**2. Antibiotic classes and multidrug‐resistant** *E. coli*

### **2.1. Antibiotic classes and their respective natures**

From the discovery of penicillin by Alexander Fleming in the early nineteenth century, approximately 20 classes of antibiotic have been discovered in time. However, only antibiotic classes that are effective against *E. coli* would be thoroughly discussed in this subsection.

### *2.1.1. β‐lactam antibiotics*

β‐Lactam antibiotics are one of the most common yet diverse classes of antibiotics and are the first class of antibiotics discovered in the 1930s. They were effective against both Gram-positive and Gram-negative bacteria and were categorized into four main groups, carbapenems, cephalosporins, monobactams, and penicillins, with each group sharing structural similarity in the β‐lactam ring within the antibiotic molecule [4]. The β‐lactam antibiotics mainly target the bacterial cell wall synthesis pathway, and are thus termed "broad spectrum antibiotics." Under normal physiological conditions, bacteria constantly renew their cell wall in order to replace broken ones. A unit of peptidoglycan cell wall consists of two subunits, the alternating *N*-acetylglucosamine and *N*-acetylmuramic acid. Each subunit contains an identical pentapeptide chain, which links both subunits together via the action of a transpeptidase, penicillin‐binding protein (PBP) [5, 6]. β‐Lactam antibiotics would act as an irreversible inhibitor toward PBP. The β‐lactam ring of the antibiotic mimics the structure of the pentapeptide chain and thus is able to bind with PBP, acylating it's active site and rendering it inactive [7, 8]. Hence, the action of β‐lactam antibiotic halts cell wall synthesis of bacteria, which eventually compromises the rigidity of the cell wall, leading to cell lysis.

## *2.1.2. Fluoroquinolone*

(CD) and noncommunicable diseases (NCD) further complicates the problem [1]. There is a two-sided role for antibiotics; and although their uncontested and unquestionable role was recognized to significantly reduce the statistics of the infectious diseases burden worldwide, their rampant use also contributed to the unexpected emergence of antibiotic resistant microorganisms attributed to over‐prescription and misuse, hence, the emergence of the multidrug resistant Enterobacteriaceae, especially *Escherichia coli* (*E. coli*). Adversely, the last line of antibiotics, colistin, which had only been recently revived since 1959 amidst the fairly new emergence of carbapenem-resistant *Enterobacteriaceae*, had been reported by Chinese researchers to be inefficacious against *E. coli* recently, in infected pigs from a farm near Shanghai, and the spread of colistin resistance had increased significantly especially in the agriculture industry over time, which may be escalated to a global scale [2, 3]. With the establishment of new resistance, the Chinese authors have emphasized the urgent need for coordinated global action in the fight against pan‐drug‐resistant Gram‐negative bacteria and one of these strategies proposed included investigation into natural products, in this case, essential oils. This chapter aims to introduce the usage of synergistic combinatorial therapy between different classes of antibiotics and essential oils against multidrug resistant *E. coli* (MDR *E. coli*) and to detail the methodologies used to establish synergism as well as the

300 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

From the discovery of penicillin by Alexander Fleming in the early nineteenth century, approximately 20 classes of antibiotic have been discovered in time. However, only antibiotic classes that are effective against *E. coli* would be thoroughly discussed in this subsection.

β‐Lactam antibiotics are one of the most common yet diverse classes of antibiotics and are the first class of antibiotics discovered in the 1930s. They were effective against both Gram-positive and Gram-negative bacteria and were categorized into four main groups, carbapenems, cephalosporins, monobactams, and penicillins, with each group sharing structural similarity in the β‐lactam ring within the antibiotic molecule [4]. The β‐lactam antibiotics mainly target the bacterial cell wall synthesis pathway, and are thus termed "broad spectrum antibiotics." Under normal physiological conditions, bacteria constantly renew their cell wall in order to replace broken ones. A unit of peptidoglycan cell wall consists of two subunits, the alternating *N*-acetylglucosamine and *N*-acetylmuramic acid. Each subunit contains an identical pentapeptide chain, which links both subunits together via the action of a transpeptidase, penicillin‐binding protein (PBP) [5, 6]. β‐Lactam antibiotics would act as an irreversible inhibitor toward PBP. The β‐lactam ring of the antibiotic mimics the structure of the pentapeptide chain and thus is able to bind with PBP, acylating

mechanisms involved.

