ANTIBIOTICS & RESISTANCE

OVERVIEW OF ANTIBIOTIC RESISTANCE

Antibiotic resistance is a phenomenon that occurs when bacteria are not killed or inhibited by usually achievable systemic concentration of an antibiotic which is supposed to prevent their harmful effect to a host. It is a global health problem that has bedeviled our health sector worldwide. There emergence and increase is gradually eroding the efficacy of drugs used in clinical medicine for the treatment of infectious diseases. Prior exposure to antibiotics places a kind of selective pressure on bacteria, thus making them develop resistance to a particular drug over time. Microbes or bacteria can also develop resistance following changes (mutation) in their DNA. Antibiotic resistance is of many types including extended spectrum beta lactamase. Extended spectrum beta lactamases (ESBLs) are a group of β – lactamase enzymes that break down and cause resistance to beta-lactam antibiotics including penicillins and cephalosporins, thus rendering them ineffective for treatment in vivo.

They are Multidrug resistant organisms, extending their resistance to both β – lactams and non- ­beta lactams like aminoglycosides and quinolones. ESBLs are plasmid-mediated and can be exchanged between bacteria via genetic transfer. They carry tremendous clinical implications since most of the drugs used in therapy are rendered ineffective by them. It is therefore pertinent that the clinical microbiology laboratories all over the world detect ESBL production in clinical isolates following the CLSI standards of detecting them. This will help to guide therapy, prevent the morbidity and mortality caused by antibiotic resistant bacteria – thereby putting us on the road map to curbing and containing antibiotic resistance in our world of today.

In recent times, antibiotic resistance of pathogens to drugs (antibiotics) directed towards the degrading properties of microbes in vivo has been on the increase both in the community and in the hospital. Antibiotics are exceptionally vital in clinical medicine for the treatment of bacterial related infections, but unfortunately bacteria are capable of developing resistance to them. Antibiotic resistance is a global health problem that has bedeviled our health sector worldwide, affecting both the developed and developing countries of the world. They make infectious diseases very difficult to treat. The emergence of antibiotic resistance is a complex problem that is driven by many interconnected factors, of which the use and misuse of antimicrobial agents (antibiotics, antiseptics, disinfectants, and preservatives) amongst other factors, is the main driving force for the development of resistance.

Antibiotic resistance occurs when bacteria change in some way that reduces or eliminates the effectiveness of drugs, chemicals or agents designed to cure or prevent the infection. Thus, the bacteria survive and continue to multiply causing harm and havoc in the patient (host) taking the drug. There has been a very great concern that the “antibiotic era” might be coming to an end – firstly, because the rate of production of new drugs has diminished greatly and, secondly, because microbes (viruses, bacteria, fungi, protozoa) are showing great inventiveness in devising mechanisms for circumventing the inhibiting and killing properties of drugs (antibiotics) directed towards them. Deaths from acute respiratory infections, diarrheal diseases, measles, AIDs, malaria and tuberculosis account for more than 85% of the mortality from infection worldwide.

Resistance of microbes to first-line drugs causing these diseases according to the WHO ranges from zero to almost 100 % and in some cases, resistance to both second – and third – line drugs is seriously compromising treatment outcome. A major example is extended spectrum β – lactamase (ESBL) – producing bacteria which is resistant to virtually all beta – lactam drugs and some non – beta lactam drugs. Antibiotic resistance though a natural biological phenomenon, has in no doubt lead to the loss in the efficacy of some important drugs (especially the beta-lactams) from our therapeutic armamentarium.  Nobody is to be blamed for this plethora of menace that is gradually eroding the efficacy of our drugs, since the introduction of every antimicrobial agent into clinical practice at one time or the other has been followed by the detection in the laboratory of strains of microorganisms that are resistant to these antimicrobial agents.

In containing antibiotic resistance in both our community and the hospital, a good and adequate routine diagnostic antimicrobial susceptibility testing in the microbiology laboratory is paramount. They should be charged with the responsibility of detecting antibiotic resistant strains of microbes (especially ESBL-producing bacteria) in the hospitals and in the community using internationally recognized protocol as outlined by the Clinical Laboratory Standard Institute, CLSI (formerly National Committee for Clinical Laboratory Standards, NCCLS) guideline. Data emanating from such studies should be made available and harnessed properly by all stakeholders in order to develop a road map for the proper control and eradication of antibiotic resistance from our world. Therefore, it is ripe for us to close the door on antibiotic resistant strains of bacteria before we wake up someday and find out that the only weapon (antibiotics) we have against bacterial related diseases have left us.

DEFINITION OF AN ANTIBIOTIC

There is no consensus to the definition of antibiotics. But it is very important that we do not displace the key points (“Killing and Inhibition”) that must be contained in any definition of an antibiotic. Abinitio, an antibiotic was originally defined as a substance produced by one microorganism, which inhibits the growth of other microorganisms. But due to the development of other synthetic or chemical methods by which drugs can be produced, there has been a modification to this definition.

Today, an “antibiotic” can be defined as a substance produced by a microorganism (wholly or partly by chemical synthesis), which in low concentrations kill or inhibit the growth of other microorganisms in vivo. The discovery of antibiotics and their subsequent application to clinical medicine is one of the outstanding scientific achievements of the twentieth century. Antibiotics – “Magic Bullets” as fondly called by many have been used for about 6 decades now to treat and cure variable series of diseases including tuberculosis (TB), pneumonia, gonorrhea, urinary tract infections (UTI’s), respiratory infections, syphilis, et cetera and they have been found to be very efficacious in this aspect. Antibacterial drugs (antibiotics) has been the most effective of all medicines and their success is reflected by their continued use in clinical medicine and the decrease in morbidity and mortality from bacterial infections over the past 50 years.

