Antibiotics and Antibiotic Resistance PDF

INTRODUCTION TO ANTIBIOTICS AND ANTIBIOTIC RESISTANCE DR. JONATHAN ASANTE DEPARTMENT OF PHARMACEUTICAL MICROBIOLOGY U.C.C Introduction- Timeline of the history of infectious diseases Before 1800: The general understanding was that diseases were caused by bad gases, which were called...

INTRODUCTION TO ANTIBIOTICS AND ANTIBIOTIC RESISTANCE DR. JONATHAN ASANTE DEPARTMENT OF PHARMACEUTICAL MICROBIOLOGY U.C.C Introduction- Timeline of the history of infectious diseases Before 1800: The general understanding was that diseases were caused by bad gases, which were called miasmas, or by an imbalance of fluids in the body. The treatment for diseases at that time included blood-letting. 1795: Alexander Gordon observed that puerperal fever was spread between patients by attending midwives and doctors. He recommended cleanliness of the attending medical staff as a measure to prevent disease from spreading Introduction-Timeline of the history of infectious diseases 1847: Ignaz Semmelweis discovered hygiene theory – that is, the importance of handwashing in preventing puerperal fever. 1854: John Snow traced an outbreak of cholera to a single contaminated water pump in London, and thus discovered the link between contaminated water and this disease. He is considered as one of the founding fathers of the discipline of epidemiology.  Timeline of the history of infectious diseases 1850–1880: Collective efforts by Louis Pasteur and Robert Koch led to the discovery that diseases are caused by micro-organisms (germ theory). 1928: Alexander Fleming discovered that a substance produced by a mould – which he called penicillin – could kill bacteria. Florey and Chain later played an important role in making the antibiotic penicillin widely available.  Introduction The discovery of antibiotics and their power to fight bacterial disease began with Alexander Fleming. He observed the mould Penicillium notatum accidentally growing on a sample of staphylococci and saw it had killed the surrounding colonies of disease-causing bacteria.  Introduction A bacterial infection can take hold so quickly because bacteria can double their numbers in such a short time unless something keeps them in check. Many bacteria are opportunistic pathogens, meaning that they can cause an infection when the defence mechanisms of the host – which could be a human, animal or plant – are weakened.   Bacterial infections have always been a major cause of mortality in humans. Introducti  Many infections are caused by bacterial on pathogens such as: tuberculosis (TB) caused by Mycobacterium tuberculosis, gonorrhoea caused by Neisseria gonorrhoeae, and whooping cough caused by Bordetella pertussis.  In the pre-antimicrobial era, a small cut that became infected with a bacterium like Staphylococcus aureus could easily spiral into a life-threatening blood stream infection (sepsis). Introduction Microbes make up around 90% of the cells in a typical human body and 10% of our body weight. Most of the microbes are in our gut and on our skin. Many microbes are beneficial, for example, helping us to digest our food. Only a tiny fraction cause disease, and are usually kept in check by our immune system. But when they aren't, microbes also help us to fight back. Definition of terms  Antibacterial: Compounds that kill or inhibit the growth of bacteria.  An antibiotic is an agent or substance that is produced by or derived from a microorganism that kills or inhibits the growth of another living micro-organism.  Antibiotic substances that are synthetic, semi-synthetic, or derived from plants or animals are, strictly speaking, not antibiotics. In this document “antibiotic” refers to an antimicrobial agent with the ability to kill or inhibit bacterial growth.’ (WHO, 2019). Definition of terms Antifungal: Compounds that kill or inhibit the growth of fungi. Antimicrobial: Compounds that kill or inhibit the growth of microbes/microorganisms. ‘An antimicrobial is an agent or substance derived from any source (micro-organisms, plants, animals, synthetic or semi-synthetic) that acts against any type of microorganism, such as bacteria (antibacterial), mycobacteria (anti-mycobacterial), fungi (antifungal), parasite (anti-parasitic) and viruses (antiviral).’ (WHO, 2019). Definition of terms Antiparasitic: Compounds that kill or inhibit the growth of parasites. Antiviral: Compounds that kill or inhibit the growth of viruses.  QUESTION: ‘ALL ANTIBACTERIALS ARE ANTIMICROBIALS.’ TRUE OR FALSE? QUE QUESTION: ‘ALL ANTIMICROBIALS ARE ANTIBACTERIAL.’ TRUE OR FALSE? What antibiotics do  Antibiotics that are used for medical treatment work through a mechanism of selective toxicity. They often also attack bacteria that are not pathogenic.  Selective toxicity refers to targeting drugs to affect only the pathogenic organism and minimise damage to the host cells and commensal microorganisms.  