciprofloxacin, TGC: tigecycline QR: quinolone Resistant, QS: quinolone sensitive S: Sensitive, I: Intermediate, R: Resistant, AmpC +: Overproduction of AmpC, ESBL+: ESBL producer.
MICs and MBCs of the different fractions of
Bacterial strain | Crude ( | Petroleum ether ( | Dichloromethane ( | Ethyl acetate ( | Aqueous ( | |||||
---|---|---|---|---|---|---|---|---|---|---|
MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | |
Ec001SGH | 80 | 80 | X | X | ND | ND | 10 | 10 | 80 | 80 |
Ec002SGH | 20 | 20 | X | X | ND | ND | 2.5 | 5 | 40 | 80 |
Ec003SGH | 10 | 10 | X | X | ND | ND | 2.5 | 5 | 20 | 20 |
Ec004SGH | 40 | 40 | X | X | ND | ND | 5 | 10 | 40 | 80 |
Ec007SGH | 80 | 80 | X | X | ND | ND | 10 | 10 | 80 | ND |
Ec010SGH | 40 | 40 | X | X | ND | ND | 5 | 5 | 80 | 80 |
Ec011SGH | 40 | 40 | X | X | ND | ND | 5 | 5 | 40 | 80 |
Ec012SGH | 40 | 40 | X | X | ND | ND | 5 | 10 | 40 | 40 |
Ec013SGH | 20 | 20 | X | X | ND | ND | 10 | 10 | 40 | 80 |
Ec016SGH | 10 | 20 | X | X | ND | ND | 2.5 | 2.5 | 20 | 20 |
Ec017SGH | 40 | 40 | X | X | ND | ND | 2.5 | 2.5 | 5 | 5 |
Ec018SGH | 20 | 20 | X | X | ND | ND | 5 | 10 | 80 | 80 |
Ec019SGH | 10 | 20 | X | X | ND | ND | 2.5 | 2.5 | 40 | 40 |
Ec020SGH | 20 | 20 | X | X | ND | ND | 2.5 | 5 | 40 | 80 |
Ec021SGH | 20 | 20 | X | X | ND | ND | 2.5 | 2.5 | 40 | 40 |
Ec023SGH | 40 | 40 | X | X | ND | ND | 5 | 10 | 40 | 80 |
Ec026SGH | 20 | 40 | X | X | ND | ND | 2.5 | 5 | 80 | 80 |
Ec030SGH | 40 | 40 | X | X | ND | ND | 5 | 10 | 40 | 80 |
Ec031SGH | 20 | 20 | X | X | ND | ND | 5 | 5 | 40 | 80 |
Ec032SGH | 10 | 20 | X | X | ND | ND | 2.5 | 5 | 40 | 80 |
Control | ND | ND | X | X | ND | ND | ND | ND | ND | ND |
MIC90 | 44 | X | N/A | 10 | 80 | |||||
Kp001SGH | 20 | 20 | X | X | ND | ND | 2.5 | 2.5 | 40 | 80 |
Kp002SGH | 20 | 20 | X | X | ND | ND | 5 | 5 | 80 | 80 |
Kp001SGH | 20 | 20 | X | X | ND | ND | 2.5 | 2.5 | 40 | 80 |
Kp002SGH | 20 | 20 | X | X | ND | ND | 5 | 5 | 80 | 80 |
Kp005SGH | 10 | 20 | X | X | ND | ND | 5 | 5 | 20 | 20 |
Kp006SGH | 20 | 20 | X | X | ND | ND | 10 | 10 | 80 | ND |
Kp007SGH | 20 | 20 | X | X | ND | ND | 5 | 5 | 80 | 80 |
Kp008SGH | 40 | 40 | X | X | ND | ND | 5 | 5 | 40 | 80 |
Kp009SGH | 40 | 40 | X | X | ND | ND | 5 | 5 | 80 | 80 |
Kp010SGH | 40 | 40 | X | X | ND | ND | 10 | 10 | 80 | 80 |
Kp013SGH | 40 | 40 | X | X | ND | ND | 20 | 20 | 80 | 80 |
Kp016SGH | 40 | 80 | X | X | ND | ND | 2.5 | 2.5 | 80 | 80 |
Control | ND | ND | ND | ND | ND | ND | ND | ND | ||
MIC90 | 40 | X | N/A | 10 | 80 |
MIC: Minimum Inhibitory Concentration, MBC: Minimum Bactericidal Concentration, ND: Not Detected, X: missing extract,
Both inhibitory and bactericidal effects of
MICs and MBCs of the different fractions of
Bacterial strain | Crude ( | Petroleum ether ( | Dichloromethane ( | Ethyl acetate ( | Aqueous ( | |||||
---|---|---|---|---|---|---|---|---|---|---|
MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | |
Ec001SGH | 80 | 80 | X | X | X | X | 2.5 | 5 | 80 | 80 |
Ec002SGH | 80 | ND | 5 | 5 | 10 | 10 | 5 | 5 | 80 | 80 |
Ec003SGH | 80 | 80 | 5 | 5 | 5 | 10 | 5 | 5 | 80 | ND |