Today, it is very difficult to imagine a society without antibiotic despite the emergence of antibiotic resistant strains of bacteria that is gradually eroding the efficacy of our therapeutic regimens. Antibiotics cure disease by killing or injuring bacteria. Some antibiotics are ‘Bactericidal’, meaning that they work by killing bacteria. Other antibiotics are ‘Bacteriostatic’, meaning that they work by stopping bacteria multiplication. Each different type of antibiotic affects different bacteria in many different ways, thus stopping their deleterious effect to the host. Some antibiotics can be used to treat a wide range of infections and are known as ‘broad-spectrum’ antibiotics. Others are only effective against a few types of bacteria and are called ‘narrow-spectrum’ antibiotics.

HISTORY OF ANTIBIOTICS

Antibiotic history dates back to 1928 when Sir Alexander Fleming discovered the antibacterial effects of the yeast, Penicillium notatum, a Fungus in his laboratory. The “antibiotic era” was ushered in through the work of several pioneering scientists including Sir Alexander Fleming, Howard Florey, Ernest chain, Selman Waksman, Albert Schatz, Paul Ehrlich to mention but a few. Sir Alexander Fleming astutely recognized that a contaminated Petri dish of Staphylococcus aureus actually contained a bacterium – killing mould, which was later deciphered to be P. notatum. His work together with that of Ernest Chain and Howard Florey in 1939 lead to the isolation, purification, and commercial production of penicillin (a beta – lactam drug widely used in clinical medicine today) in 1940.

Penicillin, a beta – lactam antibiotic is a cell wall inhibitor. Unlike other antibiotics, the penicillin does specifically affect the synthesis of peptidoglycan – which is very crucial in the synthesis of cell wall in bacterial cell. This jeopardizes the integrity and structure of bacterial cells, thereby exposing them to external pressure or harmful chemicals that finally destroy the bacterial cell. Just a few years later after the discovery of the antibacterial properties of the penicillins, the antibacterial properties of “Sulphonamides” were also noted by Paul Ehrlich in 1932. Since then, a plethora of other antibiotics were also discovered and produced synthetically and semi – synthetically, thus giving hope to clinical medicine in the treatment of bacterial related infections.

Antibiotics target different features of bacterial physiology, thus expanding the range of bacterial species that can be successfully treated with them. In the early 1940’s, the industrialization of penicillin production was quickly followed by the successful isolation and development of a large number of antibiotics that has led to most of the major classes of antibiotics in use even today, namely: Tetracycline, Chloramphenicol, Glycopeptides, Aminoglycosides, Cephalosporins, and Rifamycins. It is no doubt that the discovery and development of antibiotics and their introduction into clinical medicine, together with the introduction of such infection control measures like vaccination, use of clean water, and personal hygiene brought infectious diseases caused by bacterial species under control.

SOURCES OF ANTIBIOTICS

Before the advent of conventional medicine used in clinical medicine today for the treatment of infectious diseases, people in ancient times and even now have been using some herbal plants traditionally for centuries to combat microbes that cause diseases in man. Recently, scientists have studied and reported results that support the use of these herbal plants (Carson et al., 2006). Herbal antibiotics have been found to be milder than pharmaceutical antibiotics, and according to the Center for Disease Control and Prevention (CDC), the overuse of pharmaceutical antibiotics cause pathogens to mutate and grow stronger, giving them the chance to mount resistance to antibiotics. Some of the herbal plants used for the treatment of infectious diseases include: The Neem Tree/plant (Azadirachta indica), Ginger, Garlic, Tea Tree Plant et cetera. Antibiotics are obtained or sourced from one of the following means:

  1. MICROORGANISMS: Microorganisms are the primary source of antibiotics. Though not all antibiotics used today in clinical medicine are produced completely (wholly) from microorganisms, microorganisms still provide the parent root from which antibiotics are developed. Bacitracin and polymyxins are obtained from Bacillus species; streptomycin and tetracycline are obtained from Streptomyces species; gentamicin from Micromonospora purpurea; penicillins and cephalosporins are obtained from Penicillium and Cephalosporium species respectively.
  2. SYNTHESIS: Synthetic antibiotics are antibiotics produced from natural drugs by making a prototype (model) of the natural drug in the laboratory through chemical reactions without having to do anything with the original source of the drug. Example of a synthetic antibiotic is chloramphenicol.
  3. SEMI – SYNTHESIS: Semi – synthetic antibiotics are natural antibiotics that are modified by the removal or addition of a particular chemical group in order to increase the therapeutic effect of the drug. Here, compounds isolated from natural sources (e.g. plants or microorganisms) are used as starting materials and a fermentation process is involved in the production of such antibiotics. After which, the antibiotic is further modified by a chemical process in the laboratory. Examples of drugs produced this way include: penicillins, cephalosporins, and the antimalarial drug artemether.

TYPES OF ANTIBIOTICS

There are several classification/types of antibiotics today, which is based on bacterial spectrum of activity (whether broad or narrow) or type of activity exhibited by the agent (whether bactericidal or bacteriostatic). Some antibiotics are also classified based on their chemical structure. And this leaves antibiotics within a particular structural class to have similar patterns of effectiveness, toxicity, and allergic potential. The types of antibiotics expanded here are not exhaustive of the different classes or types of antibiotics and they include:

BETA – LACTAM ANTIBIOTICS: The beta – lactam antibiotics are a broad class of antibiotics that consist of all antibiotic agents that contains a beta – lactam ring/nucleus in its molecular structure (Figure 1). They are the oldest class of antibiotics especially the penicillins and they are produced from Penicillium and Cephalosporium bacteria. Examples of antibiotics in this class include: penicillins, cephalosporins, monobactams and carbapenems. The beta – lactam antibiotics work by inhibiting the synthesis of cell wall in bacteria. They are the most widely used group of antibiotics in clinical medicine, and they are active on both Gram positive and Gram negative bacteria. Beta – lactam antibiotics have no antibacterial activity on bacterial cells that lack cell wall e.g. Mycoplasmas. They are only effective on bacterial species that have cell wall.

