Microbio exam 3

Introduction

Antibiotics have played a major role in increasing life expectancy throughout the world. But antibiotics may soon become useless.-Their overuse and misuse have led to the development of antibiotic-resistant strains. This is especially true in hospital settings.-Indeed, as many as 60%-70% of nosocomial staphylococcus infections are the result of methicillin-resistant S. aureus (MRSA). Drug resistance is a problem that will only get worse.

ch.27 pt.3 and pt. 4

Antibiotics interfering with DNA include the antimetabolite sulfa drugs that inhibit nucleotide synthesis; quinolones that inhibit DNA topoisomerases, and a drug, metronidazole, that, when activated, randomly nicks the phosphodiester backbone. Inhibitors of RNA synthesis target RNA polymerase (rifampin and pyronins) or bind DNA and inhibit polymerase movement (actinomycin D) Different protein synthesis inhibitors target prokaryotic ribosomes. Some target the 30S subunit to cause misreading of mRNA (aminoglycosides) or prevent tRNA binding (tetracyclines). Others target the 50S subunit to inhibit translocation (macrolides, lincosamides), peptidyltransferase activity (chloramphenicol), formation of the 70S complex (oxazolidinones), or peptide exit through the ribosome exit channel (streptogramins). Still others targe aminoacyl-tRNA synthetases (mupirocin) to prevent charging of tRNAs.

Target - membranes

Antibiotics targeting membranes - Platensimycin (FA biosynthesis) - Polymixin (detergent), Daptomycin (ion channel, leaks K+)

How Does Drug Resistance Develop?

Can be acquired via horizontal gene transfer - generally thinking of plasmid transfer: -Conjugation -Transduction -Transformation -Integrons ----Integrons are assembly platforms that incorporate exogenous open reading frames through site-specific recombination and convert them to functional genes by ensuring their correct expression. -Effectors

How do bacteria acquire antibiotic resistance?

De novo - Mutations, duplications - Inherent for their protection HGT - Transformation - Conjugation - Transduction

Broad spectrum

Effective against many species

Antibiotic effectiveness imperiled as use in livestock expected to increase 67 percent by 2030

Increase due to growing # of animals raised for food, and the shift in farming practices towards intensive farming systems or factory farms.

Drugs That Affect the 50S Subunit (inhibit translocation etc)

Inhibit translocation: - Macrolides: Erythromycin and azithromycin - Lincosamides: Clindamycin - Streptogramins (cyclic peptide antibiotic) -Streptogramin A: blocks tRNA binding -Streptogramin B: blocks translocation - Blocks peptidyl transferase - Chloramphenicol: Prevent formation of the 70S ribosome initiation complex - Oxazolidinones: synthetic, broad spectrum erythromycin - Broad-spectrum antibiotic - Bacteriostatic: 20% of total world production of antibiotics (macrolides) - Virgiamysin and tylosin

tricoslan review

- ppGpp activator! - Growth inhibitor - Alters human hormone regulation = Makes other antimicrobials less effective, contributes to antibiotic resistance

How Does Drug Resistance Develop DeNovo?

ACTS ON CELL POPULATIONS - GROWTH AND SURVIVAL ADVANTAGE •Spontaneous, random mutations or induced mutations •Huge numbers of bacteria in a population •Constant rates of mutation - spontaneous resistance to a given antibiotic is one out or 107 cells •Innate

Antibiotic Resistance

About 2.8 million people in the United States are sickened each year by bacterial infections that resist conventional antibiotics 0 and at least 35,000 people die And, 223,900 cases of Clostridiodes difficile occurred in 2017, and 12,800 people died - according to the U.S. Centers for Disease Control and Prevention. (Oct 28, 2020) The World Health Organization called antibiotic resistance "a problem so serious it threatens the achievements of modern medicine."

prevent antibiotics from binding the target

Add modifying groups that inactivate the antibiotic - aminoglycosides -Three classes of enzymes are used to modify and inactivate the aminoglycoside antibiotics. -Acetyltransferase (AAC), phosphotransferase (APH), adenyltransferase (ANT) Modify the target so that it no longer binds the antibiotic. - Mutations in key penicillin-binding proteins and ribosomal proteins confer resistance to methicillin and streptomycin, respectively.

Potential new antibiotic made by an insect-linked microbe

Because microbes often generate antibacterial compounds, they present a potential source of antibiotics that humans can use. Soil microbes have already helped us create some of our best medicines. Now researchers have found another interesting source of antimicrobial compounds - bacteria that are associated with insects. - Better than soil microbes at stopping pathogens -They shield their hosts from infection New antibiotic called Cyphomycin, from a Streptomycesin the ant's microbiome - From Brazilain ant that farms fungus - Works against drug resistant fungus

The search for new antibiotics

As of June 2019, approximately 42 new antibiotics with the potential to treat serious bacterial infections are in clinical development. The success rate for clinical drug development is low; historical data show that, generally, only 1 in 5 infectious disease products that enter human testing (phase 1 clinical trials) will be approved for patients