Ec004SGH | 80 | 80 | X | X | 5 | 5 | 2.5 | 5 | 80 | ND |
Ec007SGH | ND | ND | 10 | 10 | 20 | 20 | 20 | 20 | ND | ND |
Ec010SGH | 40 | 40 | 2.5 | 2.5 | 5 | 10 | 5 | 5 | 80 | 80 |
Ec011SGH | 80 | 80 | X | X | 2.5 | 5 | 2.5 | 5 | 80 | ND |
Ec012SGH | 80 | ND | X | X | 5 | 5 | 5 | 5 | 80 | 80 |
Ec013SGH | 40 | 80 | X | X | 10 | 10 | 5 | 5 | 80 | ND |
Ec016SGH | 80 | 80 | 1.25 | 1.25 | 5 | 5 | 2.5 | 2.5 | 40 | 40 |
Ec017SGH | 20 | 40 | 10 | 10 | 10 | 10 | 2.5 | 2.5 | 20 | 20 |
Ec018SGH | 40 | 40 | 2.5 | 2.5 | 10 | 10 | 5 | 5 | ND | ND |
Ec019SGH | ND | ND | ND | ND | ND | ND | 2.5 | 5 | 80 | 80 |
Ec020SGH | 80 | 80 | 5 | 5 | ND | ND | 5 | 5 | 80 | 80 |
Ec021SGH | 40 | 80 | X | X | ND | ND | 5 | 5 | 80 | 80 |
Ec023SGH | ND | ND | X | X | ND | ND | 5 | 5 | 80 | 80 |
Ec026SGH | 40 | 80 | 2.5 | 2.5 | 5 | 10 | 5 | 5 | 80 | ND |
Ec030SGH | 80 | 80 | X | X | X | X | 5 | 5 | 80 | ND |
Ec031SGH | 40 | 80 | 2.5 | 5 | 10 | 10 | 5 | 5 | 80 | 80 |
Ec032SGH | 80 | 80 | 1.25 | 1.25 | 5 | 10 | 2.5 | 5 | 80 | ND |
Control | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
MIC90 | 80 | N/A | N/A | 5 | 80 | |||||
Kp001SGH | ND | ND | 2.5 | 5 | 10 | 10 | 5 | 10 | ND | ND |
Kp002SGH | 80 | 80 | 2.5 | 2.5 | 5 | 10 | 5 | 5 | 80 | ND |
Kp005SGH | ND | ND | 1.25 | 2.5 | 10 | 20 | 5 | 5 | 80 | ND |
Kp006SGH | 40 | 80 | 10 | 10 | 10 | 10 | 20 | 20 | ND | ND |
Kp007SGH | 80 | 80 | 1.25 | 1.25 | 20 | 20 | 5 | 10 | ND | ND |
Kp008SGH | ND | ND | X | X | ND | ND | 5 | 10 | 80 | ND |
Kp009SGH | ND | ND | 5 | 10 | 10 | 20 | 5 | 5 | 80 | ND |
Kp010SGH | 80 | 80 | X | X | X | X | 5 | 10 | 80 | ND |
Kp013SGH | ND | ND | X | X | X | X | 2.5 | 2.5 | 80 | ND |
Kp016SGH | ND | ND | 5 | 10 | 20 | ND | 10 | 10 | 80 | 80 |
Control | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
MIC90 | 80 | N/A | N/A | 11 | 80 |
MIC: Minimum Inhibitory Concentration, MBC: Minimum Bactericidal Concentration, ND: Not Detected, X: missing extract,
The inhibitory effects of
MICs and MBCs of the different fractions of
Bacteria | Crude ( | Petroleum ether ( | Dichloromethane ( | Ethyl acetate ( | Aqueous ( | |||||
---|---|---|---|---|---|---|---|---|---|---|
MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | |
Ec001SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec002SGH | ND | ND | ND | ND | ND | ND | X | X | ND | ND |
Ec003SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec004SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec007SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec010SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec011SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec012SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec013SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec016SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec017SGH | ND | ND | ND | ND | ND | ND | X | X | ND | ND |
Ec018SGH | ND | ND | ND | ND | ND | ND | X | X | ND | ND |
Ec019SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec020SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec021SGH | ND | ND | ND | ND | ND | ND | X | X | 10 | 10 |
