Review on Antibiotic Resistance: Resistance Mechanisms, Methods of Detection and Its Controlling Strategies

Antibiotic resistance (AR) is among the leading global health concerns to date. The misuse of antibiotics in the medical, veterinary and agricultural sectors, which include the inappropriate prescribing of antibiotics, their overuse in the livestock sector, and insufficient hygiene practices in hospital, all contribute to the rise of AMR. There are many mechanisms that bacteria exhibit to protect themselves from antibiotics and understanding the mechanisms by which bacteria resist antibiotics will become critical to solving the crisis. Laboratory based antibiotic resistance detection methods can determine resistance or susceptibility of an isolate against any therapeutic candidates. Appropriate use of antibiotics, vaccination, education, research, development of novel antibiotics, policy, regulations, surveillance of antimicrobial resistance and antibiotic use have a great role in minimization of antibiotic resistance. Abbreviations:


Introduction
Antibiotics are used for treatment and prevention of bacterial infection. Antibiotics played a significant role in increasing life expectancy witnessed in the second half of the 20th century.
Antibiotics transformed modern agriculture and livestock industries, the latter of which used antibiotics for prophylaxis, metaprophylaxis, treatment for infection, and as a growth promoter to enhance feed efficiency in healthy livestock [1]. Antibiotics are either cytotoxic or cytostatic to the micro-organisms, allowing the body's natural defenses, such as the immune system, to eliminate them. They are low molecular weight compounds and most of our natural products made by microorganisms or derived from natural products, which are active at low concentrations against other microorganisms but some antibiotics such as sulfa drugs and oxazolidinones do not originate from natural products [2]. Often, they act by inhibiting the synthesis of a bacterial cell, synthesis of proteins, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), by a membrane disorganizing agent, or other specific actions [3].
Antibiotic resistance is among the leading global health concerns to date. Due to an overuse of antibiotics in medicine and agriculture, antibiotic resistance mechanisms commonly emerge and threaten modern medicine by diminishing the utility of clinically relevant antibiotics. Resistance to antimicrobial agents (AMR) has resulted in morbidity and mortality from treatment failures and increased health care costs [4].
Historically, veterinarians selected antimicrobials to treat bacterial infectious diseases based primarily on past clinical experiences. However, with the increase in bacterial resistance to clinicians to empirically select an appropriate antimicrobial agent [5]. Increasing global antimicrobial resistance (AMR) is a major threat to human and animal endangering decades of improvements in health-care outcomes. It endangers modern human and veterinary medicine and undermines food safety [6]. Conventional methods for phenotypic antibiotic susceptibility tests remain essential in routine diagnostics as they directly test for bacterial growth on solid or liquid media in the presence of antibiotics. Solid media based antibiotic susceptibility tests, such as disk diffusion assay or E-test, require at least 18-22 h for bacteria to grow visibly on agar plates to be able to evaluate growth inhibition with naked eye [7]. Molecular analysis can used to investigate the presence of an antimicrobial resistance gene [8]. Appropriate use of antibiotics, vaccination, education, research, development of novel antibiotics, policy, regulations, surveillance of antimicrobial resistance and antibiotic use have a great role in minimization of antibiotic resistance [9]. Therefore, the objective of the review is to review resistance mechanisms, methods of detection and its controlling strategies on antibiotic resistance.

Antimicrobial Resistance
Antimicrobial resistance (AMR) is a complex issue of global concern. The misuse of antibiotics in the medical, veterinary and agricultural sectors, which include the inappropriate prescribing of antibiotics, their overuse in the livestock sector, and insufficient hygiene practices in hospital, all contribute to the rise of AMR. Global trade and travel are also accelerating the spread. At the same time, the development pipeline of new antibiotics has slowed, mainly due to insufficient incentives, allowing microorganisms to outpace the development of new drugs. The discovery of antibiotics meant that previously lethal infections could be cured, and surgical procedures made safer, allowing for the development of modern medicine [10].
Micro-organisms are termed 'antimicrobial-resistant' or 'drugresistant' when they are no longer inhibited by an antimicrobial to which they were previously sensitive. Such resistance is called 'acquired resistance' and is encoded by resistance genes in the DNA of the microbe. Resistance genes can arise through spontaneous mutations in the microbial DNA, but some have evolved over many years due to natural selection by natural antimicrobials in the environment. These genes can antimicrobial resistance also transfer from drug-resistant microbes to drug sensitive ones. The first drug-resistant bacterium in a clinical setting was identified in the late 1940s; only 4 years after mass treatment with penicillin had been introduced. Since then the emergence and spread of drugresistant microbes has continued to grow. Highly resistant bacteria, such as methicillin resistant Staphylococcus aurous (MRSA) and vancomycin-resistant enterococci, account for a high percentage of hospital-acquired infections [11].

