This study aimed to evaluate the antibacterial activity of flavonoids extracted from two Libyan brown algae namely Cystoseira compressa and Padina pavonica using microwave-assisted extraction method against pathogenic bacteria isolated from meat, meat products, milk and dairy products ( Staphylococcus aureus subsp. aureus (5 isolates), Bacillus cereus (3 isolates), Bacillus pumilus (1 isolate), Salmonella enterica subsp. enteric (4 isolates) and Enterohaemor-rhagic Escherichia coli O157 (EHEC O157) (4 isolates)). All of these isolates were muti-drug resistant with high MAR index. The results showed that C. compressa extract exhibited better and stronger antibacterial activities against the seventeen tested isolates with inhibition zones diameter ranged from 14 - 22 mm compared to P. pavonica extract which showed positive effect against 9 isolates with low inhibition zone ranged from 11 - 16.5 mm. Flavonoids extracted from C. compressa also displayed the best spectrum of bactericidal effect with a ratio MBC/MIC ≤ 4 obtained on all susceptible tested bacterial strains. Flavonoids and proanthocyanidins significantly contributed to the antibacterial properties. The mode of action of these active extracts is under investigation.
Seaweeds contain various bioactive metabolites which can benefit human health [
Although flavonoids are the most important polyphenolic compounds, few reports have paid attention to flavonoids from marine sources. The flavonoids are members of a class of natural compounds that recently have been the subject of considerable scientific and therapeutic interest. Flavonoids are known to contain a broad spectrum of chemicals and biological activities including antioxidant and free radical scavenging properties, antibacterial, antiviral, anticancer, anti-inflammatory, anti-allergic and also as potential therapeutic agents against a wide variety of diseases [
The importance of microbes to food products of animal origin had been demonstrated by recent outbreaks of food-borne illness associated with consumption of meat, milk and dairy products that had been contaminated with pathogenic organisms or toxins. Undesirable microorganisms constitute the primary hazard to safety, quality, and wholesomeness of food products. Recently there is urgent need to find new antibacterial agents due to the wide spread of drug-resistant bacteria [
As plants synthesized flavonoids as a response action to microbial infection; therefore it is expected that flavonoids are a good antimicrobial agent against various microorganisms. The antibacterial activity of flavonoids depends on the structures, namely on the substitutions on the aromatic rings. Antibacterial flavonoids might be having multiple cellular targets, rather than one specific site of action. One of their molecular actions is to form complex with proteins through nonspecific forces such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation. Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesion, enzymes, cell envelope transport proteins, and so forth. Lipophilic flavonoids may also disrupt microbial membranes [
The objective of this study was to assess the antibacterial activity of flavonoids extracted from two Libyan brown algae using microwave-assisted extraction method against multi-drug resistant bacterial strains.
Cystoseira compressa (C. compressa) and Padania pavonica (P. pavonica) were collected from western coast of Libya (SA1, N32 53.764 E13 20.990, SA 02, N32 53.756 E13 21.064; SA 03, N32 53.792 E13 21.070; SA 04, N32 53.804 E13 21.028; SA 05, N32 53.777 E13 20.983) between June and August 2015 (
Preliminary phytochmical tests for identification of alkaloids, anthraquinones, coumarins, flavonoids, saponins, tannins, and terpenes were carried out for all the extracts using standard qualitative methods that have been described previously [
Experiments were carried out in a domestic (Black & Decker, Model No. MZ
3000 PG, SL13YD, England) microwave oven system. Twenty five grams of the powdered plant materials were mixed with solvents (ethanol or methanol 80%) at a suitable ratio (500 ml). An intermittent microwave irradiation method was used to keep the temperature of the extraction mixtures below 80˚C [
Antibacterial activity assay was accomplished in the Department of Microbiology and Parasitology, Faculty of Veterinary Medicine; and Biochemistry Lab, Chemistry Department, Faculty of Science, University of Tripoli, Libya.