Ec023SGH | ND | ND | ND | ND | ND | ND | X | X | ND | ND |
Ec026SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec030SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Ec031SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | |
Ec032SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Control | ND | ND | ND | ND | ND | ND | X | X | ND | ND |
MIC90 | N/A | N/A | N/A | X | 20 | |||||
Kp001SGH | ND | ND | ND | ND | ND | ND | X | X | ND | ND |
Kp002SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Kp005SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Kp006SGH | ND | ND | ND | ND | ND | ND | X | X | ND | ND |
Kp007SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Kp008SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Kp009SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Kp010SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Kp013SGH | ND | ND | ND | ND | ND | ND | X | X | ND | ND |
Kp016SGH | ND | ND | ND | ND | ND | ND | X | X | 20 | ND |
Control | ND | ND | ND | ND | ND | ND | X | X | ND | ND |
MIC90 | N/A | N/A | N/A | X | N/A |
MIC: Minimum Inhibitory Concentration, MBC: Minimum Bactericidal Concentration, ND: Not Detected, X: missing extract,
Production of Extended Spectrum Beta Lactamase enzymes emerged in Gram negative bacteria and caused the infections to become more difficult to treat in view of their resistance to a wide range of antibiotics [
The present study showed that different extracts/fractions exhibited antimicrobial activity against ESBL producing
Our results show that all plant extracts, except
The low activity of the crude extracts against the tested
All aqueous extracts exerted antimicrobial activity against the majority of the tested strains. This antimicrobial activity was moderately low, which may possibly be related to the fact that most of the secondary metabolites were extracted either by petroleum ether, dichloromethane, or ethyl acetate solvents.
Extraction of secondary metabolites highly depends on using extractory techniques that depend on the chemical properties of these compounds. Water-soluble compounds and proteins can be extracted in water or polar solvents whereas water insoluble compounds can be extracted with organic solvents [
The crude extract of each plant was partitioned by extraction with different solvents in order to subfractionate the plant components according to their polarity. Solvents were applied starting by the least polar to the more polar. These solvents were selected in order to extract compounds with different polarities. Petroleum ether is known to extract the nonpolar metabolites. Dichloromethane is known to extract compounds with medium polarity, and ethyl acetate is known to extract the polar compounds [
Since most of the identified MICs, especially MICs of the crude extract and aqueous fraction, consisted of the highest concentrations tested, and since the MBC would normally be the concentrations of higher dilutions, some of the MBCs were not determined such as with
Our findings reported here show that different extracts of