Antibiotic Resistance

Antibiotic Resistance An investigation of the mechanisms of antibiotic resistance and how this medical phenomenon can be combatted in an efficient and feasible manner. Charles Jian, Navneet Natt, Judy Nguyen, Ankita Prabhakar, Swathi Palepush, Jude

The aim of this investigation is to learn about the causes and implications of antibiotic resistance in respect to science and medical research . Over the course of this study, the following questions will be kept in mind: • What causes certain bacterial species to become resistant to antibiotics? • Given current technology, how can antibiotic resistance be combatted? • Antibiotic resistance is not just a local problem; it is a rising concern on a world-wide scale. What are the exact global ramifications of this issue? • How will this issue effect scientific research and development in the long-run?

Introduction Bacteria are small prokaryotic organisms of the Monera kingdom. Bacteria are generally found in small strains that reproduce rapidly and frequently through asexual means such as binary fission. Many bacterial species are beneficial for the body and are naturally found in areas such as the intestines and digestive tract. Probiotic cultures such as Lactoba-cillus acidophilus live in symbiosis with the body. As a matter of fact, only 10% of bacteria are deemed pathogenic. They secrete toxins (chemicals) which can disrupt bodily function and create infections.

Purpose and Function of Antibiotics • Antibiotic – drug used to kill pathogenic bacteria that cause infections • Synthesized from microbes (naturally-occurring substances – i.e. Penicillin is derived from Penicillium) • Antibiotics work in one of two ways. They can: a) Dismantle the vital functions of a bacterium cell causing it to die b) Stop further bacterial growth by preventing the cells from reproducing

Purpose/Functions of Antibiotics (Cont.) • Broad-spectrum antibiotics target a wide range of bacteria • Narrow-spectrum antibiotics act against a particular bacterial species • Antibiotics can attack the: -Cell Wall -Ribosomes -Cell Membrane -Nucleic Acids

Antibiotics – Mode of Action

Cell Wall Synthesis Inhibitors Antibiotics such as penicillin belong to a class of antibiotics called Beta-lactams The cell wall is a vital physical component in bacteria Peptidoglycan – Molecule that makes up the rigid and stable cell wall structure in bacteria B-lactam enzymes prevent bacteria from producing peptidoglycan, thus inhibiting cell wall formation Lysis can occur. This is rupturing of the cell wall thus causing death to the cell

Cell Wall Synthesis Inhibitors

Protein Synthesis and Cell Membrane Inhibitors Tetracyclines are antibiotics that prevent bacteria from making essential proteins and amino acids Attach onto ribosomes Disrupt the process of protein synthesis – mRNA and tRNA can no longer create coded polypeptide chains Some antibiotics may simply bind onto phospholipids in the cell membrane causing damage to the external cellular structure Examples include polymyxins

Nucleic Acid Synthesis Inhibitors and Anti-Metabolites DNA Gyrase is an enzyme that allows the DNA structure to unzip so that it can be duplicated Quinolones prevent DNA replication from occurring by binding onto bacterial DNA gyrase Other antibiotics inhibit RNA synthesis by binding onto RNA polymerase – an enzyme that produces ribonucleic acids Metabolic activity within the bacterium can also be disrupted by attacking particular enzymes within the cell

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.

Antibiotic Resistance • After continuous exposure, a bacterial colony will be unaffected by the use of antibiotics • Strains of bacteria develop immunity to certain antibiotics thus reducing the effectiveness of the drug • Bacteria develop resistance through natural selection and are able to transfer resistance genes through gene transfer or asexual reproduction

When bacterial strains are attacked by antibiotics, mutant bacteria will survive exposure to the drug and continue to thrive As the mutant bacteria reproduce, this resistance trait will be inherited by the offspring This creates a generation of bacteria that are fully resistant to the antibiotic Relates to Darwin’s theory of natural selection – survival of the fittest and inheritance of attributes that will ensure survival of a species MUTATIONS AND NATURAL SELECTION

Bacteria species can transfer resistance genes to one another through a process of horizontal gene transfer known as conjugation • Plasmids are strands of free floating genetic information found in • prokaryotic organisms • The F factor (fertility factor) is the plasmid that will be • transferred between two bacterium cells. The plasmid will code • for resistance against a particular antibiotic • The donor cell containing the F factor is known as • being F+ while the recipient is said to be F- • A bridge (also known as a pilius) forms between the two • bacteria and the plasmid is transferred • Both bacteria now contain the F factor (F+) and are resistant

H O R I Z A N T A L T R A N S F E R G E N E

Follow the link below to view an animation demonstrating conjugation within infectious bacteria: http://www.hhmi.org/biointeractive/animations/conjugation/conj_frames.htm

ULTIMATELY ... As the use of antibiotics increases on a global scale, so does the risk of antibiotic resistance. Through the natural phenomenon of evolution, bacterial species will adapt to the effects of the medicine and these traits will be passed on to future generations of bacteria. This poses a great concern in the health and pharmaceutical industry!