Finding new antibiotics

Brute-force screening of microbes, plants, and animals Combinatorial chemistry Genome sequence analysis to identify potential bacterial molecular targets Photosensitive chemicals Interfering with quorum-sensing mechanisms CRISPR-based strategies for reversing antibiotic resistance Until recently, however, the United States has spent less than $5 million per year on conserving antibiotic effectiveness through public education, research, and surveillance, and even with recent increases in allocations to antibiotic resistance programs at the U.S. Centers for Disease Control and Prevention, the imbalance remains. As long as we inhabit a world of microbes, we must focus more resources and effort on the conservation of existing antibiotics by ̵ensuring their appropriate—and less frequent— use - while finding ways to incentivize the development of new antibiotics - using mechanisms that promote appropriate use Automated chemistry methods (combinatorial chemistry) has sped up drug discovery - 7,000,000 compounds must be screened to find a single useful clinical drug - Low hanging fruit has already been tested - Most antibiotics found this way are the same class and effectiveness

Urgent threats

Carbapenem-resistant Acinetobacter Candida auris Clostridioides difficile Carbapenem-resistant Enterobacteriaceae Drug-resistant Neisseria gonorrhoeae For CR Acinetobacer: - Estimated cases in hospitalized patients in 2017: 8,500 - Estimated deaths in 2017: 700 For CRE: - Estimated cases in hospitalized patients in 2017: 13,100 - Estimated deaths in 2017: 1,100

ch.27 pt 6 review

Bacteria can acquire resistance de novo, or via HGT Evolution acts on populations The loss and acquisition of functional modules are an important part in the processes of rapid bacterial adaptation and development of resistance. -Conjugation -Transduction -Transformation Integrons Effectors by injection

Resistance to beta-lactam antibiotics

Bacteria develop resistance to penicillin and other beta-lactam antibiotics in two ways .1) Inheritance of a gene encoding a beta-lactamase enzymethat cleaves the critical beta-lactam ring of beta-lactam antibiotics 2) Inheritance of a gene encoding an altered transpeptidase that no longer binds beta-lactam antibiotics

Antibiotics Are Classified as Bacteriostatic or Bactericidal

Bactericidal antibiotics kill target organisms. -Many drugs only affect growing cells. -Inhibitors of cell wall synthesis -Only effective if organism is building new cell wall -Example: penicillin Bacteriostatic antibiotics prevent growth of organisms. -Cannot kill organism -Immune system removes intruding microbe

global TB report

Global burden: The latest anti-TB drug resistance surveillance data show that 4.1% of new and 19% of previously treated TB cases in the world are estimated to have rifampicin- isoniazid - or multidrug-resistant tuberculosis (MDR/RR-TB). About 6.2% of MDR-TB cases have additional drug-resistance, extensively drug-resistant TB (XDR-TB). - First appeared in 2006, •In 2009, a cohort of 15 patients in Iran was reported which were resistant to all anti-TB drugs tested

Serious threats

Drug-resistant Campylobacter Drug-resistant Candida ESBL-producing Enterobacteriaceae Vancomycin-resistant Enterococci (VRE) Multidrug-resistant Pseudomonas aeruginosa Drug-resistant nontyphoidal Salmonella Drug-resistant Salmonella serotype Typhi Drug-resistant Shigella Methicillin-resistant Staphylococcus aureus (MRSA) Drug-resistant Streptococcus pneumoniae Drug-resistant Tuberculosis

World-wide production and use of antibiotics

Each year ~ 10,000 metric tons of antimicrobial agents are manufactured worldwide. b-Lactams include: - Cephalosporins, penicillins, carbapenems and others - "Other" includes: Tetracycline, aminoglycosides

Quinolones

nalidixic acid, ciprofloxacin - Block bacterial topoisomerases such as DNA gyrase, and topoisomerase IV -And so prevent DNA replication Broad Spectrum - Effective against both gram positive and gram negative bacteria - Used to treat urinary tract infections - Drug of choice for treating anthrax - Moxifloxacin - treatment of TB (gyrase resistant mechanisms developing) Quinolones also used to treat beef and poultry respiratory diseases

Antipersister and antibiofilm approaches

Kill persisters directly Prevent persister formationInterfere with biofilm formation Induce biofilm dispersal The progression of these strategies has been slow because the road to FDA approval, though necessary for safety reasons, is long (8 to 10 years) and expensive, and it can discourage some companies from investing in antibiotic discovery.

Measuring Drug Susceptibility

One critical decision a clinician must make when treating an infection is which antibiotic to prescribe for the patient. There are several factors to consider. - The relative effectiveness of different antibiotics on the organism causing the infection. - The average attainable tissue levels of each drug. - The route of administration.

Classification

StructureActionSpectrumsource

Protein Synthesis Inhibitors cont.

The differences between prokaryotic and eukaryotic ribosomes account for the selective toxicity of antibacterial antibiotics. - Recall that protein synthesis inhibitors can be classified into several groups based on structure and function. Many of these antibiotics work by binding and interfering with the function of bacterial rRNA. In general, protein synthesis inhibitors are bacteriostatic (not bactericidal). - What about the ribosomes in mitochondria?

DeNovo mutations and HGT

The presence of drug does not cause resistance, but it will kill off or inhibit the growth of competing bacteria that are sensitive. - The resistant microbe can now grow to high numbers. - Resistance genes can then be horizontally transferred to other bacteria, some of which may be pathogens. Many of these situations have conspired to produce incredibly dangerous bacteria resistant to almost every antibiotic known. ESKAPE pathogens, Term coined by the Infectious Diseases Society of America, Six highly resistant bacterial species that collectively cause about two-thirds of all U.S. nosocomial infections Enterococcus faecium Acinetobacter baumannii Staphylococcus aureus Pseudomonas aeruginosa Klebsiella pneumoniae Enterobacter sp.

Overuse, over prescribed

The presence of drug does not cause resistance, but it will kill off or inhibit the growth of competing bacteria that are sensitive. -The resistant microbe can now grow to high numbers.

