Abstract: The increased resistance to antimicrobial agents among clinical isolates is a serious problem that dramatically raises the cost of health care worldwide. Seeking alternative approaches to enhance the susceptibility of these microorganisms to killing is a major concern to researchers. One such approach is the combination of some adjuncts with antibiotics. Garlic (Allium sativum) is an herbal product traditionally used for many health-related purposes, including protection against microbial infections. This work investigates the effect of combining garlic, in sub-inhibitory concentrations, with four different antibiotics against sixteen selected multidrug resistant Gram-negative clinical isolates belonging to Pseudomonas and Acinetobacter genera. A combination of 5 and 10 mg mL-1 of garlic (for Acinetobacter and Pseudomonas, respectively) with levofloxacin, gentamicin, azithromycin and doxycycline resulted in a decrease in the antibiotics' Minimum Inhibitory Concentrations (MICs) against the isolates in the range of 4-≥32, 4-≥2048, 2-≥2048 and 2-≥128 fold, respectively. The kinetics of killing of the garlic-gentamicin combination were subsequently followed in four Pseudomonads for 24 h and a significant effect ranging between 2 and 5 log reduction in bacterial count, compared to the control, was obtained. The results show a great potential for the use of garlic as an adjunct to antibiotics for the treatment of infections caused by resistant Gram-negative strains and warrant further investigation.
INTRODUCTION
The extensive production and widespread use of antibiotics worldwide in clinical and veterinary medicine, agriculture, aquaculture, horticulture as well as other human activities has lead to the evolution of antibiotic resistance among human and animal pathogens (Aminov, 2009). Dissemination of antibiotic resistance results in thousands of deaths each year and imposes a considerable economic and social burden on health care systems (De Kraker et al., 2011).
Pseudomonas aeruginosa is a Gram-negative bacterium omnipresent in the environment, in addition to being an important human pathogen. It is a major causative agent of infections in immunocompromised patients, such as those suffering from burn wounds or receiving cancer chemotherapy. P. aeruginosa also infects the lungs of cystic fibrosis patients leading to high mortality and morbidity (Rao et al., 2011). The reason for such high cost of Pseudomonas infections lies in its intrinsic resistance to a broad spectrum of antibiotics and its arsenal of virulence factors (Hancock and Speert, 2000). Acinetobacter is yet another Gram-negative bacterium found in soil and fresh water, besides, it has become a serious culprit in causing an array of nosocomial infections (Bergogne-Berezin and Towner, 1996). Such infections are especially challenging because of the organism's high potential to rapidly develop antibiotic resistance, with the looming possibility of multidrug resistance within a few decades (Perez et al., 2007).
Garlic (Allium sativum) is an herbal product that has been known since ancient times and is used in the folklore medicine to guard against a number of infections because of its antibacterial, antifungal and antiviral properties (Block, 1985; Weber et al., 1992). The active ingredient (allicin or diallyl thiosulphinate, Fig. 1a) is produced when a garlic clove is crushed or damaged and alliin (Fig. 1b), (a precursor compound representing about 0.24% (w/w) of each garlic clove, comes in contact with the enzyme alliinase (originally present in a separate vesicle) (Block et al., 1984).
In this study we hypothesize that the use of garlic as an adjunct in antibiotic regimens will improve the antimicrobial performance to overcome the problem of increasing antibiotic resistance among clinical isolates.
| Fig. 1(a-b): | The chemical structure of (a) allicin (b) alliin |
Another objective of the current work is to follow the killing kinetics of the garlic-antibiotic combination, relative to either component alone.
MATERIALS AND METHODS
Test organisms: Sixteen Gram-negative microorganisms were used in this study. They were collected in a clinical microbiology laboratory affiliated with El-Meery tertiary teaching hospital in Alexandria, Egypt. The microorganisms were eight Acinetobacter baumannii (designated “A” isolates) and eight P. aeruginosa (designated “P” isolates) obtained from various clinical specimens.
Antibiotic powders: Levofloxacin (Amoun Pharmaceutical Company, Egypt), Doxycycline (The Nile Co. for Pharmaceuticals and Chemical Industries, Egypt) and Gentamicin (Schering Plough Corporation, Egypt) were purchased from the corresponding pharmaceutical companies. Azithromycin was obtained as a gift from Amriya Pharmaceutical Co. (Egypt).
Bacterial identification: The microorganisms were identified by means of conventional methods and included morphological, cultural properties and biochemical characteristics that were estimated by API system (BioMérieux, France). For A. baumannii isolates, growth at 44°C was used to confirm identity. The identified strains were stored at –70°C in nutrient broth (Oxoid, England) containing 20% glycerol until needed for further tests.