About 100 years ago, despite the understanding about infection prevention and spread, infections were difficult to treat, because the treatments that were available not only killed the infection, but also often killed the patient. What antibiotics do  Not all antibiotics attack bacteria in the same way: some attack the cell wall, some attack bacterial protein syntheses and some attack the DNA-replicating mechanism of bacteria.  In all cases, they tackle structures or biochemical processes that are either not found in human or animal cells, or don’t work in the same way.  Antibiotics are classified based on their chemical  What are antibiotics  Bacteria cause disease when they are able to reproduce in the body. They produce toxins, which damage tissues and organs.  But in nature, microbes can also antibiotics to protect themselves against competitors.  And one move they have is to produce antibiotics. These are compounds that allow them to kill other microbes and take all the resources that they need.  What are antibiotics?  And so the competition between microbes results in these very sophisticated antibiotic molecules.  Broad-spectrum antibiotics are those classes of antibiotics that target several bacterial species.  Antibiotics with a working mechanism that only attacks a small group of bacterial species are called narrow-spectrum antibiotics. Let’s try these Question: Antibiotics can be used to treat infections caused by: a. bacteria and viruses b. bacteria c. Viruses Que: Antibiotics: a. do not cause side-effects b. are only active against pathogens c. can be used in a prophylactic manner to prevent bacterial infections d. stimulate the body’s immune system.  Antibiotics are not selective; they inhibit or kill ‘good’ bacteria along with ‘bad’ bacteria. This can lead to common side-effects such as an upset stomach and antibiotic-associated diarrhoea caused by Clostridium difficile or other gut microbes. Role of Cancer chemotherapy: antibiotic Chemotherapy is one of the s in principal treatments for cancer; a major-side effect is that the modern immune system is weakened. medicine Opportunistic bacteria can take advantage of this situation and cause an infection. Antibiotics are important to treat bacterial infections in patients with extremely weakened immune systems due to chemotherapy. Role of antibiotics in modern medicine Surgical prophylaxis:  Antibiotics are used before certain surgical procedures to prevent infection.  For example, for orthopaedic operations where implants are put in to replace joints (such as a hip or knee), there is a possibility of micro-organisms infecting the prosthesis.  Mortality rates of surgeries performed before the discovery of antibiotics were high.  Role of antibiotics in modern medicine Food-animal production and veterinary medicine: Antibiotics are used to treat disease, prevent disease and promote growth in food-animal production. These production systems promote pathogen spread. The use of antibiotics to promote growth in food- producing animals was common historically but is now considered inappropriate. Members of the World Organisation for Animal Health (OIE) agreed in 2016 to ‘phasing out the use of antibiotics for growth promotion in the absence of risk analysis’.  Role of antibiotics in modern medicine Avoiding the use of some antibiotics in animals that should be reserved for treatment of some bacterial infections in humans.  Antibiotics- Historical development  In the late 19th century, scientists discovered that bacteria cells could be stained by chemical dyes.  This led scientists to start looking for compounds that would kill pathogenic bacteria.  The first antibiotic to be discovered was arsphenamine (Salvarsan), which was discovered in 1909 by Paul Ehrlich and Sahachirō Hata.  This was the first effective treatment for the sexually transmitted infection syphilis (caused by Treponema pallidum). Antibiotics- Historical development This was followed in 1932 by the discovery of Prontosil by a team led by Gerhard Domagk, who were systemically testing chemical dyes for their ability to kill bacteria. Prontosil is a sulphonamide antibiotic, which is effective against a number of different pathogenic bacteria and is still in use today. Penicillin, was however the first broad spectrum antibiotic. Antibiotics- Historical development While the first two antibiotics to be put into clinical usage were synthetic compounds, penicillin came from another living organism (Penicillium chrysogenum). In 1928 Alexander Fleming found that a fungus called Penicillium chrysogenum produced a compound that could kill the bacterial pathogen Antibiotics- Historical development In 1940, Howard Flory assembled a team of researchers including Ernst Chain, Norman Heatley, Edward Abraham and others to work on penicillin. They discovered the method required to extract penicillin in high enough concentrations to begin to test the effectiveness of penicillin against real infections. Antibiotics- Historical development They first showed excellent activity of penicillin against bacterial infections in mice. They then moved on to conduct the first successful clinical trials in human patients. Later, researchers and pharmaceutical companies in the USA developed the methods to enable the industrial-scale production of penicillin required for widespread clinical usage.  