How Did We Get into This Mess?

The presence of drug does not cause resistance, but it will kill off or inhibit the growth of competing bacteria that are sensitive. -The resistant microbe can now grow to high numbers. -Resistance genes can then be horizontally transferred to other bacteria—some of them pathogens. Another proposed source of antibiotic resistance is the widespread practice of adding antibiotics to animal feed. -Feeding growth-promoting antibiotics to cattle can also stimulate the spread of pathogenicity genes between bacteria.

The greatest challenge to antibiotic development

the emergence of resistance- during clinical development! *** also a lack of global monitoring

Antimicrobial drugs are classified on the basis of

•Molecular structure •Mechanism of action •Spectrum of antimicrobial activity •Broad spectrum •Narrow spectrum •Targeted pathogen •Bacteriostatic, bactericidal •Antiviral, antifungals •Source of antibiotic •Natural, synthetic, semi-synthetic •Modified antibiotics increase efficacy and (should) decrease toxicity to humans

Antimicrobial Drug Resistance cont.

•Most drug-resistant bacteria isolated from patients contain drug-resistance genes located on R plasmids - Evidence indicates that R plasmids predate the antibiotic era •Almost all pathogenic microbes have acquired resistance to some chemotherapeutic agents •A few pathogens have developed resistance to all known antimicrobial agents •Resistance can be minimized by using antibiotics correctly and only when needed •Resistance to a certain antibiotic can be lost if antibiotic is not used for several years

Practical Considerations of Differences in Cell Envelope Structure

•Outer membrane of Gram negative cells- Makes them impervious to some antimicrobial chemicals - Generally gram-negative bacteria are more difficult to inhibit or kill than gram-positive bacteria •Cell envelope can interact with human tissues and cause disease

Triclosan

Triclosan is an ingredient added to many consumer products intended to reduce or prevent bacterial contamination. - It is added to some antibacterial soaps and body washes, toothpastes, and some cosmetics—products regulated by the U.S. Food and Drug Administration (FDA). - It also can be found in clothing, kitchenware, furniture, and toys—products not regulated by the FDA. When you use a product containing triclosan, you can absorb a small amount through your skin or mouth. - A large 2008 study, which was designed to assess exposure to triclosan in a representative sample of U.S. children and adults, found triclosan in the urine of nearly 75 percent of those tested. A study reported 2,000 or more products are thought to contain triclosan, and it is also found in the environment. - Another study estimated that 96% of triclosan was used in products that end up down the drain, such as soaps and detergents. CDC detected triclosan in 58% of US Waterways.

narrow spectrum

-Effective against few or a single species

Darwinolide

- Diterpene molecule - Extracted from the Antarctic sponge Dendrilla mebranosa - Ability to kill biofilms of Methicillin-resistant Staphylococcus aureus (MRSA) Kills established MRSA in biofilms better than free-swimming (planktonic) MRSA - Prediction that this antibiotic structure could provide a unique scaffold upon which more potent anti-MRSA therapeutics can be built - Products from nature have excellent therapeutic properties!

The following aspects of a microbe's physiology are classic targets:

- cell wall synthesis -Cell membrane integrity -DNA synthesis -RNA synthesis -Protein synthesis

Dislodge an antibiotic already bound to its target.

- Ribosome protection (or rescue). Gram-positive organisms can produce proteins that bind to ribosomes and dislodge macrolide antibiotics bound near the peptidyltransferase site. Ribosomes don't operate at maximum activity RPPs bind directly, and reversibly - inhibit drug activity without permanently altering ribosomal activity RPPS can prevent drug binding or dislodge drugs that target either the 50S or 30S subunit

peptiodglycan synthesis

-However, it may be summarized in these four steps: 1.Precursors are made in the cytoplasm by the mur family of proteins. -UDP-NAG and UDP-NAM-peptide 2.They are carried across the cell membrane by a lipid carrier: bactoprenol. -The carrier is then recycled. 3.The precursors are polymerized to the existing cell wall structure by transglycosylases. 4.The peptide side chains are cross-linked by transpeptidases.

The effectiveness of antimicrobial drugs is being compromised by several alarming trends:

-Inappropriate prescriptions (viral infections) -Use of broad-spectrum instead of narrow-spectrum drugs (shotgun approach) -Use of higher cost drugs -Sale of over-the-counter antimicrobials in other countries (made in USA; w/o prescription in over 200 countries) -Over-use in livestock and poultry industry -Lack of sufficient testing before prescription

Sulfa drugs

-Known as anti-metabolites -Analogs of PABA, a precursor of folic acid -Tetrahydrofolic acid (THF) is an important cofactor needed for synthesis of nucleic acids. -Selective toxicity? High Ti? -Folic acid is supplied in our (human) diet. -Thus no folic acid synthesis to inhibit

There are three basic strategies for antibiotic resistance.

1.Keep antibiotics out of the cell. 2.Prevent antibiotics from binding the target. 3.Dislodge an antibiotic already bound to its target.

Clinically useful antibiotics

A clinically-useful antibiotic should have as many of these characteristics as possible. It should have a wide spectrum of activity with the ability to destroy or inhibit many different species of pathogenic organisms. It should be nontoxic to the host and without undesirable side effects. It should be non-allergenic to the host. It should not eliminate the normal flora of the host. It should be able to reach the part of the human body where the infection is occurring (delivery and access). It should be inexpensive and easy to produce. It should be chemically-stable (have a long shelf-life). Microbial resistance is uncommon and unlikely to develop.