Things to Consider SO IN THE GRAND SCHEME OF THINGS ... What is the real problem – the development of antibiotic resistance or the prescribing of antibiotics in the first place? It is quite clear that prescribing antibiotics often proves to be ineffective when combatting bacterial infections. It is, however, also true that without administering these medicines a patient would simply fall more ill. Taking all of this into account, is there truly a way to cure bacterial infections? Is there a trade-off present?

How are humans contributing to antibiotic resistance? • Poor medicinal practises In nations with under-qualified health professionals it is common for antibiotics to be wrongly prescribed. This may be due to uncertainty, lack of knowledge, or simply to “be on the safe side” • Overuse of antibiotics Generally people feel re-assured by taking antibiotics when they are sick, thinking that they will get better. In many developing countries, antibiotics are commercially available as over-the-counter drugs. Sometimes patient complaints will lead doctors to prescribe antibiotics despite their discretion. • Incorrect dosages Patients often discontinue antibiotics before their medication is done because they start feeling better. When this happens, surviving bacteria will reproduce to create another infection, but this time they may be tolerant to the administered antibiotic.

film Follow the link below to watch a brief video explaining antibiotic resistance: http://www.nlm.nih.gov/medlineplus/videos/news/antibiotics_100909.html

Inadequate reserve of antibiotics to treat certain conditions, diseases, and maladies Need to find new treatment options Implications of Antibiotic Resistance Global and Local Possibility of multi-resistance arising Ineffective medicines and increase in the number of diseases that do not respond to medicinal treatment Increased risks of infectious epidemics

Global Pandemic of Antibiotic Resistance • Vital Components of modern medicine will be threatened if antibiotic resistance is not tackled urgently. A global response is needed to address the rising rates of bacterial resistance caused by the use and abuse of antibiotics. Otherwise, we may return to the “per antibiotic era”. • This indicates that the effectiveness that antibiotics provide will no longer be sustainable towards diseases and other illnesses. • All antibiotic use uses up some of the effectiveness of those antibiotics, diminishing its capability for use in the future. • Antibiotics are losing their effect at an alarming pace, while the development of new antibiotics is declining. • According to the World Health Organization, the most important disease threat is from micro organisms that have become resistant to antibiotics. • One of many reasons for the rapid decline in the effectiveness of antibiotics is that antibiotics are most often over prescribed.

Antimicrobial Resistance • Since their discovery during the 20th century antimicrobial agents (antibiotics and relating medicinal drugs) have reduced the threat posed by infectious diseases. • Infections caused by resistant microbes fail to respond to treatment, resulting in a prolonged illness and a greater risk for death. • Not only in developing countries but, developed countries as well face the consequences of antimicrobial resistance. • Immunity to a certain microbe and treatment failure lead to longer periods of infectivity which increases the number of infected people moving into a community and thus exposes the general population to the risk of contacting the illness • The risk is greater in developing nations due to the hygienic conditions and as well, in developed nations due to the vast population. • In developing nations such as certain parts of Africa, if first line drugs are overused and hence ineffective, treatment options have to be switched to second or third line drugs. • These second and third line drugs are more expensive and sometimes more toxic as well. • In developed nations, the overuse of even these drugs may take us into a position in which no drugs work. • In some countries, the high costs of some of these drugs is prohibited and sometimes result in the prolonged state of diseases. • Even if pharmaceuticals industry were to step up efforts to develop new replacements drugs immediately, the main ingredients used to create high performance antibiotics may no longer be effective. • Certain trends suggest that some diseases will have no effective therapies within the next ten years.

Scientists believe that priority must be given to the most urgently needed antibiotics which are vital for the recovery of certain diseases. • In addition, the use of antibiotics must be safe guarded by regulations and practices that ensure rational use. • This is so that in the future we avoid the problem of overusing the old ones. • When a body is introduced to the same antibiotic several times, the body adjusts to its effectiveness, hence decreasing its capability for use in the future.