Figure 1: General Structure of Penicillins (Beta – Lactam Antibiotics)

MACROLIDES: The macrolides are a group of antibiotics that are characterized by possessing molecular structures that contain large (12-16 membered) lactone rings linked through glycosidic bonds with amino sugars (Figure 2). They are derived from Streptomyces bacteria, and they are bacteriostatic, binding with bacterial ribosomes to inhibit protein synthesis. Macrolides are active against most Gram positive bacteria but not against the Enterobacteriaceae. Examples of antibiotics in this category include: erythromycin, azithromycin, and clarithromycin.

Figure 2: Structure of Erythromycin, a Macrolide

FLUOROQUINOLONES: The fluoroquinolones (fluorinated – quinolones) are second – generation quinolones that are produced by the addition of a fluorine atom (molecule) on the carbon-6 (C-6) of quinolones (Figure 3).  They are synthetic antibiotics and are not sourced from microorganisms. Nalidixic acid is the first quinolone while ciprofloxacin, ofloxacin and norfloxacin are examples of fluoroquinolones. They are active on both Gram positive and Gram negative bacteria, and they are mostly used in the treatment of urinary tract infections (UTI’s). The fluoroquinolones target the DNA gyrase and topoisomerase IV enzymes of bacterial cell, leading to the inhibition of DNA synthesis or replication in them. Thus, the fluoroquinolones inhibit bacteria by interfering with their ability to make DNA. This activity makes it difficult for bacteria to multiply and cause havoc in vivo. They are used to treat most UTI’s, skin infections, and respiratory infections because of their excellent absorption in vivo.

Figure 3: General Structure of Quinolones, progenitor of Fluoroquinolones

TETRACYCLINES: The tetracyclines are a group of antibiotics that is characterized by a four cyclic ring (Figure 4). They are derived from a group of Streptomyces bacteria and they inhibit bacterial protein synthesis. Examples include oxytetracycline, doxycycline and chlorotetracycline. Tetracyclines have a wide range of activity on both Gram positive and Gram negative bacteria, thus they are broad spectrum bacteriostatic agent.

Figure 4: General Structure of Tetracyclines

AMINOGLYCOSIDES: Aminoglycoside antibiotics contain amino sugars in their structures and they possess a cyclohexane ring (Figure 5). They are derived from Streptomyces bacteria, and they are bactericidal in action, and they inhibit the synthesis of protein in bacterial cell. Examples of antibiotic in this category include gentamicin, kanamycin, tobramycin and streptomycin.

Figure 5: Structure of Streptomycin, an Aminoglycoside

MECHANISM OF ACTION OF ANTIBIOTICS

The antibiotics described above including those not described in this work are used to treat infections caused by disease causing microorganisms (bacteria). Majority of them exert a highly selective toxic action upon their target microbial cells but have little or no toxicity towards mammalian cells. These antibiotics can therefore be administered at concentrations sufficient enough to kill or inhibit the growth of infecting organisms without damaging mammalian cells. The ways by which these antibiotics exert their antibacterial activities on microbes in vivo without necessarily harming the host (patient) taking the drug is called the “Mechanism of Action of Antibiotics”. It reveals and explains the rationale behind the selective toxicity of antibiotics and how they stop the venomous effects of bacteria. Below are some of the major mechanisms of action of antibiotics:

  1. INHIBITION OF MICROBIAL CELL WALL SYNTHESIS: Peptidoglycan is a vital component of the cell wall of virtually all bacteria. But it is more pronounced in Gram positive bacteria than in Gram negative bacteria. The peptidoglycan is responsible for maintaining the shape and mechanical strength of the bacterial cell wall. If it is damaged in anyway, or its synthesis is inhibited (e.g. by antibiotics), then the shape of the bacterial cells becomes distorted and they will eventually burst (lyse) due to the high internal osmotic pressure following the influx of fluids or substances like drugs from the outside into the bacterial cell. Mammalian cell is devoid of peptidoglycan, thus antibiotics which inhibit microbial cell wall synthesis show outstanding selective toxicity. Examples of antibiotics that inhibit bacterial cell wall synthesis include: Penicillins, Bacitracin, Glycopeptides (e.g. Vancomycin), and Cephalosporins. These antibiotics stop the cross – linking of N-acetyl-glucosamine and N-acetyl-muramic acid (transpeptidation reaction) which is supposed to lead to the formation of peptidoglycan, an important component of bacterial cell wall.
  2. INHIBITION OF DNA SYNTHESIS FUNCTION: Deoxyribonucleic acid (DNA) is a vital component of the chromosome of bacterial cells as they are known to direct the activity of the cells. Antibiotics that inhibit the synthesis of DNA in bacterial cell work by interfering with the transcription stages which are very vital to the final and complete formation of bacterial DNA. These antibiotics block the DNA gyrase enzyme (Topoisomerase II) which is important to the complete synthesis of DNA in bacteria. Examples of antibiotics that inhibit bacterial DNA synthesis function include: Sulfonamides, Trimethoprim, Rifampicin, Quinolones, and Fluoroquinolones.
  3. DESTRUCTION OF MICROBIAL CELL MEMBRANE: The integrity of the cytoplasmic membrane in bacterial cell is very important for the normal functioning of all cells. Bacterial cell membranes do not contain sterols. This differentiates them from fungal and mammalian cells which do contain sterols, thus giving such antibiotics a highly selective toxicity on bacteria. Antibiotics that destroy the cytoplasmic membrane of bacterial cells cause irreversible leakage of cytoplasmic components by disturbing the integrity of the membrane. This can also impair other metabolic functions associated with the membrane. Examples of antibiotics that destroy microbial cell membrane include: Polymyxins.
  4. INHIBITION OF PROTEIN SYNTHESIS: Bacterial ribosomes are smaller than their mammalian counterparts. The ribosomes in bacterial cells are vital for the synthesis of proteins. Antibiotics that inhibit the synthesis of protein in bacterial cells act by binding to a receptor on either the 30S or 50S subunit ribosomes. This action prevents the complete reaction of translocation, thereby inhibiting the synthesis of protein in bacterial cells. Antibiotics that are protein synthesis inhibitors include: Tetracyclines, Chloramphenicol, Macrolides, and Streptomycin.