Following the success of Antibioti penicillin, researchers began to search for other cs- fungi and bacteria that Historica produced antibiotics. l As a result, in the 1950’s, 60’s and 70’s many new develop types of antibiotics were ment discovered and went into clinical use. Antibiotics- Historical development By the 1950s, the use of antibiotics had However, once we revolutionized the reached the 1990’s treatment of previously the pipeline of new untreatable infectious antibiotics dried up. diseases. 1950s 1990’s ‘The time has1967 come to close the book on infectious diseases. We have basically wiped out infection in the United States’. - William Stewart Antibiotics- Historical development How do antibiotics work? In broad terms antibiotics work either by killing the bacteria or by preventing their growth. Antibiotics that kill a bacteria are called “bactericidal” and antibiotics that prevent bacterial growth are called “bacteriostatic”. Broadly, antibiotics target: The bacterial cell wall and membrane DNA synthesis Protein production How do antibiotics work?  How do antibiotics work?   Although these broad groups How do are useful for classification antibioti purposes, different cs work? ways to affect these antibiotics work in different processes and structures. How do antibiotics work? 1. Antibiotics targeting the bacterial cell wall and membrane: Penicillin is part of the family of antibiotics known as β-lactams. β-lactam antibiotics are so called because they all share common chemical structure known as a β-lactam ring. β-lactam family includes the penicillins, cephalosporins and carbapenems - which are some of the clinically most important antibiotics. β-lactam antibioti cs How do antibiotics work? β-lactam antibiotics kill bacteria by binding to bacterial enzymes called penicillin-binding proteins (PBPs). These PBPs crosslink parts of the peptidoglycan layer of bacterial cell walls. By binding the PBPs, β-lactams stop the PBPs working to build or repair the bacterial cell wall meaning the bacterial cells break open and die. How do antibiotics work? Another family of antibiotics known as the glycopeptides which includes vancomycin also prevent the cell wall forming by inhibiting peptidoglycan synthesis. By contrast, daptomycin, a lipopeptide antibiotic, damages the cell membrane by inserting itself into the cell membrane and inducing membrane depolarization. How do antibiotics work?  Likewise, colistin, a polymyxin antibiotic used in the treatment of multidrug resistant bacteria, binds to the outer membrane causing damage to the cell membrane.  2. DNA synthesis: Three families of antibiotics How do prevent DNA synthesis through different mechanisms. antibio Sulphamethoxazole (of the sulphonamide family) and tics trimethoprim both block the activity of enzymes that are work? involved in the synthesis of folic acid. Without folic acid the bacteria are unable to synthesise the DNA bases (A, T, C, G). How do antibiotics work? The third family of antibiotics is known as the fluoroquinolones. Fluoroquinolones work by binding to an enzyme known as a topoisomerase. Topoisomerase enzymes are involved in the coiling of DNA to form the double helix shape. By binding to the topoisomerase enzyme, fluoroquinolones cause the replication of DNA to stall, meaning that the bacterial cell is unable to replicate. How do antibiotics work? 3. Protein production:  Rifampicin works by binding to RNA polymerase, an enzyme which copies DNA into RNA.  RNA is then used as the template to synthesise proteins in the ribosome. By blocking the RNA polymerase, rifampicin stops the bacteria producing proteins which are essential for life.  Many families of antibiotics including the aminoglycosides, tetracyclines, macrolides, phenicols, and lincosamides work by binding to parts of the bacterial ribosome blocking its activity. How do antibiotics work? The ribosome is the machinery that reads the RNA template and synthesises proteins (known as translation) by linking together different amino acids. Other antibiotics such as mupirocin and fusidic acid work by interfering with other bacterial enzymes that work with the ribosome during translation. Levels of bacterial response to antibiotics Susceptibility: Susceptible bacteria will die after being attacked by the antibiotic Tolerance: Tolerant colonies will not be killed but they will be unable to grow and reproduce under the presence of the antibiotic. These bacteria will be destroyed by the mechanisms of the immune system. Resistance: Resistant bacteria will continue to flourish even when exposed to an antibiotic. The medicine will be ineffective.  