Unprecedented Trends • In the past, medicine and science were able to stay ahead of this natural phenomenon through the discovery of potent new class antibiotics. Though, resisting microbes have severely increased due to some trends: • Urbanization with its associated overcrowding and poor sanitation, which greatly facilitates the spread of such diseases as tuberculosis. • Pollution, environmental degradation and changing weather patterns, which can affect the incidence and distribution of infectious disease; especially those such as malaria that are spread by insects. • The AIDS epidemic, which has greatly enlarged the population of immune compromised patient at risk of numerous infections, many of which were previously rare. • The enormous growth of global trade and travel which have increased the speed and facility with which both infectious diseases and resistant microorganisms can spread between continents. • As the number of corresponding antimicrobials have increased, so have the prevalence of resistance.

China, United States and Kuwait CHINA • With globalization booming, it is important to understand international patterns of resistance. A study was conducted in three countries which recent patterns of antibiotic resistance. • China was found to have the highest levels of antibiotic resistance. • In a study of common bacteria in China in 1999 and 2001, the mean prevalence of resistance among hospital acquired infections was as high as 41% and among that, community acquired infections was 26%. • China also has the most rapid growth rate of resistance; probably due to its vast population. • Measures have determined that hospital acquired infections are more resistant to antibiotics.

United States According to laws of Darwinism evolution, antimicrobial use creates a selection pressure on microorganisms: weak ones are killed but stronger ones may adapt and survive. Scientists have established that once a single resistance mechanism is detected, it can often allow a bacterium to resist multiple drugs. It remains unclear whether this can or cannot be reversed. The average growth rate of resistance was 8%, 15% lower than what China concluded. This may be due to the hygienic conditions of where the study took place. China has a vast number of people living in its country and hence the environment may not be as clean and communities maybe overpopulated.

There is considerably less detailed data on the antibiotic resistance in Kuwait. • Data from one single major teaching hospital concludes that the average resistance levels for all surveyed bacteria was about 27% from 1999 to 2003, higher than the 17% for the US and about the same as China, 28%. • Nonetheless, resistance seems to be growing in Kuwait, a developing country. • It is important to realise that resistant rates seems to be linked to the medicine itself. It has to determine whether the particular antibiotic has reached a potential equilibrium. The nature of antibiotic resistance is a global pandemic and if not treated quickly, it may be a untreatable issue for the future. Both developed and undeveloped nations face the same problem; how do we combat the use and abuse of antibiotics. Kuwait

Need for a Global Response • In September 2001, WHO launched the first global strategy for combating the serious problems caused by antimicrobial resistance. It is known as the WHO Global Strategy for the Containment of Antimicrobial Resistance. • The strategy recognizes that antimicrobial resistance is a global problem that must be addressed in all countries. • No single nation effective at containing the resistance within its own borders can protect itself from the importation of resistant through travel and trade- poor prescribing practices in any country now threaten to undermine the potency of vital antimicrobials everywhere. • The strategy recommends interventions that can possibly be used to slow the emergence and reduce the spread of resistance. • Organization need to be established in each nation so that they can act according to groups of people whose practices and behaviours contribute to resistance and where changes are likely to have significant impact at a both international and national level • The groups of people include consumers, prescribers, dispensers, veterinarians, managers of hospitals and diagnostic laboratories.

Global Implications of Antibacterial Resistance • The World Health Organisation has stated “As a result we are facing the possibility ofafuture without effective antibiotics. This fundamentally changes the way modern medicine is practised.”Increasing use of antibiotics in human and animal populations has led to evolution of resistant microbes. There is a growing body of evidence to indicate that, in important respects, this battle against resistance is being lost. Socio-economic impact • It has been estimated that in the EU each year there are more than two million hospitalised patients with nosocomial infection and, perhaps 175,000 deaths from infection. • A significant proportion can be attributed to antibacterial resistance, which is an increasing problem in the community as well as in hospitals. • In 1998, the Institute of Medicine estimated that the total cost to US society of antibacterial resistance was at least $5 billion annually.

What is currently being done to combat this problem?