Standard microbiological methods [
The bacterial suspensions were standardized following the CLSI guidelines for aerobic bacteria [
Susceptibility against antimicrobials was performed against all of the above mentioned isolated bacterial strains by disk diffusion method according to standard microbiological protocol (National Committee for Clinical Laboratory Standards) [
The antibiotic susceptibility patterns obtained from the standard disc diffusion procedure were used to calculate the MAR and ARI index for total number of isolates as follow:
where, y is the number of resistant isolates, n is the number of isolates and x is the number of antibiotics [
where, a is the number of antibiotics to which the isolates are resistant and b is the total number of antibiotics exposed. A MAR value ≥0.2 indicates that antibiotics are ineffective.
The antimicrobial activity of algal flavonoid extracts was performed in vitro using the “hole-plate diffusion method” [
The MIC was defined as the lowest concentration that completely inhibited the growth for 24 h. The MIC for the flavonoids extracts was determined by the macrodilution agar method. In the macrodilution agar method, a two-fold serial dilution of the flavonoids extracts was prepared in sterile freshly prepared Mueller-Hinton (M-H) broth used as diluents to achieve a decreasing concentration ranging from 500 to 20 µg/ml. Sterile cork borer of 8.0 mm diameter was used to bore well in the pre-solidified Mueller-Hinton (M-H) plates and 150 μl volume of each dilution was added aseptically into the wells made in M-H plates in triplicate that had bacteria seeded with the standardized inoculum (1.5 × 108 CFU/ ml). All the test plates were incubated at 37˚C and were observed for the growth after 24 hr [
The content of the MIC tubes and the content of the preceding tubes in the serial dilutions (2MIC, 3MIC and 4MIC) were subcultured into the MH broth. All bacterial plates were inoculated at 37˚C for 24 hours after which they were examined. MBC was the lowest concentration that completely inhibited bacterial growth. To confirm the results of MBC, 1 ml of the experimental suspensions was resubcultured in the M-H broth which were incubated at 37˚C for 18 - 24 h.
Total flavonoid content was estimated according to [
Total proanthocyanidin content was determined according to Li’s method [
Data were expressed as means ± standard deviations (SD) of triplicate determinations. All statistical analyses were carried out using SPSS V.16 (Statistical Program for Social Sciences, SPSS Corporation, Chicago, IL). Statistical differences between extract activities were determined using ANOVA followed by Least Significant Difference (LSD) testing. Differences were considered statistically significant when P < 0.05. The Pearsons correlation analysis was performed between antibacterial activity, total flavonoids and proanthocyanidin contents.
Phytochemical screening of C. compressa and P. pavonica extracts showed the presence of most important phytoconstituents (results not shown). Flavanoids were presented in noticeable amounts in both C. compressa and P. pavonica.
The high content of flavonoids in the investigated algae, drawn our interest toward application of the recently adopted microwave procedure for the efficient extraction of these phytochemicals.
A total of 17 isolates were obtained from milk, dairy products, meat and meat products. Based on standard microbiological techniques and 16S rDNA gene sequence, the isolates were identified as Staphylococcus aureus subsp. aureus (5 isolates), Bacillus cereus (3 isolates), Bacillus pumilus (1 isolates), Salmonella enterica subsp. enteric (4 isolates) and Enterohaemorrhagic Escherichia coli O157 (EHEC O157) (4 isolates) [
The 16S rDNA gene sequences of isolates are deposited at Libyan GenBank under accession numbers as S. aureus 122 (S1), S. aureus 128 (S2), S. aureus 287 (S3), S. aureus 125 (S4); S aureus 283 (S5), B. cereus 4 (B1), B. cereus 16 (B2), B. cereus 72 (B3), B. pumilus 124 (B4), S. enterica 17 (Sal 1), S. enterica 18 (Sal 2), S. enterica 19 (Sal 3), S. enterica 29 (Sal 4), EHEC O157 57 (E1), EHEC O157 55 (E2), EHEC O157 52 (E3), EHEC O157 49 (E4).