Triclosan cont.

Alters hormone regulation in animals - Human reproduction and development. Animal studies have linked triclosan to lower testosterone levels and less sperm production = developmental and reproductive toxicity. - Estrogen, androgen and thyroid hormone disruption. It also may affect the thyroid, which produces hormones essential to brain development in children. Abnormal endocrine system/thyroid hormone signaling - The estrogenic nature of triclosan allows it to bind to estrogen hormone receptors - may stimulate the growth of estrogen dependent breast cancer. - Reduces fertility. May interfere with the production of sperm, and affects sperm health and testicular development Might be harmful to the immune system - Allergies and asthma. Animal and human studies suggest that triclosan may make people more sensitive to allergens May alter the Human microbiome - Gut bacteria. A study focused on triclosan in one particular type of product: toothpaste. Might contribute to the development of antibiotic-resistant germs - One study published by a team of researchers at Stanford University found that you are more likely to carry extra weight with repeated exposure to triclosan. - As the researchers explain, triclosan "has the potential to alter both gut microbiota and endocrine function and thereby affect body weight." Antibiotic and antimicrobial research:̵some research suggests that bacteria may become immune to triclosan over time. ̵That may make it and other antimicrobials less effective.

Antibiotic potential of Cannabis sativa

An antibacterial chemical cannabinoid compound - CBG - cannabigerol (non-psychoactive) that is effective against MRSA (staph infections) in mice 18 different cannabinoids tested, all showed some antimicrobial activity CANNABIGEROL prevented the ability of bacteria to form biofilms and destroyed pre-formed biofilms and with it cells resistant to antibiotics (persister cells) Targets cell membranes of bacteria - Works with Polymixin B However, also proved toxic to host cells! Therefore the therapeutic window in rather small until it can be modified to be more specific. Research has been done in Canada for several years since cannabis became legal

Metronidazole

An example of a prodrug—harmless until activated -Metabolized by cofactors ferredoxin and flavodoxin -Effective against anaerobic bacteria and protozoa-Broad spectrum

Antibiotics Exhibit Selective Toxicity

Antibiotic must affect target organism. -But it must not affect humans. -Many have side effects at high concentration. -Chloramphenicol interferes with our ribosomes. -At high levels, it interferes with RBC development. -Some may cause allergic response. -Antibiotics are foreign substances in our bodies. -Drug should affect microbial physiology. -That does not exist, or is greatly modified, in humans. -Peptidoglycan -Differences in ribosome structure -Biochemical pathway missing in humans TI- the larger the index, the safer is the drug (antibiotic) for human use (therapuetic index)

Rise of Drug-Resistant Bacteria

Antibiotic resistance is a growing problem worldwide. -Antibiotics are overused. -Overprescribed; used in farm animal feed This exerts selective pressure for drug-resistant strains. -Resistant to multiple drugs -Multiple resistant genes on plasmids -Often have mobility to chromosome or plasmids ---transposons

review ch. 27 pt 2

Antibiotic specificity for bacteria can be achieved by targeting a process present in bacteria but not host cells; by targeting small structural differences between components of a process shared by bacteria and hosts, or by exploiting a physiolocial growth condition unique to bacteria, such as anaerobiosis .Antibiotic targets include cell wall synthesis, cell membrane integrity DNA synthesis, RNA synthesis, protein synthesis, and metabolism. Antibiotics targeting the cell wall bind to the transglycosylases, transpeptidases, and lipid carrier proteins involved with peptidoglycan synthesis and cross-linking. Carbapenemases are a class of beta lactamases active against carbapenems Bacteria develop resistance to beta lactam antibiotics in 2 ways: Inheritance of a gene encoding a beta-lactamase enzyme that cleaves the critical beta-lactam ring of beta-lactam antibiotics or Inheritance of a gene encoding an altered transpeptidase that no longer binds beta-lactam antibiotics Gram negative bacteria have more means than Gram Positive bacteria to become antibiotic resistant, such as creating an outer membrane barrier (porins) and drug efflux pumps. Beta-lactam antimicrobials bind irreversibly to penicillin binding protein (PBP - transpeptidase) Penicillins, cephalosporins, carbapenems are beta lactam antibiotics, having a structure that resembles the D-ala-D-ala of peptidoglycan (transpeptidase uses this structure to cross-link peptidoglycan) There are many antibiotics that target the various and numerous processes of cell wall synthesis. - Cycloserine, bacitracin, vancomycin, penicillin, cephalosporins, carbapenems)

Fundamentals of Antimicrobial Therapy

Antibiotics are compounds produced by one microbe that adversely affects other microbes. -However, the term "antibiotics" is also used for synthetic chemotherapeutic agents that are clinically useful but chemically synthesized. We think of antibiotics as being a recent biotechnological development, but they have actually been used for centuries. -For example, ancient remedies for treatment of wounds

Challenges of Drug Resistance and Discovery

Antibiotics are considered secondary metabolites because they often have no apparent primary use in the producing organism. -Not essential for survival -Often on plasmids -But enhance ability to survive competition Microbes prevent self-destruction by means of various antibiotic resistance mechanisms. -Example: make enzymes to disable antibiotics -Genes encoding some of these drug-resistance mechanisms have been transferred to pathogens. -HGT

The politics of antibiotic resistance = Economic Barrier

Antibiotics are not a cost-effective investment ̵ Top five antibiotics combined $6 billion in 2010 - cholesterol pill Lipitor - $5.3 billion in US - Drugs are only used for weeks at a time; compared with years for medicines that treat chronic diseases (insulin) - Within a year some resistance will develop; Doctors will not prescribe as 20% of infections develop resistance - and now the advise is to limit antibiotic treatment 9 of 10 blockbuster drugs go off patent protection within 5 years - drug makers are seeking new prescriptions for profit.