Heightening awareness of the problem – to policy-makers, health professionals and the general public. • Improved surveillance – better understanding and monitoring of the different types and amounts of bacterial resistance across the EU. This requires introduction of a common, standardised methodology to map resistance. • Prudent antibiotic use – in both human and veterinary medicine, utilising evidence-based measures. • Containing the spread of resistance - rigorous and consistent implementation of infection control methods in hospitals and the community. • Coordination – action to build collaboration and coherence in data collection, intervention strategies and policy development at Member State and EU levels. Long Term support for research and innovation However, the public health measures of antibiotic reduction, resistance surveillance and containment are not sufficient. There is also a longer-term need for sustained commitment to do research and development, which includes: • Develop novel diagnostics – rapid, simple and cheap to use at point of care, differentiating bacterial from viral infections and determining resistance profiles. • Strengthening the science base – to train the next generation in microbiology and clinical infectious disease, to understand the determinants of prescribing habits and spread of resistance, to add value to hospital-university research partnerships, and – of vital importance - to identify novel drug targets. Short term actions

Global Strategies to Combat Antibacterial Resistance • In the global scale, the World Health Organization’s Communicable Disease Surveillance and Response Branch (CSR) serves a pivotal role to take action against the antibacterial problem. • It tries to strengthen national surveillance programs by establishing international links among monitoring projects. • It also sponsors projects to support development of national programs to contain the spread of antibacterial resistance. • It also developed an antimicrobial resistance information bank (InfoBank) on surveillance activities. • InfoBank is addressed to policymakers and aims to persuade governments to take urgent action for combating antibacterial resistance. • The Alliance for the Prudent Use of Antibiotics is an organization that is also committed to reducing antimicrobial resistance on a worldwide level through the prudent use of antibiotics. • This organization provides an international forum for the exchange of ideas and brings together representatives of surveillance activities worldwide for collaboration in establishing coordinated global surveillance systems.

Many investigations have been performed to find ways of combating antibiotic resistance. However, many of the methods investigated had minimal to no effect on combating this issue. Some of the methods that have been recently discovered include: • The use of engineered viruses • These engineered viruses attack the SOS system in our body which is a bacterial DNA repair system which is activated when bacteria are exposed to certain antibiotics that cause damage in the DNA. This method has seen to be the most effective when it is used with antibiotics as it helps prevent resistance from occurring. • Bacteriophage therapy • Bacteripohage are viruses that infect bacteria. “Bacteriophages take over the host’s protein-making machinery, directing the host bacteria to make viral proteins of their own.” (Kardar) • Bacterial interference (also known as bacteriotherapy) • “Bacteriotherapy is the practice of deliberately inoculating hosts with nonpathogenic (commensal) bacteria to prevent infection by pathogenic strains.” (Kardar)*This is helpful as the virus creates an infection without stimulating the host’s immune system which helps limits the selection of antibiotic resistance. CombattingAntibiotic Resistance

Future Approaches to Combat Antibiotic Resistance

Bacterial Interference / Bacteriotherapy • To inject the host with nonpathogenic bacteria to prevent infections caused by pathogenic strains. • Nonpathogenic bacteria are also known as commensal bacteria. • In order to cause an infection or initiate a disease, the pathogenic bacteria must seek nutrients and attach itself to various sites. • These commensal bacteria compete with the pathogenic bacteria directly for nutrients and adhesion receptors (attachment sites). • This practice is advantageous because an infection can be evaded without affecting the host’s immune system. • Positive results are mainly found in gut infections, wound sites and urogenital tracts.

BACTERIOPHAGE THERAPY • Bacteriophages are viruses that infect and destroy bacteria cells • These phages take control of the host’s protein-making system as they instruct the host bacteria to make their own viral proteins • With the use of bacteriophages, pathogens can be targeted through phage DNA manipulation • Bacteriophages are highly looked at due to its natural mutational and exponential growth abilities in which would aid in reducing bacterial resistance • Phage particles are narrow spectrum agents, meaning they carry an intrinsic system allowing them to not only infect bacteria but other particular strains

Diagram of a bacteriophage

BACTERIAL VACCINES • Drugs are often developed when a bacterial genome scan takes place • In this process, specific sequences of the DNA are identified and used to stimulate a defensive response of the immune system against certain bacterial strains • This practice is considerably more practical and target-based

Cationic Peptides • These peptides contain both hydrophobic and hydrophilic properties and are found in immune systems of bacteria, plants, invertebrates and vertebrates. • Previously coevolved with commensal bacterial as well as possess characteristics against pathogenic bacteria • They encounter bacterial membranes, specifically the outer membrane and the cytoplasmic membrane