Antibiotic Sensitivity Patterns of Pathogenic IsolatesSusceptibility tests were conducted with 24 antibiotics against the tested isolates. The antibiotic profile for each pathogenic bacterium was determined using 24 commercial antibiotic discs (
The MAR index is the ratio of number of antibiotics ineffective against the organisms to the total number of antibiotics used [
Bacterium | |||||||||
---|---|---|---|---|---|---|---|---|---|
Antibiotics | B. cereus and B. pumilus | S. aureus | |||||||
B1 | B2 | B3 | B4 | S1 | S2 | S3 | S4 | S5 | |
AML | R | R | R | R | R | R | S | R | S |
AMC | R | R | R | S | S | S | S | S | S |
AMP | R | R | R | R | R | S | S | R | S |
B | R | R | R | R | I | S | S | I | S |
P | R | R | R | R | R | I | S | R | S |
ME | R | R | R | S | R | R | R | R | R |
E | I | I | R | S | I | R | R | I | R |
CN | S | S | R | S | S | S | R | S | R |
K | S | I | R | S | S | S | R | S | R |
MY | R | R | R | R | S | I | R | S | R |
TOB | S | S | R | S | S | S | R | S | R |
VA | S | S | I | S | S | S | I | I | I |
LEV | S | S | S | S | S | S | S | I | S |
DA | I | I | R | R | S | R | R | R | R |
CTX | R | R | R | R | R | R | R | R | R |
DO | S | S | I | S | S | S | R | S | R |
CIP | I | S | S | S | I | S | S | I | S |
OB | R | R | R | I | I | R | R | R | R |
F | S | S | S | S | I | R | S | R | S |
OT | I | I | R | S | S | R | R | R | R |
S | S | I | R | I | S | R | S | I | S |
TE | S | S | R | S | S | R | R | S | R |
C | S | I | S | S | S | R | S | R | S |
SXT | R | I | R | S | S | R | S | R | S |
MAR Index | 0.58 | 0.67 | 0.83 | 0.38 | 0.41 | 0.58 | 0.54 | 0.71 | 0.58 |
ARI | 0.08 | 0.03 |
R: Resistant; S: Sensitive; I: Intermediate; MAR index: Multi drug resistance; Antibiotics (µg/disc), AML: Amoxycillin 10; AMC: Amoxycillin/clavulanic acid 30; AMP: Ampicillin 10; B: Bacitracin 10; P: Penicillin 10; ME: Methicillin 5; E: Erythromycin 15; CN: Gentamycin 10; K: Kanmycin 30; MY: Lincomycin 10; TOB: Tobramycin 10; VA: Vancomycin 30; LEV: Levofloxacin 5; DA: Clindamycin 2; CTX: Cefotaime 30; DO: Doxycycline 30; CIP: Ciprofloxacin 5; OB: Cloxacillin 5; F: Nitrofurantoin 300; OT: Oxytetracyclin 30; S: Streptomycin 10; TE: Tetracycline 30; C: Chloramphenicol 30; SXT: Sulphamethoxazole/Trimethoprim 25.
Bacterium | ||||||||
---|---|---|---|---|---|---|---|---|
S. enterica | EHEC O157 | Antibiotics | ||||||
Sal 4 | Sal 3 | Sal 2 | Sal 1 | E4 | E3 | E2 | E1 | |
I | R | R | R | S | R | R | R | AML |
I | I | R | S | R | S | S | S | AMC |
S | I | R | S | R | R | R | R | AMP |
R | R | R | R | R | R | R | R | B |
I | R | R | R | R | R | R | R | P |
I | R | R | R | R | R | R | S | ME |
R | R | R | R | R | R | R | R | E |
S | S | R | I | S | R | S | R | CN |
S | I | R | I | I | I | S | I | K |
R | R | R | R | R | R | R | R | MY |