*2.1.1. β‐lactam antibiotics*

**2. Antibiotic classes and multidrug‐resistant** *E. coli*

**2.1. Antibiotic classes and their respective natures**

Fluoroquinolones is another class of antibiotics that exert their effect on both Gram‐positive and -negative bacteria. The main structural feature of this particular antibiotic class is the presence of the fluorine atom within these antibiotics. It exhibits a broad spectrum activity against a large panel of bacteria as this group of antibiotic inhibits DNA synthesis by locking both the DNA gyrase and topoisomerase IV with the DNA strand during DNA replication. This prevents the action of other enzymes such as the RNA polymerase and DNA helicase for normal DNA replication, which eventually leads to cell death [9, 10]. Commonly prescribed fluoroquinolones include ciprofloxacin, gemifloxacin, levofloxacin, and moxifloxacin, which had relatively low adverse effects.

## *2.1.3. Aminoglycosides*

Aminoglycoside is another major group of antibiotics showing enhanced potency toward Gram-negative bacteria. As the name suggests, this compound comprises of sugar units bounded to an amino group. Aminoglycosides exhibit high potency as well as a broad spectrum of action as it disrupts protein synthesis by binding only to the prokaryotic 30S ribosomal subunit, which then impairs the proofreading mechanism during protein translation [11, 12]. This disruption produces dysfunctional proteins, either due to misreading or premature termination, and eventually causes cell death. Even though aminoglycosides are specific toward prokaryotic ribosome, toxicity had been observed and reported in mammalian cells when a high dosage was applied [13]. Hence, aminoglycosides are only prescribed during life-threatening infections. Commonly prescribed aminoglycosides includes amikacin, gentamicin, and streptomycin.

### *2.1.4. Nitrofurans*

Nitrofurans are a highly potent antibiotic class, which contain a furan ring and a nitro group. They are only used against urinary tract infections, especially when the infection is caused by an antibiotic-resistant pathogen. This is due to the high metabolism rate of the liver in partially breaking down the ingested nitrofuran. The remaining nitrofuran is then concentrated in the urinary bladder and thus suitable to be used in urinary tract infection, enabling targeted delivery [14]. High potency of nitrofuran is contributed by its diverse mode of actions when used against bacteria. In the presence of bacterial nitroreductases, nitrofuran is converted into reactive intermediates such as peroxynitrite and nitric oxide, which attack the bacterial ribosome, thus halting the protein synthesis in bacteria [15]. It was also reported that these reactive intermediates of nitrofuran can attack bacterial DNA as well as acting as a quorum sensing inhibitor [16, 17]. Due to the attribute of their multiple‐action mode, resistance toward nitrofurans has yet to be observed in pathogens. Nonetheless, the exact mechanism of nitrofuran has yet to be fully understood. Nitrofurantoin is the common form of nitrofuran, which is prescribed generally.

*E. coli*, which has the ability to cleave β‐lactam antibiotic, rendering it nonfunctional [29]. Antibiotic target modification had also been observed in MDR *E. coli*. Penicillin-binding protein (PBP), a transpeptidase, which links peptidoglycan subunit together, is the main target of the β‐lactam antibiotic. It has been observed that isolated PBP from MDR *E. coli* had conformational differences when compared to nonresistant strains of *E. coli*. This slight conformational change prevents effective binding of β‐lactam antibiotic but allows the trans-

Essential Oils: The Ultimate Solution to Antimicrobial Resistance in *Escherichia coli*?

http://dx.doi.org/10.5772/67776

303

**3. Synergistic potential of essential oils and antibiotics: challenges**

The emergence of multidrug-resistant pathogens, especially *E. coli*, have caused an interest shift from the onerous development of novel classes of antibiotics to the more straightforward application of synergism or combinatory therapy in the hope of reviving the efficacy and effectiveness of existing antibiotics. Quite a number of publications regarding the usage of essential oils and antibiotics as a combinatory therapy have indicated great success, with significant reductions in the dosage of antibiotics required to completely annihilate multidrug‐resistant pathogens [31–36]. Despite this, the usage of essential oils as a component for combinatory treatment posed a few challenges in its application. For instance, solubility of the hydrophobic essential oil in the aqueous medium is one of the greatest challenges faced. To solve this problem, emulsifiers such as dimethyl sulfoxide (DMSO) and polysorbate 80 (Tween 80) had been used to increase the solubility of essential oils in the aqueous medium. This would ensure maximum contact between the test organism as well as the test compound used throughout the experiment [37]. The concentration of such emulsifiers should also be taken into consideration as high concentration would cause toxicity to the test organism, resulting in false positivity during testing. For example, usage of DMSO at a concentration of more than 4% would reduce the viability of *Salmonella paratyphi* A, *Staphylococcus epidermis*, *Shigella flexneri*, *Vibrio cholerae,* and *Pseudomonas oleo‐ vorans* to less than 50% [38]. To better address the solubility issue, there is need to standardize the method used to determine synergism. The broth microdilution method has been shown to be the most accurate when compared to other susceptibility tests such as the disk diffusion and agar dilution methods, which are less informative [39]. In order to further maximize solubility, the incubation parameter should be standardized to shake at 200 rpm to ensure the formation of consistent emulsion, a crucial attribute in indicating the solubil-