Outer Membrane • Interacts with the negatively charged side, believed to displace the magnesium ions in the bacteria (magnesium ion serves its purpose in neutralizing the charge of the membrane) • Cationic peptides alter the membrane structure by directly attaching itself to the negatively charged membrane lipopolysaccharide (known as LPS) or neutralize the charge over an area of the membrane • The peptides then transfer across the outer membrane

Cytoplasmic Membrane • This membrane is also highly negatively charged which allows the peptides to insert itself parallel to the membrane lipids and fold into the membrane-bound structure • Ultimately, this kills the bacterial cell because the peptides attack various sites present of cellular polyanions such as nucleic acids once it has penetrated into both membranes

The different ways bacteria adapt to antibiotics (see diagram) can be countered directly. That is, using, for example, a beta-lactamase inhibitor in conjunction with an antibiotic based around the beta-lactam ring. The inhibitor would prevent the enzyme from breaking down the antibiotic.

However, when bacteria find alternate ways around the antibiotic, such as an alternate metabolic pathway, it may be difficult to a) isolate the new change, and b) implement drugs to attack the new pathway. As well, the bacteria may not have the same alternate pathways, further contributing to the difficulty of countering this method. Efflux pumps pump the antibiotic substance out of the cell before it can cause any damage. A pump inhibitor, perhaps following the same concepts as the proton pump inhibitor, may counteract the effects of the efflux pumps. Sadly, these solutions are only temporary; inevitably the bacteria will develop new ways around these methods, such as a beta-lactamase-inhibitor-inhibitor, or an enzyme to break down the pump inhibitor, etc. As well, since most drugs must be taken orally, drug designers must take into account the effects of the human metabolism on the drug, as well as side effects the metabolism may cause. This also affects bioavailability of both the drug and the countering substance. Is there a way to increase the bioavailability of a drug?

Nanotechnology? Current antibiotics are administered via oral (most often) or intravenous solution. This spreads the antibiotic throughout the entire body, and may contribute to antibiotic resistance by killing off commensal bacteria (in the gut, for example). It also causes unwanted side effects, for example gastro-intestinal problems such as diarrhea if the commensal bacteria in the intestines are killed. A “solution” would be to find a more accurate way of targeting where to deliver the antibiotic; if the infection is localized, for example in the case of respiratory infections, urinary tract infections, etc., then precisely delivering the drug to where it is needed would minimize drug interference by and to the other parts of the body. How can this be accomplished?

Perhaps we can enclose the drug in a buckyball (fullerene), (made of carbon, and not damaging to the body) where it is chemically inert until it reaches the site of the infection. An ultrasound pulse or an electric shock or some other impulse can be used to break open the fullerenes, and release the drug directly to the site. This drastically improves bioavailability of antibiotic; new avenues of antibiotics can be developed that do not have to take into account the body metabolizing the drug first.

Continuous Culture The majority of the antibiotics we use today are derived from other organisms, be they fungi or even other bacteria. One way to “counteract” drug resistance is to culture another antibiotic-producing organism in conjunction with the resistant bacteria. For example, the bacteria Streptomyces griseus produces streptomycin, which Escherichia coli can be resistant to. We can provide environments for these two organisms to compete, and let evolution do its work.

Although it is likely that many of the S. griseusenvironments will succumb to competition from E. coli, some will inevitably succeed in developing a new substance to counteract thedrug resistant E. coli. The “failed” trials can be disposed of via UV light, or combustion (for example). The new substance can be “mass-produced” by isolating the gene that produces the antibiotic, then using bacteriophages to splice the gene into other bacteria. The substance can then be tested for effectiveness and harmful side effects on humans. Eventual trial-and-error can develop a new drug. The downside of this method is the time and resources involved for a new drug, which bacteria may become resistant to in a short period of time. For now, it probably is not economically feasible.

FINAL NOTE Antibiotic resistance cannot be solved by a “miracle drug”. It is caused by evolution, the process of natural selection, which we cannot hope to stop. The primary way to prevent resistance is, as always, to take the drugs prescribed correctly, and to raise public awareness about the importance of doing so. As well, drug disposal should also be more regulated; many “expired” pills are simply poured down the drain. Until hunter-killer nanomachines, for example, can independently select and hunt down rogue bacteria (which may create some unexpected adaptations), we can only try to stem back the tide by continuously developing new substances to use against these persistent organisms.

DID YOU KNOW • Antibiotics have an annual market ranging from 7 to 22 billion dollars, classifying it as the third largest selling type of drug. • An estimation of approximately 4 to 5 billion dollars of the total amount show to be antibiotic-resistant bacteria

Antibiotic Resistance