A continuation of the previous article concerning antibiotic resistance and essential oils. Here are some of the pathways resistant organisms use:
THE PREVALENCE OF RESISTANCE MODIFYING CAPABILITY BY ESSENTIAL OILS
Antimicrobial agents are classified based on their principal mechanisms of action. These mechanisms include interference with cell wall biosynthesis (β-lactams and glycopeptides agents), inhibition of bacterial protein synthesis (marcolides and tetracyclines), interference with nucleic acid synthesis (fluroquinolones and rifampin), inhibition of a metabolic pathway (trimethoprim-sulfamethoxazole, and disruption of bacterial membrane structure (polymyxins and daptomycin) [9]. Three main targets of antibiotics are cell wall, protein and nucleic acids biosynthesis. Throughout the years since antibiotic was introduced, bacteria have acquired various resistances to survive in the deluge of antibiotics. The mechanisms vary and make the work of mitigating the spread of resistance more challenging.
Β-lactam antibiotics are the commonest treatment for bacterial infections. This antibiotic class consists of four major groups: penicillins, cephalosporins, monobactams and carbapenems. Hydrolysis of β-lactam compounds by β-lactamases is the most widespread mechanism of bacterial resistance against this class of antibiotic. Capability of Staphylococcus aureus in producing penicillinase (a form of β-lactamase) to destroy penicillin G was reported within two years after the antibiotic was first introduced [44]. The occurrence of MRSA is a classic example of the redundancy of new antimicrobials with regards to only one species.
Beta-Lactamase Inhibition
The β-lactamases are the major defense of gram-negative bacteria against β-lactam antibiotics. Under the selective pressure of β-lactams, bacteria produce a vast array of β-lactamases. Bacteria respond with a plethora of “new” β-lactamases – including extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC enzymes, and carbapenem-hydrolyzing β-lactamases (carbapenemases) with variable successes [45]. Resistance is typically mediated by the expression of plasmid-encoded β-lactamases, such as TEM-1, TEM-2, or SHV-1, which hydrolyze and inactivate these drugs. However, investigations into essential oils/antibiotics combination against beta-lactamase producers are currently limited, there is one report on the synergistic effects of oregano essential oil in combination with fluroquinolones, doxycycline, lincomycin, maquindox and flofenicol against ESBL-producing E. coli suggesting the possibility that essential oils might function as the ESBLs inhibitor [38].
Bacterial Efflux Pump Inhibition
Most of the antibiotics need to be transported across the cell membrane and subsequently achieve an effective concentration in the cytoplasm to exert inhibitory effects on bacteria. It was demonstrated by Walsh that the overproduction of protein pumps at the bacterial membrane facilitates the pumping out of the drug to be faster than it can diffuse in to keep intra-bacterial drug concentrations below the therapeutic level [8]. The pumps are highly conserved and are chromosomally encoded elements while multidrug resistance efflux pumps are variants of membrane pumps in all microorganisms in response to the external environment. They are able to efflux a large range of compounds including synthetic antibiotics that were not present in the natural ecosystems before their synthesis by humans. Thus, it is believed that bacteria will not easily resist compounds which are natural as compared to the synthetic compounds (the later classes of antibiotics). Related study on phytochemicals addresses this hypothesis and they have been proven to be potent antimicrobial agents. The ability of essential oils in inhibiting the multidrug efflux pump reveals its potential in broader spectrum of pump-inhibitory activity against multi-drug resistant organisms [10]. Lorenzi et al. (2009) evaluated that general component in the essential oil of Helichrysum italicum not only reduces chloramphenicol resistance of the multi-drug resistant Enterobacter aerogens that overexpresses efflux pumps but also modulates the intrinsic resistance of the wild-type control strain and other gram-negative bacteria [46].
Cell Wall and Membrane Disturbance
The bacterial cell wall biosynthetic machinery remains one of the most promising niches for antibiotic targets. However, the impermeable nature of the gram-negative envelope and presence of multiple efflux pumps in combination with other resistance mechanisms contribute to the difficulty of this task. Clinical resistance to β-lactams in gram-negative bacteria is often coupled with reduced outer membrane permeability. As the secondary constituents of the aromatic plants, essential oils are known to contain wide ranges of polyphenols and terpenoids. These phenols possess a strong binding affinity to different molecular structures such as protein or glycoproteins due to their large lipophilicity. Hence, they have great affinities for cell membranes and exhibit high potential to permeate through cell walls, leading to the leakage of cell contents [28, 47]. The ability of tea tree oil (Melaleuca alternifolia) acting as membrane permeabilizer leading to loss of chemiosmotic control in both gram-positive and gram-negative bacteria was elucidated by Cox et al. (2000) [48]. Later, the biological damage of tea tree oil on cell ultra-structures (i.e. cytoplasm and cytoplasmic membrane) was also studied with the aid of electron microscopy [49, 50].
