Enhancement of Antimicrobial Activity of Four Classes of Antibiotics Combined
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.
Received: March 10, 2012;
Accepted: March 15, 2012;
Published: June 26, 2012
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.
|| 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.
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.
||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
||Time-kill curve showing the bactericidal activity of garlic-gentamicin
combination against A 15
||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.
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 garlics 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.
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.
1: Aminov, R.I., 2009. The role of antibiotics and antibiotic resistance in nature. Environ. Microbiol., 11: 2970-2988.
2: Ankri, S. and D. Mirelman, 1999. Antimicrobial properties of allicin from garlic. Microbes Infect., 1: 125-129.
CrossRef | Direct Link |
3: Bergogne-Berezin, E. and K.J. Towner, 1996. Acinetobacter spp. as nosocomial pathogens: Microbiological, clinical and epidemiological features. Clin. Microbiol. Rev., 9: 148-165.
PubMed | Direct Link |
4: Bjarnsholt, T., P.O. Jensen, T.B. Rasmussen, L. Christophersen and H. Calum et al., 2005. Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infectin. Microbiology, 151: 3873-3880.
Direct Link |
5: Block, E., 1985. The chemistry of garlic and onions. Sci. Am., 252: 114-119.
Direct Link |
6: Block, E., S. Ahmad, M.K. Jain, R. W. Crecely, R. Apitz-Castro, and M.R. Cruz, 1984. (E,Z)-Ajoeno: A potent antithrombotic agent from garlic. J. Am. Chem. Soc., 106: 8295-8296.
7: Cai, Y., R. Wang, F. Pei and B.B. Liang, 2007. Antibacterial activity of allicin alone and in combination with β-lactams against Staphylococcus spp. and Pseudomonas aeruginosa. J. Antibiot., 60: 335-338.
8: Cellini, L., E.D. Campli, M. Masuli and S. Di Bartolomeo and N. Allocati, 1996. Inhibition of Helicobacter pylori by garlic extract (Allium sativum). FEMS Immunol. Med. Microbiol., 13: 273-277.
9: Ciofu, O., B. Giwercman, S.S. Pedersen and N. Hoiby, 1994. Development of antibiotic resistance in Pseudomonas aeruginosa during two decades of antipseudomonal treatment at the Danish CF Center. Apmis, 102: 674-680.
10: CLSI., 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A7. 7th Edn., Clinical and Laboratory Standards Institute, Wayne, PA
11: Cutler, R.R. and P. Wilson, 2004. Antibacterial activity of a new, stable, aqueous extract of allicin against methicillin-resistant Staphylococcus aureus. Br. J. Biomed. Sci., 61: 71-74.
12: De Kraker, M.E., P.G. Davey and H. Grundmann, 2011. Mortality and hospital stay associated with resistant Staphylococcus aureus and Escherichia coli bacteremia: Estimating the burden of antibiotic resistance in Europe. PLoS Med., Vol. 8
13: Hancock, R.E.W. and D.P. Speert, 2000. Antibiotic resistance in Pseudomonas aeruginosa: Mechanisms and impact on treatment. Drug Resistance Updates, 3: 247-255.
CrossRef | PubMed | Direct Link |
14: Iwalokun, B.A., A. Ogunledun, D.O. Ogbolu, S.B. Bamiro and J. Jimi-Omojola, 2004. In vitro antimicrobial properties of aqueous garlic extract against multidrug-resistant bacteria and Candida species from Nigeria. J. Med. Food, 7: 327-333.
Direct Link |
15: Jonkers, D., E. van den Broek, I. van Dooren, C. Thijs, E. Dorant, G. Hageman and E. Stobberingh, 1999. Anti-bacterial effect of garlic and omeprazole on Helicobacter pylori. J. Antimicrob. Chemother., 43: 837-839.
16: Kharazmi, A., H.O. Eriksen, G. Doring, W. Goldstein and N. Hoiby, 1986. Effect of Pseudomonas aeruginosa proteases on human leukocyte phagocytosis and bactericidal activity. Acta Pathol. Microbiol. Immunol. Scand., 94: 175-179.
17: Perez-Giraldo, C., G. Cruz-Villalon, R. Sanchez-Silos, R. Martinez-Rubio, M.T. Blanco and A.C. Gomez-Garcia, 2003. In vitro activity of allicin against Staphylococcus epidermidis and influence of subinhibitory concentrations on biofilm formation. J. Applied Microbiol., 95: 709-711.
18: Perez, F., A.M. Hujer, K.M. Hujer, B.K. Decker, P.N. Rather and R.A. Bonomo, 2007. Global challenge of multidrug-resistant Acinetobacter baumannii. Antimicrob. Agents Chemother., 51: 3471-3484.
CrossRef | PubMed |
19: Rao, J., F.H. Damron, M. Basler, A. Digiandomenico, N.E. Sherman, J.W. Fox, J.J. Mekalanos and J.B. Goldberg, 2011. Comparisons of two proteomic analyses of non-mucoid and mucoid Pseudomonas aeruginosa clinical isolates from a cystic fibrosis patient. Frontiers Microbiol., Vol. 2.
20: Shuford, J.A., J.M. Steckelberg and R. Patel, 2005. Effects of fresh garlic extract on Candida albicans biofilms. Antimicrobial Agents Chemotherapy, Vol. 49.
21: Weber, N.D., D.O. Andersen, J.A. North, B.K. Murray, L.D. Lawson and B.G. Hughes, 1992. In vitro virucidal effects of Allium sativum (garlic) extract and compounds. Planta Med., 58: 417-423.
CrossRef | PubMed | Direct Link |