FEATURES OF ANTIBIOTICS

Antibiotics have some specific characteristics that distinguish them from other agents that are used for the treatment of microbial infections. Some of these features are as stated below:

  1. Selective Toxicity: Antibiotics must be selectively toxic as they dissipate their antibacterial properties in vivo. This means that antibiotics should kill or inhibit pathogens without causing any harm or damage to the host (patient) in anyway.
  2. Spectrum of Activity: Antibiotics should have a reasonable spectrum of activity (broad or narrow), showing efficacy over a given or wide variety of bacteria.
  3. Antibiotics should not eliminate the normal microbial flora of the body as they exert their antibacterial properties in vivo.
  4. They should be specific in their action. Thus, antibiotics should be readily directed to the site of infection where their effect is highly needed in the body.
  5. Antibiotics should not be too costly so that their prescription should not be biased or based on their price or cost.
  6. Antibiotics should not be easily neutralized or excreted from the body until it has performed its function. Thus, they should be chemically stable in vivo.
  7. Microorganisms should not become easily resistant to them.

ANTIBIOTIC RESISTANCE

Antibiotic resistance is a phenomenon that occurs when bacteria are not killed or inhibited by usually achievable systemic concentration of an antibiotic (drug) with normal dosage schedule and/or fall in the minimum inhibitory concentration ranges of the drug in question. It occurs when bacteria change in some way that reduces or eliminates the effectiveness of drugs or other agents designed to cure or prevent the infection. Thus the bacteria survive and continue to multiply causing more harm in the host taking the drug. Antibiotic resistance is a natural phenomenon and it is exacerbated following the prior administration of an antibiotic.

According to the World Health Organization (WHO) the introduction of every antimicrobial agent into clinical practice has been followed by the detection in the laboratory of strains of microorganisms that are resistant, i.e. able to multiply in the presence of drug concentrations higher than the concentrations in humans receiving therapeutic doses. Microorganisms harbor resistance genes which encode various mechanisms that allow them to resist the killing or inhibitory effects of specific antibiotics directed towards them. These mechanisms also offer resistance to other antibiotics or antimicrobial agents of the same class and sometimes to several different antimicrobial classes.

The problem of antibiotic resistance is a vital topic and a global health problem that has received increasing attention over the last two decades. It is certainly not a new topic neither was it unpredictable. When antibiotic resistance occurs, it is the microbe (bacterium) that is resistant, not the antibiotic nor the patient or host taking it. Species of bacteria that are normally resistant to penicillin for example, can develop resistance to these drugs either through mutation (vertical transmission) or through acquisition from other bacteria of resistance genes (horizontal transmission). This dual means of acquiring resistance explains why the resistance trait can spread rapidly and replace a previously drug – susceptible population of bacteria. Antibiotic resistance is on the rise threatening our ability to treat infectious diseases and even some of those that cause most deaths in the past (e.g. tuberculosis).

Diseases such as tuberculosis, which once thought to be under control, are becoming increasingly difficult to treat as medicines become less effective – steadily depleting the arsenal of drugs available for their treatment. Antibiotic resistant bacteria were first discovered soon after the medicinal use of penicillin began. The first signs of antibiotic resistance were actually observed in 1940, five years before penicillin became commercially available to the public. In that year, the first observed bacterial enzyme (beta-lactamase) that destroyed penicillin was described. This was the first observed evidence of bacterial resistance to an antibiotic action. Therefore, the history of antibiotic resistance coincided with the history of antibiotics themselves.

The number of antibiotics belonging to various families, their varied mode of action and the number of bacteria in which antibiotic resistance has been documented suggests that, in principle, any microbe could develop resistance to any antibiotic. Antibiotic resistance is one of the biggest challenges that bedevil our health sector worldwide. Resistance of microbes to antibiotics has been documented not only against antibiotics of natural and semi – synthetic origin, but also against purely synthetic compounds (such as the fluoroquinolones) or those which do not even enter the cells (such as vancomycin). And unfortunately, the discovery and development of newer antibiotics have not kept pace with the emergence and rate at which bacteria develops and mount resistance to antibiotics. Thus, the rate at which microbes are developing resistance to antibiotics is much faster than the rate at which the drugs are developed to curb the problem.

TYPES OF ANTIBIOTIC RESISTANCE

Bacteria have evolved to survive in diverse environments. They survive exposure to harsh chemicals including antibiotics, and they also survive difficult growth conditions. They have learned to “detoxify” harmful substances e.g. antibiotics. Antibiotic resistance can either be intrinsic or acquired.

INTRINSIC (INNATE) RESISTANCE: Some bacteria are said to possess innate/intrinsic resistance against antibacterial action put forward by antibiotics. They mount a great ingenuity in devising means or ways of neutralizing the killing or inhibiting action of antibiotics directed towards them. This innate form of antibiotic resistance in bacteria shows the different variations in the structure of the cell envelope of the organism, which allows them to mount resistance against drugs. It is a vertical means by which bacteria acquire resistance. Intrinsic or innate form of antibiotic resistance can occur by any one of the following route:

  • Spontaneous mutation in the chromosomal DNA of bacteria.
  • Accumulation of several point mutations in bacteria.
  • An evolutionary process occurring only under selective pressure e.g. prior exposure of bacteria to antibiotics.

ACQUIRED (PHENOTYPIC) RESISTANCE: This type of antibiotic resistance is acquired by bacteria from the environment or other microorganisms by one of the means of genetic transfer (conjugation, transformation, and transduction). In acquired/phenotypic resistance, the bacteria acquire reduced susceptibility to antibiotics through adaptation to growth within a specific environment. Acquired resistance is a horizontal means by which bacteria become resistant to antibacterial properties of antibiotics. This form of antibiotic resistance can be achieved in bacteria by one of the following route:

  • Resistance can be maintained on horizontal mobile elements like plasmids, integrons and transposons.
  • Resistant genes can be transferred among bacteria through means of genetic transfer.
  • Resistance genes can be integrated into the bacterial chromosome or can be maintained in an extra chromosomal state (e.g. plasmids).