Anti-Quorum Sensing
The role of quorum sensing is well known in microbial pathogenicity and antibiotic resistance. Quorum sensing is responsible for motility and swarming patterns, biofilm formation and stress resistance based on the signaling of molecules. An example of a well-studied molecule in this area is acylated homoserine lactones (AHLs). The Gram-negative bacteria use AHLs for signaling whereas the Gram-positive bacteria use modified oligopeptides. The crucial role of quorum sensing on so many essential aspects of the bacterial ecology makes this an interesting process to target to control persistent infections due to antimicrobial resistance.
Screening of potential quorum-quenching activities often involves biosensors and bioluminescene production or inhibition. Some of the common biosensors include Chromobacterium violaceum CV026 and N-acyl homoserine lactone producing E. coli [51-54]. Rose, geranium, lavender, clove and rosemary oils were found to be the QS inhibitors in the sensor strains Chromobacterium violaceum CV026, Escherichia coli ATTC 31298, Chromobacterium violaceum (CV12472 and CVO26) and Pseudomonas aeruginosa (PAO1) respectively [52, 55].
Sensitivity of Gram-Positive and Gram-Negative Bacteria Towards Essential Oils
Generally, essential oils are more efficacious towards gram-positive than gram-negative bacteria [56, 57]. It has been hypothesized that the presence of lipopolysaccharide encompassing the bacterial peptidoglycan layer has restricted the diffusion of hydrophobic compounds into the cytoplasm [58]. However, not all studies of essential oils have concluded that gram-positives are more susceptible [22, 59]. Interestingly, in a study carried out by van Vuuren et al. (2009), combination of the essential oil of Rosmarinus officnalis with ciprofloxacin against gram-positive bacteria gave an antagonistic profile while Rosmarinus officnalis/ciprofloxacin against gram-negative bacteria displayed a favorable synergistic profile [39]. The prevalent antagonistic interaction test against S. aureus also suggested that natural therapies using essential oils should be monitored carefully when combined with antibiotics. Since only inadequate sample sets have been studied, more exhaustive investigations would warrant a better potentiating profile of essential oils as resistance modifiers of antibiotics in clinical applications.
THE PREVALENCE OF RESISTANCE MODIFYING CAPABILITY BY ESSENTIAL OILS
Antimicrobial agents are classified based on their principal mechanisms of action. These mechanisms include interference with cell wall biosynthesis (β-lactams and glycopeptides agents), inhibition of bacterial protein synthesis (marcolides and tetracyclines), interference with nucleic acid synthesis (fluroquinolones and rifampin), inhibition of a metabolic pathway (trimethoprim-sulfamethoxazole, and disruption of bacterial membrane structure (polymyxins and daptomycin) [9]. Three main targets of antibiotics are cell wall, protein and nucleic acids biosynthesis. Throughout the years since antibiotic was introduced, bacteria have acquired various resistances to survive in the deluge of antibiotics. The mechanisms vary and make the work of mitigating the spread of resistance more challenging.
Β-lactam antibiotics are the commonest treatment for bacterial infections. This antibiotic class consists of four major groups: penicillins, cephalosporins, monobactams and carbapenems. Hydrolysis of β-lactam compounds by β-lactamases is the most widespread mechanism of bacterial resistance against this class of antibiotic. Capability of Staphylococcus aureus in producing penicillinase (a form of β-lactamase) to destroy penicillin G was reported within two years after the antibiotic was first introduced [44]. The occurrence of MRSA is a classic example of the redundancy of new antimicrobials with regards to only one species.
Beta-Lactamase Inhibition
The β-lactamases are the major defense of gram-negative bacteria against β-lactam antibiotics. Under the selective pressure of β-lactams, bacteria produce a vast array of β-lactamases. Bacteria respond with a plethora of “new” β-lactamases – including extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC enzymes, and carbapenem-hydrolyzing β-lactamases (carbapenemases) with variable successes [45]. Resistance is typically mediated by the expression of plasmid-encoded β-lactamases, such as TEM-1, TEM-2, or SHV-1, which hydrolyze and inactivate these drugs. However, investigations into essential oils/antibiotics combination against beta-lactamase producers are currently limited, there is one report on the synergistic effects of oregano essential oil in combination with fluroquinolones, doxycycline, lincomycin, maquindox and flofenicol against ESBL-producing E. coli suggesting the possibility that essential oils might function as the ESBLs inhibitor [38].