How do microorganisms actually resist antimicrobial actions? Antibi What enables them to otic do this? resista How do previously susceptible bacteria gain nce resistance? How is antimicrobial resistance detected in bacterial populations? Antimicrobial resistance All antibiotics work by disrupting a critical function in the bacterial cell. Bacteria can change their biochemistry in order to adapt to these antibiotics and prevent the antibiotics from damaging the cell. Today, more and more of our antibiotics are becoming less effective. What went wrong? Antimicrobial resistance Antimicrobial resistance (AMR) has seriously compromised the usefulness of antibiotics in the war against microbes. Antimicrobial resistance is the ability of a microorganism to survive and multiply in the presence of an antimicrobial agent that would normally inhibit or kill this particular kind of organism.   AMR is just one of the many adaptive traits that resilient bacterial subpopulations may Antimicro possess or acquire, enabling them bial to out- compete and out-survive resistance their microbial neighbours and overcome host strategies aimed against them.  The rate at which antibiotic resistance often develops and how quickly it spreads across the globe and among different species of bacteria, is alarming. AMR As a result of sequential, cumulative acquisition of resistance traits against different antibiotics, more bacterial pathogens with multiple-drug resistance are being reported worldwide. AMR As a consequence, many bacterial organisms, including major human and animal pathogens such as Mycobacterium and Salmonella species, have become resistant to antibiotics which were previously quite efficacious The first antibiotic resistance mechanism described was that of penicillinase. Its presence and activity was first reported by Abraham and Chain in 1940 shortly after its discovery Antibiotic resistance Resistance to single antibiotics became prominent in organisms that encountered the first commercially produced antibiotics. The most notable example is resistance to penicillin among staphylococci, specified by an enzyme (penicillinase) that degraded the antibiotic. Antibiotic resistance Some of the most problematic MDR organisms that are encountered currently include: Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae bearing extended- spectrum β-lactamases (ESBL) Vancomycin-resistant enterococci (VRE) Methicillin-resistant Staphylococcus aureus (MRSA) Vancomycin-resistant MRSA, and extensively drug-resistant (XDR) Mycobacterium tuberculosis Antibiotic resistance To survive in the presence of an antibiotic, bacterial organisms must be able to disrupt one or more of the essential steps required for the effective action of the antimicrobial agent. No single mechanism of resistance is considered responsible for the observed resistance in a bacterial organism. In fact, several different mechanisms may work together to confer resistance to a single antimicrobial agent. Antibiotic resistance Antibiotic resistance in bacteria can be intrinsically expressed or may be acquired Intrinsic resistance refers to the innate ability of bacteria to resist the effects of an antimicrobial agent through its innate functional or structural characteristics, without the need for mutation or gain of extra genes. Antibiotic resistance Intrinsic resistance  Generally, bacteria mediate intrinsic resistance by two major mechanisms: 1. By membrane impermeability and inaccessibility 2. Extrusion of antibiotics through chromosomally- encoded efflux pumps. Antibiotic resistance Intrinsic resistance 1. By membrane impermeability and inaccessibility  Gram-negative bacteria are resistant to vancomycin because their extra outer membrane prevents a large molecule like vancomycin entering the cell.  Gram-negative bacteria and some Gram-positive bacteria also have structures called porins Act as pores through which molecules including nutrients can pass through the membrane into the cell. Antibiotic resistance Intrinsic resistance 2. Extrusion of antibiotics through chromosomally-encoded efflux pumps.  Another mechanism of intrinsic resistance in Gram-negative bacteria is due to the presence of efflux pumps.  These pump antibiotics out of the cell thereby preventing them reaching lethal concentrations. Antibiotic resistance Intrinsic resistance  Efflux pumps often work along with reduced permeability of the cell membrane to mediate intrinsic resistance.  Some of these efflux pumps are classified as multidrug-efflux pumps as they provide resistance to a number of different classes of antibiotics.  Eg MexAB-OprM efflux system again in P. aeruginosa  fluoroquinolones, tetracyclines, phenicols, macrolides and β-lactams. Different mechanisms of intrinsic resistance Antibiotic resistance Intrinsic resistance In some intrinsically resistant bacteria the chemical properties or the size of porins exclude certain antibiotics. The number of the porins that is expressed (e.g. lower numbers) in the membrane is also thought to contribute to intrinsic resistance. Antibiotic resistance  In addition to the intrinsic mechanisms of resistance, bacterial pathogens can acquire genes and mutations that mediate resistance to antibiotics.  