MODE OF TRANSFER OF RESISTANCE

Antibiotic – resistant bacteria owe their drug insensitivity and ingenuity in developing resistance against our therapeutic regimens to resistance genes which they harbor or possess. It is these genes that resistant bacteria transfer to non – resistant susceptible strains, thus compounding the problem of antibiotic resistance. Below are some of the major ways through which bacteria pass on their antibiotic resistance genes to susceptible non – resistant bacteria:

1. CONJUGATION: Conjugation is the form of gene transfer and recombination in bacteria through which genetic materials are transferred from one bacterium to another through a direct cell – to – cell contact. It is the most important genetic transfer mechanism by which bacteria transfer their antibiotic resistance genes to susceptible bacteria. Conjugation is mediated by a particular kind of circular DNA called a plasmid, which replicates independently of the chromosome. Many plasmids carry genes that confer resistance to antibiotics. When two bacterial cells are in close proximity to each other, a hollow bridge like structure known as the “pilus” forms between the two cells. This allows a copy of the plasmid as it is duplicated (in the donor cell known as the F+ cell) to be transferred from the donor bacterium or F+ bacterium to the recipient bacterial cell known as the F- bacterium. After successful conjugation and transfer of genetic material, the F- bacteria becomes F+ bacteria. This process called conjugation enables a susceptible bacterium to acquire resistance genes to a particular antibiotic.

Illustration of conjugation.

2. TRANSDUCTION: Transduction is the transfer of genetic material between bacteria by bacteriophages (bacterial viruses). Here, antibiotic resistance genes are incorporated into a phage capsule which is later injected into another bacterium. In the process of transduction, bacterial DNA is transferred from one bacterium to another inside a virus that infects bacteria. These viruses are called bacteriophages or phage. When a phage infects a bacterium, it essentially takes over the genetic process of the bacteria to produce more phage. During this process, bacterial DNA may inadvertently be incorporated into the new phage DNA. Upon bacterial death and lyses or breaking apart, these new phage goes on to infect other bacteria. This brings along genes from previously infected bacterium into the recipient bacterium. These genes might contain advantageous genes such as antibiotic resistance genes, which will leave the recipient bacterium resistant to a particular antimicrobial agent (e.g. antibiotics).

Illustration of transduction, showing the lytic and lysogenic cycles of viruses or phages.

3. TRANSFORMATION: Transformation is a mode of genetic transfer in bacteria in which a piece of free DNA (genetic materials) is taken up by a bacterium and integrated into the recipient genome. During this process, genes are transferred from one bacterium to another as “naked” DNA. When bacterial cells die and break apart, DNA can be released into the surrounding environment. Other bacteria in close proximity can scavenge this free floating DNA and incorporate them into their own DNA. This incorporated DNA can contain advantageous genes such as antibiotic resistance genes and benefit recipient bacterial cells.

MECHANISMS OF ACQUIRING RESISTANCE

An antibiotic has to go through a number of steps in order to exert its antibacterial action in vivo. They have to come into contact with the host taking them before ever their antibacterial properties can be dissipated. First, the antibiotic has to enter the host cell, and once inside the cell, the antibiotic has to remain stable and accumulate to killing or inhibitory concentrations enough to deactivate invading microorganisms (or bacteria). In some cases, the antibiotic need to be activated to an active form and finally it has to locate and interact with its target (s) in order to exert its action. Any alteration in any one or more of these processes can render the cells of the bacteria resistant to the antibiotic directed against it.

This is what happens whenever a bacterium mounts resistance to antibiotics which was supposed to inactivate their degrading effects to the host, hence the need for the mechanism of acquiring resistance by bacteria. In addition to this, the increased dissemination and prevalence of resistance is an outcome of natural selection and should be viewed as an expected phenomenon of the “Darwinian” biological principle of “survival of the fittest”. In any large population of bacteria, a few cells will be present which posses traits that enable them to survive in the presence of harmful substances (e.g. antibiotics), in this case the ability of the bacteria to fade off or evade the action of the antibiotic directed against them.

Susceptible organisms (i.e. those lacking the advantageous traits like antibiotic resistance genes) will be eliminated, leaving behind the remaining resistant population of bacteria. With long time antibiotic in use in a given environment, the bacteria communities will change dramatically, with more resistant organisms increasing in proportion. This can result in a situation where the antibiotic is needed; it may not be effective to treat what was once and easily treatable infection. In this scenario, bacteria are now unaffected by the antibiotic because of selective pressure posed on them by initial, “unnecessary”, and long time antibiotic usage in a particular population. Below are some of the mechanisms employed by bacteria to mount resistance against antimicrobial agents (antibiotics):

1. Antibiotics resistance by influx-efflux systems: Certain bacteria can often become resistant to antimicrobial agents through a mechanism known as “efflux”. Efflux pumps are pumps found in bacteria cells, which help them to export antimicrobial agents (e.g. antibiotics) and other compounds out of the bacterial cell. The antibiotics enter the bacteria through chemical channels called “porins”, and then it is pumped out again by the efflux pumps. By actively pumping out the antibiotic and other harmful substances out of the cell, the efflux pumps prevents the intracellular accumulation of the antimicrobial agent that is necessary to exert optimal antibacterial activity inside the bacterial cell.

Bacterial cells have an inherent (natural) capacity to restrict the entry of small molecules (e.g. antibiotics) that destabilizes its internal metabolism. This is what the cell wall and outer cell membranes in both Gram positive and Gram negative bacteria respectively do. The ability of bacteria to restrict the entry of material into its internal environment is more pronounced in Gram negative bacteria unlike in Gram positive bacteria which are devoid of “outer membrane” that the former possess. The “outer membrane” is a first – line defense (protection) mechanism in Gram negative bacteria, and its absence in Gram positive bacteria is the reason why Gram positive bacteria is highly sensitive to antibiotics.