Increasing Antibiotic Resistance in the World

Antibiotics have played a major role in increasing life expectancy throughout the world. But antibiotics may soon become useless. - Their overuse and misuse in medicine and farming have led to the development of antibiotic-resistant strains of microorganisms. This is especially true in hospital settings: 50%-60% of hospital-acquired (nosocomial) staphylococcus infections are caused by methicillin-resistant S. aureus (MRSA). Drug resistance is a problem that will only get worse over time.

RNA Synthesis Inhibitors

Antibiotics that inhibit transcription are bactericidal and most active against growing bacteria. Rifampin -Binds to the beta subunit of RNA polymerase -Prevents the elongation step of transcription -Treatment for tuberculosis and leprosy Actinomycin D -Prevents the initiation step of transcription -Binds to DNA from any source (intercalates) -Thus, not selectively toxic -Used as anti-cancer drug

Antibiotics by target

Cell Wall Cell membrane Nucleic acid synthesis - DNA - RNA - Metabolic pathway Protein Synthesis What are some other targets - potentially? - FtsZ? Divisiome? - MreB?

CRE (carbapenem resistant enterobacteriaceae)

Cell wall synthesis inhibitors - Vancomycin - Cycloserine - Bacitracin - Beta lactam antibiotics - target PBP Penicillins Cephalosporins carbapenems Etc - CARBAPENEMASES

Cephalosporin Generations (x linking in cell wall)

Cephalosporins(beta lactam antibiotic) •Produced by fungus Cephalosporium •Same mode of action as the penicillins •Broader spectrum •Typically more resistant to b-lactamases •Fewer allergic reactions •Modified in laboratory so are 'semi-synthetic' •Currently account for a majority of all antibiotics administered •generations of cephalosporins exist based on their antibacterial activity - "Cef, Ceph, Kef" •Commonly used to treat gonorrhea •ceftriaxone

review ch. 27 pt 5

Certain microbes make antibiotics to eliminate competitors in the environment and prevent self-destruction by means of various antibiotic resistance mechanisms. Genes encoding some of these drug resistance mechanisms have been transferred to pathogens. Antibiotic resistance involves 3 basic strategies: 1) Keep antibiotic out of the cell by destroying the drug, reducing permeability, or pumping the drug out. 2) prevent the antibiotic from binding to its target by altering the target or the drug. 3) Knock the drug off its target. Multidrug resistance efflux pumps use promiscuous binding sites to bind antibiotics of diverse structure.

Feb 2019 - triclosan contributes to antibiotic resistance

Chemical Added to Consumer Products Impairs Response to Antibiotic Treatment Triclosan added to toothpaste and mouthwash to kill bacteria inadvertently makes such cells stronger Triclosan exposure may inadvertently drive bacteria into a state in which they are able to tolerate normally lethal concentrations of antibiotics—including those antibiotics that are commonly used to treat urinary tract infections (UTIs). Cipro In 2017, the U.S. Food and Drug Administration cited both safety concerns and lack of efficacy when it recommended against adding triclosan to consumer soaps, - but these guidelines have not discouraged companies from adding it to other products. "Triclosan is very stable. It lingers in the body and in the environment for a long time Normally, one in a million cells survive antibiotics, and a functioning immune system can control them. But triclosan was shifting the number of cells. Instead of only one in a million bacteria surviving, one in 10 organisms survived after 20 hours. Now, the immune system is overwhelmed." Triclosan increased tolerance to a wide breadth of antibiotics,

Scientists Have Grown Microbes on Actual Rock Bits From Mars

Chunks of meteorites dislodged from Mars travel through Solar System and crash on Earth. Scientists ground up a small piece of the Martian Black Beauty meteorite, and used it to grow extremophile microbes. Ancient life on Mars probably was an extremophile - a chemolithotroph as the atmosphere was rich in CO2 - Metallosphaera sedula, a thermoacidophilic Archaean found in hot, acidic volcanic springs. Electron microscopy demonstrated that the microbe used and transformed the material to build cells, left behind biomineral deposits clearly from the growth on the meteorite breccia.

Carbapenemases

Class of beta lactamases active against carbapenems - Carbapenems are mostly immune to hydrolysis by other enzymes Bacteria with carbapenemases have been reported in every US state Carbapenemases are most commonly associated with carbapenamase-producing Enterobacteriaceae (CPE) - CRE = carabamenamase resistant Mortalitly rate may be as high as 50% because few treatment options remain for infections with these organisms Carbapenemases encoded by genes on large transmissible plasmids - HGT There are many types of carbapenemases, each with a unique resistance profile. - Currently, Klebsiella pneumoniae carbapenease (KPC) is most common in the US

Measuring Drug Susceptibility - Kirby Bauer

Clinical labs can receive up to 100 or more isolates in one day, so individual MIC determinations are impractical. The Kirby-Bauer assay tests strain sensitivity to multiple antibiotics. - Uses a series of round filter paper disks impregnated with different antibiotics. - A dispenser delivers up to 12 disks simultaneously to the surface of an agar plate covered by a bacterial lawn. - During incubation, the drugs diffuse away from the disks into the surrounding agar and inhibit growth of the lawn. Diameter of zone of inhibition reflects relative sensitivity.