In some cases, bacteria may acquire multiple mechanisms of resistance to the same antibiotic  In multidrug resistant bacteria, they acquire resistance to multiple classes of antibiotics.  Intrinsic resistance ORGANISMS NATURAL MECHANISM RESISTANCE AGAINST: Aerobic bacteria Metronidazole Inability to anaerobically reduce drug to its active form Gram-negative Vancomycin Lack of uptake resulting from bacteria inability of vancomycin to penetrate outer membrane Klebsiella spp. Ampicillin (a beta- Production of enzymes (beta- lactam) lactamases) that destroy ampicillin before the drug can reach the PBP targets Enterococci All cephalosporins Lack of PBPs that effectively bind and are inhibited by these beta lactam antibiotics Antibiotic resistance The mechanisms of resistance can be broken down into the following: 1. Enzyme inactivation and modification 2. Modification of the antibiotics target site 3. Overproduction of the target 4. Replacement of the target site 5. Efflux and reduced permeability Mechanisms of antibiotic resistance 1.i Enzyme inactivation:  One of the first mechanisms of resistance to be discovered was resistance to penicillin.  Penicillin resistant strains of S. aureus were found to have acquired an enzyme known as a β-lactamase (originally known as a penicillinase). Mechanisms of antibiotic resistance 1.i Enzyme inactivation:  β-lactamase enzymes target the β-lactam ring in β- lactam antibiotics.  The β-lactamase enzyme breaks this ring open, preventing the antibiotic from binding to their target. Mechanisms of antibiotic resistance β-lactamase enzyme degrading penicillin into fragments; chemical structure of penicillin with the β-lactam ring highlighted. Mechanisms of antibiotic resistance 1.i Enzyme inactivation: β-lactamases are a family of enzymes found in many bacterial pathogens. They have different activities, meaning some will work against specific members of the β-lactam family, while others will not. Mechanisms of antibiotic resistance 1.i Enzyme inactivation:  Certain members of the β-lactamase family, known as carbapenemases, are the most problematic because they break down all members of the β-lactam family of antibiotics,  Including carbapenems, severely limiting treatment options. Mechanisms of antibiotic resistance 1.ii Enzyme modification:  Two other mechanisms of resistance are mediated by bacteria acquiring enzymes.  Firstly, bacteria can acquire enzymes that chemically modify the target of the antibiotic in the bacteria by adding additional chemical groups. Mechanisms of antibiotic resistance 1.ii Enzyme modification: E.g. the erm (erythromycin ribosomal methylation) gene that provides resistance against macrolide antibiotics like erythromycin. This enzyme methylates the part of the ribosome, which is the target of erythromycin. Mechanisms of antibiotic resistance Mechanisms of antibiotic resistance  The second type of enzyme acts by chemically modifying the antibiotic itself  This prevents the antibiotic binding to its target site.  E.g. aminoglycoside-modifying enzymes such as N- acetyltransferases, which add an additional acetyl group (CH3CO) to aminoglycoside antibiotics such as kanamycin. Mechanisms of antibiotic resistance Mechanisms of antibiotic resistance The mechanisms of resistance can be broken down into the following: 1. Enzyme inactivation and modification 2. Modification of the antibiotics target site 3. Replacement of the target site 4. Overproduction of the target 5. Efflux and reduced permeability  2. Modification of the Mechani antibiotic target site sms of Mutations can lead to modification of the target site of the antibiotic. antibioti If mutation occurs at a location of a gene that encodes for a protein that is c the target of an antibiotic, then sometimes these mutations mean that the antibiotic can no longer bind resistan to the target. This means that the bacteria with the ce mutation will have a growth advantage and will survive the antibiotic while the rest of the population will die. Mechanisms of antibiotic resistance 2. Modification of the antibiotic target site E.g. resistance to penicillin in Streptococcus pneumoniae penicillin binding proteins (PBP). Mechanisms of antibiotic resistance 2. Modification of the antibiotic target site  Similarly resistance in many bacterial pathogens to fluoroquinolone antibiotics such as ciprofloxacin is mediated by mutations in the DNA gyrase and DNA topoisomerase IV genes, which are the target of ciprofloxacin. Mechanisms of antibiotic resistance The mechanisms of resistance can be broken down into the following: 1. Enzyme inactivation and modification 2. Modification of the antibiotics target site 3. Replacement of the target site 4. Overproduction of the target 5. Efflux and reduced permeability Mechanisms of antibiotic resistance 3. Replacement of the target site Another similar mechanism of resistance