Bacterial Efflux Pump Inhibition
Most of the antibiotics need to be transported across the cell membrane and subsequently achieve an effective concentration in the cytoplasm to exert inhibitory effects on bacteria. It was demonstrated by Walsh that the overproduction of protein pumps at the bacterial membrane facilitates the pumping out of the drug to be faster than it can diffuse in to keep intra-bacterial drug concentrations below the therapeutic level [8]. The pumps are highly conserved and are chromosomally encoded elements while multidrug resistance efflux pumps are variants of membrane pumps in all microorganisms in response to the external environment. They are able to efflux a large range of compounds including synthetic antibiotics that were not present in the natural ecosystems before their synthesis by humans. Thus, it is believed that bacteria will not easily resist compounds which are natural as compared to the synthetic compounds (the later classes of antibiotics). Related study on phytochemicals addresses this hypothesis and they have been proven to be potent antimicrobial agents. The ability of essential oils in inhibiting the multidrug efflux pump reveals its potential in broader spectrum of pump-inhibitory activity against multi-drug resistant organisms [10]. Lorenzi et al. (2009) evaluated that general component in the essential oil of Helichrysum italicum not only reduces chloramphenicol resistance of the multi-drug resistant Enterobacter aerogens that overexpresses efflux pumps but also modulates the intrinsic resistance of the wild-type control strain and other gram-negative bacteria [46].
Cell Wall and Membrane Disturbance
The bacterial cell wall biosynthetic machinery remains one of the most promising niches for antibiotic targets. However, the impermeable nature of the gram-negative envelope and presence of multiple efflux pumps in combination with other resistance mechanisms contribute to the difficulty of this task. Clinical resistance to β-lactams in gram-negative bacteria is often coupled with reduced outer membrane permeability. As the secondary constituents of the aromatic plants, essential oils are known to contain wide ranges of polyphenols and terpenoids. These phenols possess a strong binding affinity to different molecular structures such as protein or glycoproteins due to their large lipophilicity. Hence, they have great affinities for cell membranes and exhibit high potential to permeate through cell walls, leading to the leakage of cell contents [28, 47]. The ability of tea tree oil (Melaleuca alternifolia) acting as membrane permeabilizer leading to loss of chemiosmotic control in both gram-positive and gram-negative bacteria was elucidated by Cox et al. (2000) [48]. Later, the biological damage of tea tree oil on cell ultra-structures (i.e. cytoplasm and cytoplasmic membrane) was also studied with the aid of electron microscopy [49, 50].
Anti-Quorum Sensing
The role of quorum sensing is well known in microbial pathogenicity and antibiotic resistance. Quorum sensing is responsible for motility and swarming patterns, biofilm formation and stress resistance based on the signaling of molecules. An example of a well-studied molecule in this area is acylated homoserine lactones (AHLs). The Gram-negative bacteria use AHLs for signaling whereas the Gram-positive bacteria use modified oligopeptides. The crucial role of quorum sensing on so many essential aspects of the bacterial ecology makes this an interesting process to target to control persistent infections due to antimicrobial resistance.
Screening of potential quorum-quenching activities often involves biosensors and bioluminescene production or inhibition. Some of the common biosensors include Chromobacterium violaceum CV026 and N-acyl homoserine lactone producing E. coli [51-54]. Rose, geranium, lavender, clove and rosemary oils were found to be the QS inhibitors in the sensor strains Chromobacterium violaceum CV026, Escherichia coli ATTC 31298, Chromobacterium violaceum (CV12472 and CVO26) and Pseudomonas aeruginosa (PAO1) respectively [52, 55].
Sensitivity of Gram-Positive and Gram-Negative Bacteria Towards Essential Oils
Generally, essential oils are more efficacious towards gram-positive than gram-negative bacteria [56, 57]. It has been hypothesized that the presence of lipopolysaccharide encompassing the bacterial peptidoglycan layer has restricted the diffusion of hydrophobic compounds into the cytoplasm [58]. However, not all studies of essential oils have concluded that gram-positives are more susceptible [22, 59]. Interestingly, in a study carried out by van Vuuren et al. (2009), combination of the essential oil of Rosmarinus officnalis with ciprofloxacin against gram-positive bacteria gave an antagonistic profile while Rosmarinus officnalis/ciprofloxacin against gram-negative bacteria displayed a favorable synergistic profile [39]. The prevalent antagonistic interaction test against S. aureus also suggested that natural therapies using essential oils should be monitored carefully when combined with antibiotics. Since only inadequate sample sets have been studied, more exhaustive investigations would warrant a better potentiating profile of essential oils as resistance modifiers of antibiotics in clinical applications.
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