S | S | R | R | I | R | I | R | TOB |
R | R | R | R | R | R | R | R | VA |
S | S | R | S | R | S | S | S | LEV |
R | R | R | R | R | R | R | R | DA |
I | I | I | S | I | I | I | I | CTX |
R | R | R | R | S | R | S | R | DO |
S | S | R | S | R | R | S | S | CIP |
R | R | R | R | R | R | R | R | OB |
S | I | S | S | S | I | S | I | F |
R | R | R | R | R | R | R | R | OT |
R | R | R | R | S | R | R | R | S |
R | R | R | R | S | R | R | R | TE |
S | S | R | S | S | S | S | S | C |
S | S | R | S | S | R | S | S | SXT |
0.67 | 0.96 | 0.75 | 0.63 | 0.67 | 0.88 | 0.63 | 0.75 | IndexMAR |
0.14 | 0.11 | ARI |
R: Resistant; S: Sensitive; I: Intermediate; MAR index: Multi drug resistance; Antibiotics (µg/disc), AML: Amoxycillin 10; AMC: Amoxycillin/clavulanic acid 30; AMP: Ampicillin 10; B: Bacitracin 10; P: Penicillin 10; ME: Methicillin 5; E: Erythromycin 15; CN: Gentamycin 10; K: Kanmycin 30; MY: Lincomycin 10; TOB: Tobramycin 10; VA: Vancomycin 30; LEV: Levofloxacin 5; DA: Clindamycin 2; CTX: Cefotaime 30; DO: Doxycycline 30; CIP: Ciprofloxacin 5; OB: Cloxacillin 5; F: Nitrofurantoin 300; OT: Oxytetracyclin 30; S: Streptomycin 10; TE: Tetracycline 30; C: Chloramphenicol 30; SXT: Sulphamethoxazole/Trimethoprim 25.
(
C. compressa | P. pavonica | ||
---|---|---|---|
DIZ (mm) | DIZ (mm) | MAR Index | |
S. aureus isolates | |||
S1 | 18.5 ± 1.5c | 13.5 ± 1.5b | 0 |
S2 | 14 ± 1d | 11.5 ± 0.5c | 0 |
S3 | 20.5± 0.5b | 11.5 ± 0.5c | 0 |
S4 | - | - | 1 |
S5 | - | - | 1 |
ARI | 0.41 | ||
B. cereus isolates | |||
B1 | 22 ± 2a | - | 0.5 |
B2 | 20.5 ± 0.5b | 16.5 ± 0.5a | 0 |
B3 | 17.5 ± 0.5c | - | 0.5 |
B4 | - | - | 1 |
ARI | 0.5 |
S1: S. aureus 122; S2: S. aureus 128; S3: S. aureus 287; S4: S. aureus 125; S5: S aureus 283; B1: B. cereus 4; B2: B. cereus 16; B3: B. cereus 72; B4: B. pumilus 124. Different letters indicate statistically significant differences between groups (P < 0.05).
C. compressa | P. pavonica | ||
---|---|---|---|
Bacterium | DIZ (mm) | DIZ (mm) | MAR Index |
S. enterica isolates | |||
Sal 1 | 31 ± 1a | 27.5 ± 0.5a | 0 |
Sal 2 | 17.5 ± 0.5c | - | 0.5 |
Sal 3 | 17 ± 1c | - | 0.5 |
Sal 4 | 24.5 ± 0.5b | 14.5 ± 0.5b | 0 |
ARI | 0.5 | ||
EHEC O157 isolates | |||
E1 | 17 ± 0c | - | 0.5 |
E2 | 16.5 ± 1.5c | - | 0.5 |
E3 | 15 ± 0d | - | 0.5 |
E4 | 16 ± 0c | - | 0.5 |
ARI | 0.5 |
Data are expressed as the mean ± standard deviation (SD) of three replicates. Sal 1: S. enterica 17; Sal 2: S. enterica 18; Sal 3: S. enterica 19; Sal 4: S. enterica 29; E1: EHEC O157 57; E2: EHEC O157 55; E3: EHEC O157 52; E4: EHEC O157 49. Different letters indicate statistically significant differences between groups (P < 0.05).
22 mm compared to P. pavonica extract which was observed positive effect against 9 isolates with low inhibition zone ranged from 11 - 16.5 mm.