Antimicrobial susceptibility testing: The in vitro antimicrobial activity of the four antibiotics, alone and combined with garlic, against the tested isolates was determined by the broth microdilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2006). Microtiter plates, containing two-fold serial dilutions of each antibiotic in Luria-Bertani (LB) medium (Bioshop, Canada) in addition to 5 or 10 mg mL-1 of garlic (for Acinetobacter and Pseudomonas, respectively), were inoculated with each organism to yield the appropriate density (5x105 CFU mL-1) in a final volume of 180 μL per well. The plates were then incubated for 24 h at 37°C. To determine the MIC of the antibiotics alone, the same procedure was used except that garlic was replaced with distilled water. The MIC was defined as the lowest concentration of the antibiotic completely inhibiting the growth of the organism as detected by the unaided eye. Susceptibility rates were determined following CLSI breakpoints.
Biocidal activity of garlic-gentamicin combination against selected strains: In vitro bactericidal activity of garlic-gentamicin combination against three Acinetobacter and four Pseudomonas isolates was evaluated using time-kill assays according to CLSI M26-A protocol with some modifications to suit the test conditions. Probe tubes contained garlic (5 or 10 mg mL-1 for Acinetobacter and Pseudomonas, respectively), gentamicin (4 μg mL-1 for all isolates, except for A13, A16 and P25 2 μg mL-1, corresponding to the next to last inhibitory concentration as determined from antimicrobial susceptibility testing) or garlic-gentamicin combination (at the previous concentrations). Tubes were inoculated with the test microorganisms (106 CFU mL-1) and incubated aerobically in a shaking water bath at 37°C for 24 h. Aliquots were removed from each tube and serial dilutions were plated in duplicates onto LB plates after 0, 2.5, 5 and 24 h of incubation. Colony counts were determined after 24 h of incubation at 37°C. Bactericidal activity was defined as a 3 log10 reduction in the bacterial count compared with the initial inoculum at zero time. In each case, a control lacking gentamicin and garlic was included in the procedure.
RESULTS
Susceptibility testing: In vitro antimicrobial activity of levofloxacin, gentamicin, azithromycin and doxycycline, alone and in combination with a sub-inhibitory concentration of garlic, was determined by broth microdilution technique against sixteen clinical isolates belonging to the Pseudomonads and Acinetobacter species and the findings are summarized in Table 1.
| Table 1: | Distribution of fold decrease in the Minimum Inhibitory Concentration (MIC) of levofloxacin, gentamicin, azithromycin and doxycycline, against the sixteen tested isolates, as a result of combining with garlic |
| Fig. 2a: | Time-kill curve showing the bactericidal activity of garlic-gentamicin combination against A 15 |
| Fig. 2b: | Time-kill curve showing the bactericidal activity of garlic-gentamicin combination against P3657 |
Garlic was initially tested alone for its antimicrobial activity against a large number of clinical Pseudomonas and Acinetobacter isolates. As expected, garlic demonstrated great potency against the majority of the tested isolates and its inhibitory concentration for each isolate was recorded (data not shown). As for the combination, generally, more promising results were obtained with Acinetobacter than with Pseudomonas isolates. 65.7% of the combinations tested against Pseudomonas species demonstrated variable enhancement in the activity of the antibiotics with the isolates P25 and P3657 being the most inhibited in response to gentamicin-garlic, followed by levofloxacin-garlic combination. On the other hand, a higher percentage, amounting to 90.6%, of garlic-antibiotic combinations showed even greater inhibitory effect on Acinetobacter species, compared to the effect of antibiotics alone. Two isolates, A17 and A18, were particularly highly affected by all four antibiotic-garlic combinations.
The two antibiotics whose antimicrobial activities were most highly influenced by the combination with garlic were azithromycin and gentamicin, showing shifts in MIC values ranging between≥2 and≥2048 fold against the tested clinical isolates. This was best evidenced in the finding that 37.5 and 25% of the Acinetobacter isolates (A17, A18, A22 and A17, A18) displayed≥2048 fold increase in their susceptibility to gentamicin and azithromycin, respectively, after combining with garlic. Doxycycline and levofloxacin came next with MIC shifts ranging between≥2 and≥128 fold, with the highest shifts observed against A17 in case of doxycycline and P414 and P3657 in case of levofloxacin.
Time-kill studies: The in vitro bactericidal activity of the garlic combination with gentamicin was then tested using the time-kill assay against seven clinical isolates displaying MIC decrease due to the combination, the effect can be seen in the representative examples shown in Fig. 2a and b. The effect was bacteriostatic at the best with the Acinetobacter showing regrowth after 24 h to counteract the inhibitory effect seen at shorter times of exposure to garlic or the combination. Garlic enhanced the antibacterial activity of gentamicin against Pseudomonas, an effect that was more pronounced at 24 h with 3.6, 4.2, 4.77 and 2.1 log reduction in viable count of P3657, P414, P410 and P25 isolates, respectively, compared to the control at the same time interval (data not shown). At shorter exposure times (2.5 h) and to a lesser extent (5 h), the effect of garlic on the activity of gentamicin was generally less pronounced.