This is because there is no form of security to protect its peptidoglycan. The “influx – efflux” system in bacteria has to do with the entry and partial accumulation of harmful substances like antibiotics within the cytoplasm of a bacterium and the subsequent exit or removal of these harmful substances from the bacterial cell through efflux pumps. The “efflux system” in bacterial cell pumps out the antibiotics that finally made their way into the cytoplasm of the bacterium, thereby preventing their intracellular accumulation. The most well studied efflux system is in Escherichia coli and with this mechanism in place; bacteria can easily mount resistance to antibiotics directed against them.

2. Antibiotic resistance by chemical alteration of antibiotics in vivo: Some antibiotics (e.g. the Nutrofuran family used to treat UTI’s) need to be activated in vivo before ever they can reduce (bring out) their antibacterial activity against a given pathogen to which they were meant to attack and deactivate. Such antibiotics are activated in vivo by being reduced by a specific enzyme (gene). Only then can they be able to elicit their biological properties in vivo. For example, antibiotics in the Nitrofuran family like nutrafurantoin are reduced by cellular reductase enzymes encoded by nfsA and nfsB Any mutation in these genes can eventually lead to a nitrofuran resistance. In contrast to this, the chemical alterations of some antibiotics (e.g. Beta – lactams) in vivo by enzymes (e.g. Beta – lactamases) can inactivate the biological activity of these antibiotics, thereby leading to resistance.

3. Antibiotic Resistance due to Target Alterations: Most pathogens have the ability to alter target (s) of antibiotics in their cell. These alterations in the target of the drugs occur in such a way that the toxic effect of the antibiotic on the target pathogen is countered. A typical example is the alteration that occurs in penicillin – binding – proteins (PBPs) in bacteria. The PBPs are transpeptidases which catalyze the cross-linking reaction between two stem peptides, each linked to adjacent N-acetyl-muramic acid residues of the peptidoglycan backbone. This reaction confirms and gives rigidity to the bacterial cell wall. Penicillin exerts its antibacterial activity by binding to the PBPs, thereby preventing the cross-linking of N-acetyl-muramic acid and N-acetyl-glucosamine that will eventually lead to the formation of a very rigid bacterial cell wall. But alterations in this target (PBPs) due to a mutational change in the organism can mount antibiotic resistance on the bacterial cell.

4. Antibiotic resistance due to non – heritable states of bacteria: The non – heritable form of antibiotic resistance posed by bacteria to antibiotics has to do with some physiological states in which bacteria exist in, and which are not heritable or transferred by other organisms. These non – heritable physiological states of bacteria includes: Persistence State, Swarming State, and Biofilm State; and they are known to render bacteria insensitive to antibiotics. Such physiological states are expressed by bacteria in a transient state, and they are reversible and non – heritable. In such states, bacteria are said to be antibiotic tolerant. These non – heritable states of bacteria are as follows:

A. PERSISTENCE STATE: In this state, bacteria exist in a small fraction of slow or non – growing, antibiotic tolerant cells called persisters. They exist in this form and remain insensitive to harmful substances like antibiotics.

B. BIOFILM STATE: Biofilms are organized structures in which many bacterial species exist in. In biofilms, bacterial cells of several species are embedded in a self – produced exo – polysaccharide matrix. These structures are highly organized and they permit the transport of nutrients and metabolic wastes in and out of the matrix structure. Biofilms have a very high tolerance to high concentrations of antibiotics.

C. SWARMING STATE: Swarming is a form of multicellularity in many bacterial species, and it is characterized by the migration of highly differentiated bacterial cells (swarm cells) on semi – solid surfaces. This gives them some form of protection from antibiotics as, they are known to remain in close contact with one another and they also migrate as a group in this form.

Diagram showing the mechanisms by which bacteria evade (dodge) antibacterial activities of antibiotics. From: https://www.reactgroup.org

GENETIC BASIS OF ANTIBIOTIC RESISTANCE

Genetic resistance of microbes to antibiotics is due to a chromosomal mutation in the bacterial DNA or acquisition of antibiotic resistance genes on plasmids or transposons from other bacteria. Bacteria are extremely ingenious in becoming resistant to antibiotics directed towards them because they are able to regulate their drug resistance genes over time. This is often exacerbated by prior antibiotic usage especially irrational drug use. In this case where bacteria regulate their drug resistance genes, antibiotics used against them for treatment is inadvertently rendered useless in vivo. The genetic basis for antibiotic resistance may include: the acquisition and further expression of new DNA by horizontal gene transfer or mutations in cellular genes or acquired genes that alter antibiotic target sites on the bacteria. The genetic alterations (mutation) mediate a diversity of biochemical mechanisms of resistance that benefits the bacteria, and in turn makes the antibiotic less efficacious in vivo. These mechanisms may include:

  1. Enzymatic inactivation of the antibiotic.
  2. Target substitutions, amplification or modifications bypassing the binding of the antibiotic, or reducing the affinity of the bacteria for the drug.
  3. Barriers such as efflux pumps which reduce the access of the antibiotic to the target site of the bacteria.

FACTORS THAT CONTRIBUTE TO ANTIBIOTIC RESISTANCE

A great many factors contribute to the antibiotic resistance that we now face in both the community and in the hospital. Most of these factors have to do with both human behavior and activities while the other factors are contributed by the microbes themselves. The first among these factors that contribute to the development of antibiotic resistance is “Natural selection”. Microbes, over time, are capable of adapting in ways which increases their ability to survive in a changing environment. Bacterial genomes represent a large natural pool of diverse genetic information that can be accessed under appropriate selection pressures, using a variety of gene acquisition and dissemination mechanisms“. Human application of toxic agents on massive scale activates these genetic systems to promote survival of the microbial population.