The highest activity was recorded with C. compressa extract against S. aureus 287, and B. cereus 4 with zones of inhibition 20.5 and 22 mm respectively (P < 0.05). Moderate activity was obtained against S. aureus 122, B. cereus 16 and B. cereus 72 with zones of inhibition 18.5, 20.5 and 17.5 respectively. Weak activity was obtained against S. aureus 128 with zones of inhibition of 14 mm (
The MAR and ARI indices of the tested extracts were reported in
The minimal inhibitory concentration (MIC) of flavonoids extracted from C. compressa and P. pavonica were determined for various tested organisms. The MICs ranged from 31.25 μg/mL to 125 μg/mL and 62.5 μg/mL to 500 μg/mL, respectively. The MICs and MBCs of the extracts are presented in
The flavonoids extract of C. compressa had the lowest MIC value, 31.25 μg/ ml and the lowest MBC value 62.5 μg/ml against S. aureus 122 and S. enterica 17; MIC value of 62.5 μg/ml and MBC value of 125 μg/ml against B. cereus 4
C. compressa | P. pavonica | |
---|---|---|
MBC/MIC | ||
S. aureus isolates | ||
S1 | 2 | 2 |
S2 | 2 | 2 |
S3 | 4 | 3 |
B. cereus isolates | ||
B1 | 2 | - |
B2 | 3 | 2 |
B3 | 2 | - |
S. enterica isolates | ||
Sal 1 | 2 | 2 |
Sal 2 | 4 | - |
Sal 3 | 2 | - |
Sal 4 | 4 | 3 |
EHEC O157 isolates | ||
E1 | 4 | - |
E2 | 4 | - |
E3 | 4 | - |
E4 | 4 | - |
S1: S. aureus 122; S2: S. aureus 128; S3: S. aureus 287; S4: S. aureus 125; S5: S. aureus 283 ; B1: B. cereus 4; B2: B. cereus 16; B3: B. cereus 72; B4: B. pumilus 124; Sal 1: S. enterica 17; Sal 2: S. enterica 18; Sal 3: S. enterica 19; Sal 4: S. enterica 29; E1: EHEC O157 57; E2: EHEC O157 55; E3: EHEC O157 52; E4: EHEC O157 49.
Algae | TFC* | Proanthocyanidin** |
---|---|---|
C. compressa | 110.92 ± 11.38a | 0.24 ± 0.0a |
P. pavonica | 70.08 ± 2.42b | 0.072 ± 0.0b |
Each value is represented as mean ± SD (n = 3). Means with the same letter are not significantly different at P < 0.05. *: Expressed as mg Rutin/g, ** Expressed as mg Catechin/g.
and B. cereus 72, MIC value of 125 μg/ml and MBC value of 250 μg/ml against S. aureus 128 and S. enterica 19, MIC value of 62.5 μg/ml and MBC value of 187.5 μg/ml against B. cereus 16, while MIC value of 62.5 μg/ml and MBC value of 250 μg/ml against S. enterica 29. On the other hand, the highest MIC value of 125 μg/ml, and the highest MBC value of 500 μg/ml due to C. compressa extract were noted for EHEC O157 57, EHEC O157 55 and S. enterica 18 (
The flavonoids extract of P. pavonica showed the lowest MIC value of 62.5 μg/ml and the lowest MBC value of 125 μg/ml against S. enterica 17; MIC value of 125 μg/ml and MBC value of 250 μg/ml was recorded against B. cereus 16; MIC value of 250 μg/ml and MBC value of 500 μg/ml was noted against S. aureus 122 and S. aureus 128; MIC value of 250 μg/ml and MBC value of 750 μg/ml were recorded against S. enterica 29 while the highest MIC value of 500 μg/ml, and the highest MBC value of 1500 μg/ml was obtained against S. aureus 287 (
Flavonoids extracted from C. compressa also displayed the best spectrum of bactericidal effect with a ratio MBC/MIC ≤4 obtained on all tested bacterial strains (
Flavonoids including proanthocyanidin were determined in tested algae. Their
levels in C. compressa extracts were remarkably higher than their counterpart in P. pavonica (P < 0.05). The amount of flavonoids in C. compressa was 110.92 mg/g Rutin equivalent while in P. pavonica was 70.08 mg/g Rutin equivalent (
In order to examine possible association between antibacterial activity and flavonoids and proanthocyanidin content in algae extracts, correlation coefficient (R2) was evaluated.