DISCUSSION
The development of bacterial resistance to antibiotics is a severe problem that highlights the importance of developing new strategies to limit the different mechanisms of resistance (Ciofu et al., 1994). Garlic (Allium sativum) an essential food ingredient, has established strong antibacterial, antifungal and antiviral actions. Allicin (diallyl thiosulphinate) is the main constituent of garlic showing antimicrobial activity and it is generated by the enzyme alliinase when garlic is crushed (Weber et al., 1992; Ankri and Mirelman, 1999). Garlic extract has demonstrated a great antibacterial activity for controlling methicillin-resistant Staphylococcus aureus and other pathogens (Cutler and Wilson, 2004). Garlic extract also affected Escherichia, Salmonella, Staphylococcus, Streptococcus, Klebsiella, Proteus, Clostridium, Mycobacterium and Helicobacter species as previously demonstrated (Cellini et al., 1996).
The present study investigates the potential of using garlic as an adjunct to antibiotic therapy, in an attempt to overcome the increased tolerance among infectious microorganisms towards these antibiotics. The tested antibiotics belong to four different classes with mechanisms of action inhibiting protein synthesis or affecting nucleic acids.
The antimicrobial activity, in terms of MIC, of the four studied antibiotics was tested alone and in the presence of sub-inhibitory concentration of garlic, using broth microdilution method as it was more convenient than the agar diffusion technique in this particular situation. When combined with antibiotics, garlic was found to enhance the antibacterial activity of the tested antibiotics to variable degrees against Acinetobacter and Pseudomonas species. A previous report was in accordance showing a synergistic antibacterial effect when garlic extract and tobramycin were combined (Shuford et al., 2005). In another study, allicin was found to enhance the antibacterial activity of cefazolin, oxicillin and cefoperazone at sub-inhibitory concentrations (Cai et al., 2007).
A possible explanation could be blocking of quorum sensing and communication systems of the microorganism as a result of garlic treatment rendering P. aeruginosa sensitive to the action of antibiotics and suggesting a means to reduce the virulence and control P. aeruginosa infections (Bjarnsholt et al., 2005). Moreover, garlic appears to alter the structure and integrity of the outer surface of microbial cells as well as decrease their total lipid content (Iwalokun et al., 2004) which allows better access and subsequent inhibition of the tested antibiotics to their respective targets either protein or nucleic acid. Another probable mechanism for the enhancement of antimicrobial activity of antibiotics when combined with garlic could be the well-established garlic’s antibiotic function, which is likely to inhibit bacterial attachment. Evidence for this last mechanism was provided by a previous study that showed that sub-MICs of allicin might play a role in the prevention of adherence of Staphylococcus epidermidis to microtiter plates (Perez-Giraldo et al., 2003). Similarly, Shuford et al. (2005) demonstrated that fresh garlic extract inhibited growth of Candida albicans in its planktonic, adherent and sessile phases. In addition, the administration of garlic is expected not only to reduce the persistence of biofilms, a structure that plays a major role in increasing antimicrobial resistance, but also to inhibit the expression of bacterial virulence determinants that actively degrade components of the defense system (Kharazmi et al., 1986). Nevertheless, in the current work, some tested combinations were ineffective. This agrees with the findings of Jonkers et al. (1999) who found no effect of garlic on amoxicillin or clarithromycin against Helicobacter pylori suggesting the changeable effect of garlic on the antibacterial activity of antibiotics depending on the strain tested.
In time-kill assays conducted in the current study, the potential bactericidal effect of garlic-gentamicin combination was followed against selected clinical isolates for 24 h. Garlic was found to enhance the activity of gentamicin against P. aeruginosa particularly at 24 h, yet the effect was not bactericidal as the combination usually showed a count reduction of less than 3 logs, compared to the count at zero time. This comes in contrast with the findings of Shuford et al. (2005) who found that the superior activity of garlic occurred at 1 versus 48 h of treatment and this probably relates to the half-life of fresh garlic extract at 37°C and would be an important consideration in the development of in vivo uses. However, data obtained by Shuford and colleagues (Shuford et al., 2005) somewhat agree with our results for the Acinetobacter and may imply that a genus related factor might be involved. Another possible explanation for the little or no effect observed with the Acinetobacter could be the development of biofilm. These findings are consistent with other works that found that the in vitro activity decreases as the biofilm phenotype develops (Bjarnsholt et al., 2005).
The results at hand show great promise and merit thorough investigation to further document and determine the exact mechanism of action of the different components of garlic extract in a synergistic combination with antibiotics. This is the aim of our next research project since the current work is one in a series conducted to elucidate the combined effect of garlic and antibiotics in overcoming antibiotic resistance among bacterial cells.
ACKNOWLEDGMENTS
We are indebted to the Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University for providing the facilities to conduct the current work and to Atos-pharma for supplying garlic powder.