As a result, each and every antibiotic will have a finite lifetime depending on the magnitude and nature of its use; thus the development of antibiotic resistance is inevitable. The basic idea here is that, as pathogens encounter large amounts of antibiotics, those that have little or no resistance to the antimicrobial agent are killed off en masse, while on the other hand, those that already possess some measure of resistance due to previous encounters with the drug or random mutation in their genome, have a much higher likelihood of survival. Once the weaker bacteria have been destroyed by the antibiotics, the remaining resistant organisms will continue to thrive. Though the process of natural selection takes generations to occur, but in bacteria, those generations are produced in a matter of hours or days rather than years or decades in other organisms. In addition to the above factors, other contributing factors to the development of antibiotic resistance in bacteria include:

  • Overuse of antimicrobial agents especially unwisely.
  • Excessive use of antibiotics in livestock and animal feeds.
  • Poor patient compliance towards drug regimens.
  • Self medication that defies a doctor’s prescription.

IMPACT AND COST OF ANTIMICROBIAL RESISTANCE ON PUBLIC HEALTH AND THE ECONOMY OF A NATION

The impact and cost of antibiotic resistance on the public health and economy of a nation are enormous. It is therefore imperative that we optimize the use of antibiotics in both our communities and hospitals in order to curtail or abate the development of bacterial resistance which is gradually eroding our therapeutic armamentarium. Due to the selection pressure caused by antibiotic use, a large pool of resistant genes has been created and this resistance places an increased burden on the society in terms of high mortality, morbidity, and cost of treating infections that they cause.

Patients infected with drug resistant organisms are more likely to have ineffective therapy, longer duration of hospital stay, need of treatment with broad spectrum antibiotics that are most toxic and more expensive than their counterparts. All these factors increases the cost of treatment for an individual patient; and on a national or global scale, its effect on the economy can be colossal. Antibiotic resistance drives up health care cost, thereby increasing the severity of disease and death rates of some infections since not all patients can afford the hospital bill due to the economic situation around. Ineffective treatment due to antimicrobial resistance will eventually culminate to increased human suffering, lost productivity, and often death.

CONTROL OF ANTIBIOTIC RESISTANCE

Antibiotic resistance can be controlled by one of the following methods:

  • Hand washing as a measure of infection control.
  • Review of antibiotic use in hospitals.
  • Updating of clinicians, nurses, pharmacists, and even patients on rationale antibiotic use.
  • Good personal hygiene in both the hospital and in the community.
  • Restriction of human medicine in livestock and animal feeds.
  • Patronage of over – the – counter (OTC) drugs by patients for self medication without doctor’s prescription should be discouraged.
  • Patients should always endeavour to take full course of their drugs when under any medication.

ANTIMICROBIAL SUSCEPTIBILITY TESTING

Antimicrobial susceptibility testing (AST) is a test that is used to determine the specific antibiotic/drug that can be used to inhibit or kill a microbial cell. It helps to guide a medical doctor on the best drug of choice and dosage to be prescribed for a given infection. Antibiogram is another name for antimicrobial susceptibility testing. AST is a very significant protocol in the clinical microbiology laboratory because it is the basis upon which drug prescription can be made for a patient suffering from a given ailment. It is worthy of note that antibiogram is carried out only on known and identified pathogens. It is wrong to carry out AST on non-pathogenic strains of microbes including commensals isolated from patients specimens. Though viruses, fungi, and protozoa can also be identified and characterized in the clinical microbiology laboratory, the apparatus with which to undertake AST on these pathogens are not routinely available in the laboratory as it is with bacterial pathogens that can be routinely identified and tested for antibiogram.

The main aim or reason of testing pathogenic microorganisms for their susceptibility to antibiotics is to be able to detect any form of drug resistance in the pathogens being tested, and also to select the appropriate drugs to which the pathogens will be susceptible to in the course of therapy. Due to increase in the morbidity and mortality rate of infectious diseases caused by drug resistant pathogens, AST should be a routine in every clinical m microbiology laboratory so as to keep the development and spread of drug resistant microbes at bay. The resistance of microbes to some empirical antimicrobial agents portends grave danger to the health sector and even to the economy of any nation due to increase in the cost of treatment and the possibility of treatment failures that may result from therapy.

Thus antibiogram should continue to be an integral part of the practice of clinical microbiology laboratories around the world, and it should always be performed on individual pure isolates of pathogenic microorganisms so that therapy can be properly guided. The introduction of several antibiotics into clinical medicine coupled with the increasing rate of the emergence of resistant pathogens necessitates the need to routinely carry out AST in the clinical microbiology laboratory in order to sustain the shelf-life of these drugs. Factors that can affect the result of antimicrobial susceptibility testing include: inoculum size of the test organism, pH, temperature, moisture and effects of thymidine or thymine.

The results of antibiogram are usually reported as minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC), and each of these results are further categorized as susceptible, intermediate or resistant depending on the inhibition zone diameter (IZD) produced by the tested drug on the test pathogen. MIC is the lowest concentration of a drug/antibiotic that can inhibit the growth of a microorganism while MBC is the lowest concentration of an antibiotic that can kill a microorganism.. According to the Clinical and Laboratory Standard Institute (CLSI), AST is carried out in the laboratory by any of the following methods:

1. Disk diffusion method: Disk diffusion method is a simple and practical AST that is widely carried out in the clinical microbiology laboratory. It requires no special equipment to carry out, and it is one of the oldest means of testing a wide range of bacterial pathogens for susceptibility to drugs. The Kirby-Bauer method is a typical example of disk diffusion method. Disk diffusion method is carried out by applying a bacterial inoculum (adjusted to 0.5 McFarland turbidity standards) on a Mueller-Hinton or nutrient agar plate. The plate is allowed for some minutes, after which commercially available paper disk(s) containing known concentration of antibiotics is aseptically applied to the plate. The plate is incubated, and the IZD around each of the disk is measured to the nearest millimeter using a caliper or meter rule. Basically the IZDs obtained are compared to known breakpoints of the tested drugs as per the CLSI criteria in order to know if the organism is susceptible, intermediate or resistant to the drugs being tested.