is to gain an additional copy of the gene that encodes a protein that still retains activity (e.g., the antibiotic can’t bind to it) in the presence of the antibiotic. E.g., Staphylococcus aureus becomes resistant to most β-lactam antibiotics such as penicillin by this mechanism. Mechanisms of antibiotic resistance 3. Replacement of the target site  Methicillin-resistant Staphylococcus aureus (MRSA) becomes resistant by gaining an extra copy of penicillin binding protein 2, which is the target of β-lactam antibiotics. Mechanisms of antibiotic resistance  This additional version known as penicillin binding protein 2a (PBP2a) can still function in the presence of β-lactam antibiotics. Mechanisms of antibiotic resistance The mechanisms of resistance can be broken down into the following: 1. Enzyme inactivation and modification 2. Modification of the antibiotics target site 3. Replacement of the target site 4. Overproduction of the target 5. Efflux and reduced permeability Mechanisms of antibiotic resistance 4. Overproduction of the target. Bacteria can also overproduce the target of the antibiotics. Thus, there is an excess of the protein target of the antibiotics compared to the antibiotic itself. This means that there is enough of the target protein for it to continue its role in the cell in presence of antibiotics. This is a mechanism of resistance to trimethoprim in Escherichia coli and Haemophilus influenzae. Mechanisms of antibiotic resistance The overexpression is sometimes found in combination with mutations that lower the ability of the antibiotic to bind to its target. Mechanisms of antibiotic resistance The mechanisms of resistance can be broken down into the following: 1. Enzyme inactivation and modification 2. Modification of the antibiotics target site 3. Replacement of the target site 4. Overproduction of the target 5. Efflux and reduced permeability  Mechanisms of antibiotic resistance 5. Efflux and reduced permeability.  In addition to the intrinsic resistance mediated via reduced permeability and efflux pumps, bacteria can acquire additional efflux pumps that specifically pump a single type of antibiotic.  E.g. TetA efflux pumps that specifically pump tetracycline from the cell.  Mechanisms of antibiotic resistance 5. Efflux and reduced permeability.  Also, permeability of the cell can be altered by the acquisition of mutations in porins.  These mutations can include porin loss.  Modification of the size or conductance of the porin channel  OR a lower expression level of a porin. Mechanisms of antibiotic resistance Ultimately efflux pumps and reduced permeability, lower the intracellular antibiotic concentration in the bacterial cell by either exporting the antibiotic or by not allowing its importation, respectively. Mechanisms of antibiotic resistance 5. Efflux and reduced permeability.  Efflux pumps are variants of membrane pumps possessed by all bacteria, both pathogenic and non- pathogenic  They move lipophilic or amphipathic molecules in and out of the cells.  Some are used by antibiotic producers to pump antibiotics out of the cells as fast as they are made.  Thus, they constitute an immunity protective mechanism for the bacteria to prevent being killed by their own chemical weapons  Mechanisms of antibiotic resistance Reduced permeability has been observed in:  Pseudomonas aeruginosa against imipenem (a beta- lactam antibiotic)  Enterobacter aerogenes and Klebsiella spp. against imipenem  Vancomycin intermediate-resistant S. aureus or VISA strains with thickened cell wall trapping vancomycin  Many Gram-negative bacteria against aminoglycosides  Many Gram-negative bacteria against quinolones Mechanisms of antibiotic resistance Extrusion via efflux pumps has been observed in:  E.coli and other Enterobacteriaceae against tetracyclines  Enterobacteriaceae against chloramphenicol  Staphylococci against macrolides and streptogramins  S. aureus and Streptococcus pneumoniae against fluoroquinolones Chromosomal mutation and recombination Random errors in DNA replication can occur during clonal reproduction Results in a clonal progeny that will inherit replication “errors” in their DNA. These errors are known as mutations. Chromosomal mutation and recombination Just by chance, some of these mutant cells can be resistant to antibiotics. for example, if mutations alter the antibiotic target (or through other mechanisms). A 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. Chromosomal mutation and recombination Resistance through mutations in chromosomal DNA is the main driver of acquired resistance in certain bacterial species. Such as Mycobacterium tuberculosis and Helicobacter pylori, or for particular antibiotics, especially synthetic agents such as fluoroquinolones and oxazolidinones. Antibiotic-resistant mutations can be transmitted “vertically” by clonal reproduction. “horizontally” via recombination. Chromosomal mutation and recombination Bacteria can exchange DNA using recombination mediated by mechanisms that include:  Transduction (the transfer of DNA from one cell to another by bacteriophage)  Transformation (uptake of exogenous DNA from the surrounding environment) OR  Conjugation (transfer of DNA from one bacterium to another via cell-to- cell contact).  