Another challenge faced when using essential oils in combinatorial therapy would be the volatility of essential oils. It has been well documented that essential oils consist of 20–60 compounds, which are highly volatile, but none of which are actually lipid in nature [40]. Thus, with the solubility problem solved, volatility of essential oils is the next problem to tackle in order to achieve accurate determination of synergism in combinatorial therapy. Volatility of essential oils can be affected by several factors. For instance, exposure to light can accelerate the degradation as well as volatility of essential oils. It has been demonstrated that in the presence of light, the autoxidation process of essential oil was accelerated, leading to the loss

peptidase to carry out its normal physiological function [30].

ity of essential oils.

### *2.1.5. Polymyxin*

Polymyxin is a lipopeptide antibiotic that had been sidelined previously due to its high toxicity against mammalian cells. However, the emergence of multidrug-resistant pathogens has caused a resurgence in the use of polymyxin in treatments for bacterial infections as last resort. Polymyxin consists of a cyclic peptide bounded to a long hydrophobic fatty acid tail and it targets mainly Gram‐negative bacteria [18, 19]. Potency is only targeted toward Gram‐negative bacteria due to their mode of action, whereby the fatty acid tail of the antibiotic specifically targets and binds to the lipid moiety of a lipopolysaccharide, Lipid A that can only be found in Gram‐negative bacteria. This results in the insertion the cyclic peptide of the antibiotic into the cell membrane, thus compromising its integrity and increasing the permeability of the cell membrane. This eventually causes cytoplasmic leakage and leads to cell death [20–22]. Commonly prescribed polymyxin includes colistin and Polymyxin B.

### **2.2. Antibiotic resistance mechanisms in MDR** *E. coli*

The introduction of antibiotics as therapeutic agents to treat bacterial infection or as a growth promoter in molecular engineering had adversely propelled bacterial evolution, forcing bacteria to develop resistance mechanisms in order to survive within an antibiotic‐filled environment. This gave rise to multidrug-resistant (MDR) pathogens, especially *E. coli* as they are commensal microorganisms and often used as the model bacteria in research. The emergence of MDR *E. coli* has posed a great threat toward the survivability of mankind, thus, the indepth understanding of the strategies used by MDR *E. coli* to bypass antibiotic treatment is necessary to address this issue.

MDR *E. coli* exhibits the ability to resist multiple antibiotics simultaneously due to the acquisition of several genes that confer abilities such as antibiotic inactivation, multidrug efflux pump, target modification, or overproduction and reduction of cell membrane permeability. The multidrug efflux pumps are energy‐dependent and have been reported to be overexpressed in the presence of antibiotics, helping it to expel antibiotics that had successfully permeated into the cell [23]. The multidrug efflux pumps indicated low specificity enabling the removal of antibiotics beyond the same class, rendering the antibiotics ineffective. For instance, efflux pump AcrAB‐TolC of RND family is able to expel β‐lactam antibiotic, fluoroquinolones, tetracycline, and glycylcycline [23–26]. Furthermore, MDR *E. coli* can alter their outer membrane permeability by modifying the structure of porins and/or reduce or stop their expression, which would be ultimately responsible for antibiotic access into the cell [27]. It has been reported that porins observed in MDR *E. coli* had narrower channels when compared to normal strains, which prevents the antibiotics from entering the cell [28]. MDR *E. coli* had been reported to be able to deactivate antibiotic with the production of antibiotic‐targeting enzymes. β‐Lactamase is one example of enzymes produced by MDR *E. coli*, which has the ability to cleave β‐lactam antibiotic, rendering it nonfunctional [29]. Antibiotic target modification had also been observed in MDR *E. coli*. Penicillin-binding protein (PBP), a transpeptidase, which links peptidoglycan subunit together, is the main target of the β‐lactam antibiotic. It has been observed that isolated PBP from MDR *E. coli* had conformational differences when compared to nonresistant strains of *E. coli*. This slight conformational change prevents effective binding of β‐lactam antibiotic but allows the transpeptidase to carry out its normal physiological function [30].