Illustration of susceptibility test plate of a Pseudomonas aeruginosa isolate, showing different levels of susceptibility and resistance of the test organism to the test antibiotics (as detected by disk diffusion method)

2. Agar dilution and diffusion method: The agar dilution and diffusion susceptibility test is usually performed using the epsilometer (E) test strip. E-test is an exponential gradient susceptibility testing procedure that combines both dilution and diffusion techniques in the determination of antibiogram. It is a quantitative testing method that makes use of an inert test strip that is incorporated with predefined antimicrobial agents that produces a symmetrical inhibition ellipse after incubation. E-test can be used to determine the antibiogram of both fastidious and non-fastidious bacterial pathogens.

3. Dilution susceptibility method: Dilution susceptibility test is used to determine the minimal amount of an antimicrobial agent that will be required to either kill or inhibit the growth of a bacterial pathogen. They are mainly used to determine the MICs and MBCs of empirical drugs/antibiotics on specific bacterial pathogens unlike the disk diffusion method that determine the IZD of a drug. Examples of dilution tests include: broth dilution, agar dilution, and macro-broth dilution methods. The antimicrobial agents are usually diluted in either broth or agar and tested at a two-fold serial dilution in order to determine their susceptibility pattern in relation to specific bacterial pathogens. Today, there exist several automated and rapid methods of conducting antibiogram tests in the clinical microbiology laboratory. Some of these methods include epsilometer (E)-test, Vitek MIC test, the Sensititre ARIS 2X Automated Systems, BD Phoenix Automated Microbiology Systems, and so on.

McFarland Turbidity Standards

McFarland Turbidity Standards are series of standards which are used as references to adjust the turbidity of bacterial suspensions during antimicrobial susceptibility testings so that the actual amount of inoculum size used will be within a certain limit and be known at the same time. They are used as reference standards in the preparations of suspensions of bacterial cultures. McFarland turbidity standards are used to know the actual number of bacteria that is present in a liquid culture or suspension by comparing the suspension to a known standard. Basically, McFarland turbidity standards are composed or made up of three main ingredients which are barium chloride and concentrated tetraoxosulphate (vi) acid. McFarland turbidity standards is one of the easiest method of estimating or determining the actual amount of the test bacteria required to undertake an antimicrobial susceptibility testing.

PROTOCOL FOR MINIMUM INHIBITORY CONCENTRATION (MIC): 2-FOLD SERIAL DILUTION

Minimum inhibitory concentration (MIC) is defined as the minimum amount or concentration of antibiotics and/or drug that is required to kill or inhibit the growth of a pathogen either in vivo or in vitro. The procedure involved in determining the MIC of antimicrobial agents against a test organism in vitro is succinctly enumerated as follows:

  1. Set up 5 test tubes or more in a test tube rack and label them tube A, tube B, tube C, tube D and tube E as the case may be.
  2. Set up positive and negative control tubes. Negative control tube contains the bacterial culture and the Mueller-Hinton (MH) broth and remains turbid. Positive control tube is clear and contains only the MH broth.
  3. Add 0.5 ml of Mueller-Hinton (MH) broth to each of the tubes (A-E). This should be done with a sterile pipette.
  4. Add 0.5 ml of the test antibiotic (from a stock concentration of 250 mg/ml) to tube A. Mix the solution properly. Upon dilution tubes A, B, C, D & E will be 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, 3.125 mg/ml and 1.563 mg/ml.
  5. From tube A, transfer 0.5 ml of the diluted antibiotic solution to tube B.
  6. Repeat this process until all the tubes have been covered.
  7. Dispense or discard 0.5 ml of the diluted antibiotic solution from tube E.
  8. Transfer 0.5 ml of bacterial suspension (adjusted to 0.5 McFarland turbidity standards) to each of the tubes (labeled A-E). Mix the content of each tube properly.
  9. Incubate the tubes in the test tube rack at 37oC overnight.
  10. After incubation, check each of the tubes for turbidity or cloudiness. Ensure not to shake the tubes when doing so.
  11. Look out for the tube without turbidity or cloudiness. This tube without cloudiness or turbidity is the MIC of the antibiotic for the test bacteria. Absence of cloudiness is indicative of the inhibitory effect of the test agent against the bacteria in broth culture.

PROTOCOL FOR DISK DIFFUSION TEST

The disk diffusion test provides a qualitative evaluation of bacterial growth inhibition by a given antibiotic. In the disk diffusion test, the antibiotic concentrations are created by diffusion of the drug through a paper disk containing known concentration of the drug. The zone of inhibition produced after incubation of the agar plate is used to determine the susceptibility or resistance profile of the test bacteria to the antibiotic qualitatively. Disk diffusion technique involves the following simplified steps:  

  1. Inoculate the test bacteria (adjusted to 0.5 McFarland turbidity standards) on agar plate. This process is carried out by swabbing the suspension of the test bacteria on the entire surface of the agar plate.
  2. Aseptically place the disk on the swabbed agar plate.
  3. Allow plates for about 10-20 min for pre-diffusion of the drug to occur. Antibiotic disks containing defined concentration of drugs are commercially available. But susceptibility disks can be prepared in the laboratory if the researcher so chooses.
  4. Incubate the plate at 37oC for 24 hrs or overnight as the case may be.
  5. After incubation, check the plates for zones of inhibition.
  6. Measure the inhibition zone diameter (IZD) using a meter rule. The unit of the IZD should be in millimeter (mm).
  7. Record the readings or values obtained according to the CLSI criteria, and report the bacteria as susceptible, intermediate or resistant to the test antibiotic(s).

REFERENCES

World Health Organization (WHO). Strategy for Containment of Antimicrobial Resistance. WHO/CDS/CR/DRS/2001.2.

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Jacoby G.A and Munoz-Price L.S (2005). Mechanisms of Disease: The New β – lactamases. N Engl J Med, 352:380-91.

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Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Ejikeugwu Chika, Iroha Ifeanyichukwu, Adikwu Michael and Esimone Charles (2013). Susceptibility and Detection of Extended Spectrum β-Lactamase Enzymes from Otitis Media Pathogens. American Journal of Infectious Diseases. 9(1):24-29.

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Ashutosh Kar (2008). Pharmaceutical Microbiology, 1st edition. New Age International Publishers: New Delhi, India.

 

 

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