Chromosomal mutation and recombination  Some bacterial species, such as Helicobacter pylori, recombine quite frequently  Others, such as Mycobacterium tuberculosis, do not recombine.  Recombination can bring multiple mutations all at the same time from the donor to the recipient.  Mutations acquired by bacteria are thus the result of vertically inherited mutations and horizontally acquired mutations via recombination. Plasmids Plasmids are small pieces of circular extrachromosomal DNA in bacteria. They are self-replicating Some can integrate into the chromosome. They can transfer from one cell to another via cell- to-cell contact (conjugation) Plasmids Plasmids are important because they are mobile and can move between different bacterial cells Including strains of the same species or even between different bacterial species. Plasmids carry multiple genes, in some cases disease- causing and antibiotic resistance genes. How plasmids are maintained in bacterial populations How plasmids are maintained in bacterial populations In the presence of an antibiotic, all bacterial cells except for the ones carrying the plasmid will be killed. Thus, the plasmid confers an adaptive advantage and antibiotic-resistant cells are able to grow and multiply. How plasmids are maintained in bacterial populations Over time, the new bacterial population will be made up of mostly (or entirely) antibiotic-resistant plasmid-carrying cells The plasmid (and thus antibiotic resistance), that has now increased in prevalence, may be transferred to other bacterial populations via conjugation. Transposons Autonomous mobile genetic elements. Typically found integrated in the host chromosome. Contain genes that enable them to integrate and excise (transposition) from the chromosome. Many transposons carry genes that may provide some benefit to the host cells, such as antibiotic resistance.  Biological resistance refers to Biological changes that result in the Versus organism being less susceptible to a particular antimicrobial agent Clinical than has been previously Resistanc observed. e When antimicrobial susceptibility has been lost to such an extent that the drug is no longer effective for clinical use, the organism is then said to have achieved clinical resistance. Selection In the presence of antibiotics, minor resistant bacterial populations have a selective growth advantage and proliferate while the others die out. Inappropriate use or misuse of antibiotics could lead to selective amplification of resistant populations Considered as one of the main reasons for the emergence of drug resistant bacterial pathogens. Selecting for resistance Selection A close relationship between antibiotic usage and resistance levels. Another factor leading to rapid spread of resistance is human-to-human transmission (Rapid spread of MRSA). Usage of antibiotics in livestock Detecting antimicrobial resistance Antimicrobial susceptibility testing methods are in vitro procedures used to detect antimicrobial resistance in individual bacterial isolates. Because laboratory detection methods can determine resistance or susceptibility of an isolate against an array of possible therapeutic candidates, antimicrobial susceptibility testing results can be a useful clinical guideline in selecting the best antibiotic treatment option for each particular patient. These same methods can also be used for monitoring the emergence and spread of resistant microorganisms in the population. Points to consider when deciding whether or not to conduct antimicrobial susceptibility testing Clinical relevance of the isolate Purity of the isolate Logical panel of antimicrobial agents to be tested (i.e., do not include antibiotics to which the isolate is known to have intrinsic resistance) Availability of test methodology, resources, and trained personnel Standardization of testing Valid interpretation of results Cost efficiency Effective means to communicate results and interpretation to end-users AST Because of the required culture time, antimicrobial susceptibility testing may take several days, which is not ideal particularly in critical clinical cases demanding urgency. An antibiogram is a compiled susceptibility report or table of commonly isolated organisms in a particular hospital, farm, or geographic area, which can serve as a useful guideline in therapy before actual culture and susceptibility data becomes available for reference. AST Some examples of antibiotic sensitivity testing methods are:  Dilution method (broth and agar dilution method)  Disk-diffusion method  E-test  Automated methods  Mechanism-specific tests such as beta-lactamase detection test and chromogenic cephalosporin test  Genotypic methods such as PCR and DNA hybridization methods