Triclosan:

Contributes to Antibiotic resistance Disrupts Hormone regulation (thyroid hormones and more) Disrupts Immune system dysregulation Skin cancer?

NUCLEIC ACID SYNTHESIS AND INTEGRITY

DNA synthesis and integrity - Quinolones gyrase - Metronizazole Prodrug - Sulfonilamides or sulfa drugs Folic acid synthesis RNA synthesis inhibitors - Rifampin, Actinomycin D

Target - DNA

DNA synthesis inhibitors - Quinolones (fluroquinolones) Inhibit gyrase Ciprofloxacin Anthrax and TB - Metronidazole - prodrug Sulfa drugs - Sulfonamides, Trimethoprin Analogs of precursor of folic acid THF needed for nucleic acid synthesis - Pathway bacteria have, humans do not

Keep antibiotics out of the cells

Decrease membrane permeability across the outer membrane - Gram negative bacteria can express outer membrane porins with pores too narrow to allow drug penetration Destroy the antibiotic before it gets into cell -The beta-lactamaseenzymes specifically destroy penicillins. (beta lactam antibiotics) Pump the antibiotic out of the cell -Using specific transporters and transport complexes. -Able to get drugs out faster than they can enter -Of particular concern are multidrug resistance (MDR) efflux pumps (ABC export systems) -Can export many different kinds of antibiotics with little regard to structure -Similar strategy is used in cancer cells.

How does antibiotic resistance develop?

Evolving, Sharing, Drug resistant genes De novo antibiotic resistance develops through gene duplication and/or mutations. Antibiotic resistance also can be acquired via horizontal gene transfer (conjugation, transduction, and transformation ). - There is a diversity of mobile genetic elements - The loss and acquisition of functional modules are an important part in the processes of rapid bacterial adaptation and development of resistance. A study in 2015 found that antibiotic-resistant microbes occur naturally in uncontacted Amazon communities.

Scientists Found a Compound That Kills 98% of a Drug-Resistant Bacteria

Found from Antarctica ocean organism - a sponge!Most infections are in biofilms... Kills staph in biofilms that MRSA construct...̵Biofilm-penetrating anti-MRSA agent! Around 80,000 MRSA in US each year, 11,000 people die from MRSA complications

The Search for New Antimicrobial Drugs

Long-term solution to antimicrobial resistance relies on the development of new antimicrobial compounds and antibiotic conservation - Modification of current antimicrobial compounds is often productive

ch. 27 pt 8 review

Measures to counter antibiotic resistance include synthetically altering the antibiotic, using combination antibiotic therapy, and adding a chemical decoy. Persister cells in a population have stopped actively growing, making them tolerant to bactericidal antibiotics. The quest to discover novel antibiotics includes designing candidate antimicrobial compounds to interact with and inhibit the active site of known microbial enzymes, and screening previously uncultured microbes for new antibiotics. Potential targets for new antimicrobials include persister cells, proteins expressed on in vivo (virulence proteins), and pathways contributing to biofilm formation and dispersal.

Looking for microbial life on Mars

One of the focuses of Perseverance Rover is astrobiology, or the study of life throughout the universe. Rock core samples in metal tubes will be collected, looking for tell-tale signs of microbial life that may have lived on Mars billions of years ago. - Future missions will return the samples to Earth Jezero Crater was once the site of a large lake with a river delta. Biosignatures may be found in the crater shorebed, or the rim of shoreline sediments The Mars scientist investigated the ancient life in Australia's outback - The carbonate minerals preserve on Earth preserve some fossilized life. Perseverance cameras and lasers will document the plasma cloud for chemical composition. On the turret at the end of its arm, has X-ray beams (PIXL). SHERLOC has laser to detect concentrations of organic molecules and minerals that form in water environments Together, SHERLOC and PIXL will provide high-resolution maps of elements, minerals, and molecules in Martian rocks and sediments, enabling astrobiologists to assess their composition and determine the most promising cores to collect.

Post antibiotic era

Overuse and misuse of antibiotics Lack of global cooperation/monitoring Lack of pharmaceutical company investment

Beta-lactam antibiotics

Penicillins Cephalosporins Carbapenems Monobactams Beta-lactams are a class of antimicrobial compounds whose target is penicillin-binding protein (PBP) - usually is transpeptidase PBPs (transpeptidases) mediate cross-linking of peptidoglycan and are irreversibly inhibited by beta-lactams. (a)Beta-lactam antimicrobials bind irreversibly to penicillin binding protein (PBP), preventing bacteria from synthesizing the cell wall. (b)Beta-lactamaseenzymes interact with beta-lactams but form only a temporary bond, leading to drug inactivation.

Beta-Lactam Antibiotics (cell wall)

Penicillins, cephalosporins, carbapenems -The beta-lactam ring chemically resembles the -D-Ala-D-Alapiece of peptidoglycan. -This molecular mimicry allows the drug to bind transpeptidase and transglycosylase (which is why the proteins are called penicillin-binding proteins).-PBP-Thus, preventing their activities and halting synthesis of the chain

Antibiotics targeting membranes

Platensimycin - New structural class of antibiotic Selectively inhibits a bacterial enzyme central to Fatty Acid synthesis. - Broad-spectrum (gram+), - effective against MRSA and vancomycin-resistant enterococci (VRE) No toxicity No potential for development of resistance (!)