Bacterial Resistance Mechanisms
Bacteria have a remarkable genetic plasticity that allows them to respond to a wide array of environmental threats, including the presence of antibiotic molecules that may jeopardize their existence. Bacteria's are sharing the same ecological niche with antimicrobial-producing organisms have evolved ancient mechanisms to withstand the effect of the harmful antibiotic molecule and, consequently, their intrinsic resistance permits them to thrive in its presence [12]. There are many mechanisms that bacteria exhibit to protect themselves from antibiotics and understanding the mechanisms by which bacteria resist antibiotics will become critical to solving the crisis. Misuse of antibiotics may contribute to the development of resistant bacteria; an incomplete course of antibiotics risks not entirely eradicating the colony thus allowing the development of resistant bacteria. Mechanisms of drug resistance fall into several broad categories, including active efflux pumps, drug inactivation/alteration, modification of drug binding sites/targets, changes in cell permeability resulting in reduced intracellular drug accumulation, biofilm formation and others [13,14].

Efflux Pumps
Figure 1: Bacterial efflux system A, system for antibiotic pumping out of the cell; B, antibiotic interfering with ribosomes in protein biosynthesis [17].
Efflux pumps are transporter proteins involved in the removal of toxic substances from the interior of the cell to the external environment. Efflux pumps in bacteria are major contributors to drug resistance; they extrude a broad spectrum of antibiotics to the exterior of the organism ( Figure 1) [17]. Hence, infections caused by these pathogens can be difficult to treat [15]. intrinsic or acquired. The intrinsic efflux mechanism of resistance is chromosomally encoded and is activated by environmental signals or by mutation in regulatory genes [16].

Antibiotic Inactivation
Bacteria uses several mechanisms of rendering antimicrobials inactive such as the enzymatic hydrolysis of antibiotics, group transfer and redox process. The production of β-lactamases that hydrolyze the β-lactam ring of penicillins is the classical example of antibiotic inactivation. The enzymes can often be excreted by the bacteria, inactivating antibiotics before they reach their target within the bacteria. The second mechanism of antibiotic inactivation involves enzyme mediated structural alteration of the drug via transfer of a functional group such as an acyl, ribosyl, phosphoryl or thiol group. The modified antibiotic is unable to bind to the target due to the resultant change in the structure and the reaction is irreversible [18]. The third mechanism of antibiotic inactivation is by redox reaction [19].

Target Modification
Modification of the antibiotic target site makes the antibiotic unable to bind properly. Microorganisms cannot evade antimicrobial action by dispensing with them entirely because of the vital cellular functions of the target sites. In this mechanism, bacteria found ways to alter the targets of antimicrobial agents. The classical example of drug target modification is the staphylococcal mechanism of variously altering the Penicillin Binding Protein (PBP) which is the target of β-lactam antibiotics [20].

Reducing Entry of Antimicrobial Agents
Mostly antimicrobial compounds always require access into the bacterial cell to reach their target site. Porin channels are the passageways by which antibiotics normally cross the bacterial outer membrane. Some bacteria protect themselves by prohibiting these antimicrobial compounds from entering to their cell walls [21].

Mutation
Mutation is a spontaneous change in the DNA sequence within the gene that may lead to a change in the trait which it codes for ( Figure 2) [14]. A single base pair change may lead to a corresponding change in one or more of the amino acids for which its codes, which can then change the enzyme or cell structure that consequently changes the affinity or effective activity of the targeted antimicrobials. In prokaryotic genomes, mutations frequently occur due to base changes caused by exogenous agents, DNA polymerase errors, deletions, insertions and duplications [22]. c) The antibiotic is able to bind to and destroy the target wild-type bacteria.

d)
After mutation occurs, the binding site is altered, and the antibiotic is unable to bind to the mutant bacteria and is unable to kill it. These bacteria will proliferate creating a new resistant colony [14].

Biofilm Formation
Complex microbial communities containing bacteria and fungi are called biofilms. Microorganisms synthesize and secrete a protective matrix that attaches the biofilm firmly to a living or non-living surface. A biofilm can be described as bacteria embedded in a thick, slimy barrier of sugars and proteins. The biofilm barrier protects the microorganisms from external threats. The high cell density in biofilms increases the absolute numbers of resistant mutants that can be selectable under antimicrobial pressure [23].