There were no correlations between the level of flavonoids with antibacterial activity against B. cereus isolates and S. enterica isolates whereas no correlation was observed between proanthocyanidin content and antibacterial activity against EHEC O157 isolates. In addition, a positive correlation was found between each of flavonoids and proanthocyanidin contents in algae extracts with antibacterial activity against S. aureus isolates and EHECO157 isolates (R2 = 0.646, 0.99 respectively).
Antimicrobial resistance is a growing problem and a public health threat. To overcome infections caused by multidrug-resistant strains, new classes of antimicrobials should be developed. Algae may offer an alternative source of antimicrobial agents with significant activities against pathogens with less risks of adverse effect encountered with synthetic antibiotics. Moreover, a number of antibiotics have lost their effectiveness due to the abuse of their applications and the evolution of resistant strains of microorganisms [
Marine organisms including macroalgae are a rich source of structurally novel biologically active metabolites [
Flavonoids belong to the large group of secondary metabolites called polyphenols with variable phenolic structures and are found to be the most important natural substances responsible for their bioactivity. Flavonoids have been reported to exhibit a wide range of bioactivity including antioxidants, antibacterial, antiviral, anticancer, anti-inflammatory, anti-allergic and also as potential therapeutic agents against a wide variety of diseases [
It has been stated that antibacterial activity depends on algal species, the efficiency of the extraction method, and the resistance of the tested bacteria [
The extraction method plays an important role in the overall effect of natural antimicrobial products. Active compounds have been extracted and purified using different extraction methods including ultrasonication assisted extraction and soxhlet extraction [
Antibiotics provide the main basis for the therapy of bacterial infections. However, the high genetic variability of bacteria enables them to rapidly evade the action of antibiotics by developing antibiotic resistance. As resistance becomes more common, there becomes a greater need for alternative treatments. However, despite a push for new antibiotic therapies there has been a continued decline in the number of newly approved drugs [
The antibacterial activity of crude extracts of C. compressa and P. pavonica are well known [
The minimum concentration necessary to kill an organism should be equal to or greater than the MIC for that microbe [
The different rates of inhibition activities appear to be directly related to the qualitative and quantitative amount of the extracted flavonoids content in tested algae. The amount of flavonoids in C. compressa was 110.92 mg/g Rutin equivalent which was higher than that reported by [
Our results are in agreement with several previous findings demonstrating greater activity of flavonoids extracts towards Gram-positive bacteria compared to Gram-negative bacteria [
To the best of our knowledge, there are no reports concerning the comparative antibacterial activity analysis for investigated flavonoids extracted from algae. However, many studies have been done on flavonoids extracted from plants [
Flavonoids have proven to be antibacterial agents, especially the ones with hydrophobic substituent such as prenyl groups, alkylamino chains, alkyl chains, and nitrogen or oxygen containing heterocyclic moieties [
The exact mode of action mechanism of natural antimicrobials is still not fully understood. However, different mechanisms for different antimicrobial groups have been reviewed [
This study concluded that the flavonoids isolated from Libyan algae showed antibacterial potentiality against multi-drug resistant Gram positive and negative bacterial isolates including MRSA. However, whether such extracts will act as effective antibacterial agents in vivo remain to be investigated and the study of mechanisms of actions is necessary prior to their application.
This study was partially supported by a grant provided by the Libyan Authority for Research, Science and Technology (LARST). Authors are grateful to Veronica Papini, a technician in Istituto Zooprofilattico Sperimentale della Lombardia e dell’ Emilia Romagna, Brescia, Italy, who performed the partial sequecing of 16S rDNA.
Authors have declared that no competing interests exist.
Alghazeer, R., Elmansori, A., Sidati, M., Gammoudi, F., Azwai, S., Naas, H., Garbaj, A. and Eldaghayes, I. (2017) In Vitro Antibacterial Activity of Flavonoid Extracts of Two Selected Libyan Algae against Multi-Drug Resistant Bacteria Isolated from Food Products. Journal of Biosciences and Medicines, 5, 26-48. http://dx.doi.org/10.4236/jbm.2017.51003