Drugs That Affect Bacterial Membrane Integrity

Poking holes in a bacterial cytoplasmic membrane is an effective way to kill bacteria. Gramicidin -Cyclic peptide produced by Bacillus brevis -Inserts into a membrane as a dimer -Forms a cation channel, through which ions leak Polymyxin -Polypeptide produced by Bacillus polymyxa -Destroys cell membrane, just like a detergent -Used only topically Daptomycin -Lipopeptide produced by Streptomyces roseosporus -Forms an ion channel that leaks potassium ions -Very effective against MRSA

Target - protein synthesis

Protein synthesis inhibitors - Aminoglycosides Streptomycin and gentamycin Neurotoxicity and nephrotoxicity - Tetracycline Broad spectrum Animals! Macrolides Erythromycin Chloramphenicol Oxazolidinones

Target - RNA

RNA synthesis inhibitors - Rifampin Binds to RNA polymerase - Actinomycin D Intercalates in DNA, inhibits initiation

HGT via integrons

Recently, multidrug resistance has been attributed to the presence of highly-mobile gene expression elements called integrons. Integrons are genetic elements that contain a site-specific recombination system able to integrate, express and exchange specific DNA elements, called gene cassettes Environmental resistomeis a source of resistance genes - evolution

Modified Porins or PBPs, Efflux pumps and carbapenemases

Resistance mechanisms intrinsic acquired

Directly countering Drug Resistance

Several strategies are being used: prudent use and innovative strategies 1) Dummy target compounds that inactivate enzymes that create resistance -Clavulanic acid -Used in conjunction with penicillins -Clavulanic acids is a Beta lactam with no antimicrobial effect; competitively binds to beta lactamases 2) Alter antibiotic's structure so that it sterically hinders access of modifying enzymes -Modify gentamicin (aminoglycoside) to amikacin -But resistance already to amikacin 3) Linking antibiotics is another strategy currently being used to limit resistance. -A quinolone-oxazolidinone hybrid antibiotic has dual modes of action. -Thus, it can limit development of drug resistance. However, multidrug resistance efflux pumps, integron cassettes, and plasmids w/ multiple antibiotic resistance genes can overwhelm linked antibiotic approach

Drugs That Affect the 30S Subunit - block tRNA binding

Tetracyclines: consist of four fused cyclic rings -Block the binding of charged tRNAs to the A site of the ribosome -Include doxycycline -Can interfere with bone development -Passes through breast milk -Can affect tooth development -Permanent yellowing of teeth -Broad spectrum-Inhibits almost all Gm+ and Gm - bacteria-Gastrointestinal disruption -Widespread development of resistance -Widespread use in humans and animals

Carbapenems

The carbapenems are beta-lactam antimicrobials and one of the last lines of defense against multidrug resistant Enterobacteriaceae. - Carbapenems are extended spectrum penicillins that are not affect by most beta-lactamase enzymes Reserved for severe or high-risk infections caused by multidrug resistant bacteria Carbapenems play a critically important role in our antibiotic armamentarium. Of the many hundreds of different β-lactams, carbapenems possess the broadest spectrum of activity and greatest potency against Gram-positive and Gram-negative bacteria. As a result, they are often used as "last-line agents" or "antibiotics of last resort" when patients with infections become gravely ill or are suspected of harboring resistant bacteria

Protein Synthesis Inhibitors

The differences between prokaryotic and eukaryotic ribosomes account for the selective toxicity of antibacterial antibiotics. - Recall that protein synthesis inhibitors can be classified into several groups based on structure and function. Most of these antibiotics work by binding and interfering with the function of bacterial rRNA. In general, protein synthesis inhibitors are bacteriostatic (not bactericidal). - What about the ribosomes in mitochondria? Aminoglycosides Streptomycin and gentamycin Tetracycline - Broad spectrum - Animals! Macrolides -Erythromycin Chloramphenicol Oxazolinadizones

Review ch. 27 pt 1

The importance of antimicrobials in treating disease was recognized in the early 1940's. Some antimicrobials are naturally produce by living organisms (antibiotics); others are synthetically made through chemical engineering. Selective toxicity is the ability of an antibiotic to attack a unique component of microbial physiology that is missing or distinctly different from eukaryotic physiology. However, antibiotic side effects (host toxicity) can limit the clinical usefulness of an antimicrobial agent. Antibiotic spectrum of activity is the range of microbes affected by a given drug. Bactericidal antibiotics, or antimicrobials, kill microbes; bacteriostatic antibiotics inhibit microbial growth; and some antimicrobial ages are initially inactive until converted by the body to an active agent. An antibiotic's spectrum of activity and the infectious agents susceptibility to the antibiotic are critical points of information required before an antibiotic therapy is prescribed. Minimal inhibitory concentration (MIC) of a drug, when correlated with average attainable tissue levels of the antibiotic, can predict the effectiveness of an antibiotic in treating disease. MIC is measured by tube dilution techniques, but it can be approximated by the Kirby-Bauer disk diffusion technique.