Detecting Methods of Antimicrobial Resistance
Antimicrobial susceptibility testing methods are in vitro procedures used to detect antimicrobial resistance in individual bacterial isolates. Those laboratory-based detection methods can determine resistance or susceptibility of an isolate against any therapeutic candidates. Those methods can also be used for monitoring the emergence and spread of resistant microorganisms in the population [24]. Guidelines and recommendations for these are continuously updated by certain organizations worldwide, those which specify antimicrobial testing methods and interpretative criteria for veterinary pathogens are: the CLSI in the USA, OIE in EU and CDS-AST in Australia [25]. Among the available tests, agar disk-diffusion and the broth micro dilution methods are the two most commonly used methods in veterinary laboratories. Some examples of antibiotic sensitivity testing methods are dilution method (broth and agar dilution method), disk-diffusion method, E-test, automated methods and genotypic methods [26].

Disk-diffusion method
The application of commercially available drug-impregnated filter paper disks to the surface of an agar plate that has been inoculated to confluence with the organism of interest is called disk diffusion [27]. Disk diffusion is also known as Kirby-Bauer  Test tubes or wells containing increasing concentrations of each antibiotic to be tested, from 0.0312 to 512 µg/ml, are inoculated with a fixed volume of nutrient broth containing a standard concentration of bacteria. The concentration of the antibiotic in each tube is double that in the previous tube [30]. In the broth dilution assay, an antimicrobial is added to a culture tube of nonselective broth medium at different concentrations [31]. Tubes are incubated under optimum conditions for the test microorganism from 16 to 24 hours. Antimicrobial effect could be determined by spectro-photometry or by plating counting [32].
The Agar Dilution Method: Agar dilutions are most often prepared in petri dishes and have advantage that it is possible to test several organisms on each plate [33]. In the agar dilution method, the antimicrobial agent is incorporated into the agar medium with each plate containing a different concentration of the agent. The inoculum can be applied rapidly and simultaneously to the agar surfaces using an inoculum replicating apparatus. Mueller-Hinton agar is prepared from a dehydrated base. The advantages of agar dilution testing include the reproducible results and satisfactory growth of most nonfastidious organisms. Agar dilution testing generally is not performed in routine clinical laboratories but can be ideal for regional reference laboratories or research laboratories that must test large numbers of isolates [34].

Epsilometer Test (E-Test)
Epsilometer test (E-test) is an 'exponential gradient' method of determination of antimicrobial resistance. The E-test is developed to provide a direct quantification of antimicrobial susceptibility of microorganisms. This is a quantitative method that applies both the dilution of antibiotic and diffusion of antibiotic into the medium. The device consists of a predefined, continuous, and exponential gradient of antibiotic concentrations immobilized along a rectangular plastic test strip [35]. The principle of E test The cost the E test is little more when compared to disk diffusion method. However, the E test is simple, accurate, and reliable and is also used to determine the Minimum Inhibitory Concentration (MIC) of antifungal agents and antimycobacterial agents [33].

Automated Instrument Methods
There are a variety of commercially available automated systems available to help reduce the technical time required to perform and record routine sensitivity tests. For example, the results of disk sensitivity tests and breakpoint sensitivity tests can be read using a camera interfaced to a computer system. Other Systems utilize liquid cultures and detect the effect of antibiotics on the rate of bacterial growth through measurement of turbidity (nephelometry) or the production of CO 2 . These automated systems can significantly shorten the necessary incubation time [36]. There are different types of automated instruments (Micro Scan Walk away, BD phoenix, Trek Sensititere and Vitek 1 and Vitek 2). The advantage of using this test is increased reproducibility, decreased labor costs and issued rapid results but the disadvantage is they are not available widely in developing country including Ethiopia [37].

Molecular Methods for Detection of Antimicrobial Resistance
Molecular characterization of the genetic mechanism(s) underlying a given phenotypic result, obtained by traditional antimicrobial sensitivity testing, is now an integral part of many clinical investigations in relation to bacterial infections, whether in humans or animals. In some cases, when phenotypic results are too time-consuming, non-conclusive, or unavailable, molecular analysis can be used to investigate the presence of a given gene.

Strategies to Minimize Antibiotic Resistance
Antibiotic-resistant bacteria are spread globally in the same way as other bacteria. This means they can be transferred between people, animals and foodstuffs, and they can spread in our environment. These links between the various sectors mean that efforts to combat antibiotic resistance must be made from a broad perspective. Prudent use of antibiotics in healthcare, animal health and agricultural settings is essential to slow the emergence of resistance and extend the useful lifetime of effective antibiotics.

Conclusion
Antibiotic resistance is rising to dangerously high levels in all parts of the world. New resistance mechanisms are emerging and spreading globally, threatening our ability to treat common infectious diseases. Infections caused by antibiotic-resistant germs are difficult, and sometimes impossible, to treat. Therefore, prudent use of antibiotics in healthcare, animal health and agricultural settings is essential to slow the emergence of resistance and extend the useful lifetime of effective antibiotics.