Measuring Drug Susceptibility - MIC

The minimal inhibitory concentration (MIC) is the lowest concentration of the drug that prevents growth. - Varies for different bacterial species - Test by serial dilution of antibiotic - Lowest concentration with no growth = MIC Note: Culture may still contain living (but dormant) organisms! Plate MIC culture without antibiotics to see if colonies form No colony growth = minimal lethal concentration (MLC)

The Golden Age of Antibiotic Discovery

The modern antibiotic revolution began in 1928 with the discovery of penicillin by Alexander Fleming. -A contaminating mold had inhibited the growth of Staphylococcus aureus colonies on a plate. -The mold was identified as Penicillium notatum. -Penicillin was purified in the early 1940s by Howard Florey and Ernst Chain. -Has saved millions of lives since! Gerhard Domagk (1930s) - Discovered sulfa drugs (sulfanilamide) Inactive until converted by body to active agents Analogs of PABA, a precursor of a vitamin needed for DNA synthesis Precursor to folic acid, which is needed for growth Sulfanilamide inhibits one of the enzymes that converts PABA into folic acid

AR Threats report

The report lists 18 antibiotic-resistant bacteria and fungi into three categories based on level of concern to human health—urgent, serious, and concerning—and highlights: Estimated infections and deaths since the first report in 2013 Aggressive actions taken Gaps slowing progress The report also includes a Watch List with three threats that have not spread resistance widely in the U.S. but could become common without a continued aggressive approach. Antibiotic resistance is a growing problem worldwide-Antibiotics are overused, misused-Overprescribed; used in farm animal feed This exerts selective pressure for multiple drug-resistant strains-Streptococcus pneumoniae-Resistant to multiple drugs

CDC's first antibiotic resistance threat report in 2013

The situation is dire indeed: According to the newest data, more than 2.8 million people in the United States experience an infection from antibiotic resistant bacteria each year. Moreover, these "superbugs" cause 35,000 deaths per year in the country. "We need to better understand how antibiotic use in both humans and animals is related to growing antibiotic resistance — the concept is One Health, where the health of humans, animals, and plants [is] all linked and interdependent." Dr. Jesse Jacob "We need research to find new drugs but can't rely on a pipeline of new drugs alone to solve this problem, since resistance eventually happens to all drugs."

Measuring Drug Susceptibility - E test

The time required to evaluate antibiotic effectiveness can be reduced by using a strip test that avoids the need for dilutions. The MIC is the point at which the elliptical zone of inhibition intersects with the strip.

"Farmaceuticals"

Today, ~80 percent of all antibiotics used in America are given NOT to people, but to livestock.

Bacterial acquire resistance from competitors

Transformation with a twist Some bacteria inject a toxic cocktail (called effectors) into their competitors causing cell lysis and death. - Using type VI secretion system - Like a syringe Then, by integrating the released genetic material (with drug resistant genes) the predator cell can acquire antibiotic resistance. (transformation) This contributes to MDR pathogens in hospitals

How does it interfere?

Triclosan's dirty weapon: ppGpp (guanosine tetraphosphate - ALARMONE) - Makes cells less sensitive to antibiotics ppGpp is a cell growth inhibitor - is an alarmone (a.a. stress response) - Shuts down many biosynthetic pathways - Diverts resources away from growth and towards survival Don't slow growth before you try to kill the cells! - Bactericidal antibiotics kill by targeting specific biosynthetic pathways - ppGpp curtails biosynthesis - Bactericidal antibiotic become ineffective

Other Antibiotics That Inhibit Synthesis of the Cell Wall

Vancomycin: binds ends of peptides -Prevents action of transglycosylases and transpeptidases -Same step as penicillin, but different activity Cycloserine:inhibits formation of the D-Ala-D-Ala dipeptide precursor Bacitracin:blocks the lipid carrier-Disaccharide subunits do not reach periplasm

Why is our knowledge of antibiotic resistance so difficult to implement?

What is the interaction between science and society? - Is it a pharma conspiracy? - Society's short attention span? - What population is most at risk? •In 1990, 19 companies developed antibiotics, •now only 4

Biofilms, Persisters, and the Mystery of Antibiotic Tolerance

Why do some infections return after bactericidal antibiotic treatment is discontinued? The reason is a subpopulation of dormant organisms, called persister cells, that arise within a population of antibiotic-susceptible bacteria. -The stalled metabolism of persisters renders them tolerant to bactericidal antibiotics during treatment. -Persister cells can be found in any biofilm or population of late exponential-phase cells. -Tolerance provides antibiotic resistance at the price of not growing

Aminoglycosides

contain a cyclohexane ring and amino sugars -Cause the translational misreading of mRNA -Are unusual in that they are bactericidal -Include streptomycin and gentamicin -Neurotoxicity and nephrotoxicity -Considered reserve antibiotics for when other antibiotics fail

Link between persister cells and toxin-antitoxin modules

hipA and hipB genes in E. coli - HipA - toxin - HipB - antitoxin neutralizes Hip A Less stable than HipA - HipA becomes active if antitoxin degrades or synthesis lags HipA then phosphorylates and inactivates glutamyl-tRNA synthesase no charged Glu-tRNA, ribosomes stall ALARMONE ppGpp made, activating stringent response Antitoxin proteins are degraded, toxins unleashed!mRNA nucleases activated, cell stops growing = dormancy (and antibiotic tolerance) Tolerance provides antibiotic resistance at the price of not growing.

Antibiotic Overuse leads to resistant populations

livestock feed / injection humans, livestock, pets, aquaculture, crops

Antimicrobial Drug Resistance

•The use of antibiotics in medicine, veterinary medicine, and agriculture selects for the spread of R plasmids •Antibiotics used in agriculture as supplements to animal feed since 1946 - >70% of all antibiotics used in US is fed to healthy farm animals (25 million pounds of antibiotics per year) - Drug-resistant bacteria developed at livestock facilities can reach human population through food, environment or direct contact - Any biochemical cycle that depends on microbes that may be killed by runoff, unused antibiotics fed to livestock.