MCB6937 Exam 1

*KNOW* Fluoroquinolone

Broad Spectrum, Bactericidal antibiotic Ciprofloxacin is one of the MOST WIDELY used antibiotics Novobiocin -GyrB inhibitor (different from quinolones that target GyrA) -taken off market due to adverse reactions in people -isolated from Actinobacteria Streptomyces in 1950s Inhibit DNA replication Targeting bacterial gyrases does NOT interfere with Human DNA -However, fluoroquinolone still controversial due to severe toxicity to humans

*KNOW* What PBP does

D-Ala-D-Ala is substrate of bacterial cell wall transpeptidase PBP (Penicillin Binding Protein) PBP -involved in cell wall synthesis -transpeptidase catalyzes cross-linking between adjacent sugar glycan chains -REMOVES terminal D-Ala residue from one of the peptidoglycan Summary: -PBP catalyzes crosslinking of neighboring Lipid 2 molecules of peptide sidechains by REMOVING D-Ala -in both Gram- and Gram+ Lipid 1 = NAM Lipid 2 = NAM + NAG (peptidoglycan)

*KNOW* Running out of treatment options

Estimated that any single antibiotic is ONLY useful for about 50 years Means antibiotics discovered in "Golden Age" are at end of their effectiveness -Also means we need another 20 classes of antibiotics to treat infections for next 50 years (but only 2 have been developed so far) As of now, destines towards era where infections that were once treatable will become deadly

*KNOW* Central Dogma of Molecular Biology

Describes process of information flow from genes to proteins -Replication of DNA -Transcription of DNA to RNA -Translation of RNA into proteins

*KNOW* Extensive drug development process eliminates toxic candidates

Despite extensive screening, almost all drugs that are developed have some side effects

*KNOW* Neomycin

Discovered by Waksman -not readily absorbed through digestive system and is highly toxic (one of the MOST TOXIC of all the aminoglycosides) -"Neosporin" -use restricted to topical application -can lead to loss of hearing, even when topically applied over wound

*KNOW* Streptomycin

Discovered in 1944 by Selman Waksman Pioneered isolation of aminoglycosides from Streptomyces -1st effective medicine against Tb

*KNOW* Kanamycin

Discovered in 1956 by Hamao Umezawa (also 1st to make penicillin in Japan, discovered over 70 antibiotics and 40 cancer therapeutics) -provided treatment option for already streptomycin-resistant Mycobacteria Tb

*KNOW* Gentamicin

Discovered in 1963 by Marvin Weinstein -relatively less side effects than other aminoglycosides (commonly used to treat eye infections) Both *Kanamycin* and *Gentamicin* bind to 30S subunit -interfere with initiation complex -misread RNA -leads to arrest of protein synthesis

*KNOW* Adverse Drug Reacts (ADR)

Estimated number of ADRs in hospitalized patients causes over 100K deaths annually ADR 4th leading cause of death right after malpractice -100K - 400k death in US

What is this antibiotic? Figure: many 6 member-rings, F, and one 3-member ring/triangle

Fluoroquinolone

*KNOW* Narrow vs Broad Spectrum

Narrow -kills specific organism -prescribed when bacteria are known Broad -affects broad range of Gram+ and Gram- -prescribed when causative agent not known

*KNOW* The number of Antibiotic Resistant infections increases each year

Number of bacterial infections has remained constant (~14 million a year) -Number of Antibiotic Resistant Infections have been constantly increasing -Means the number of bacterial infections resistant to antibiotics is increasing

What is the function of the penicillin binding protein (PBP)?

To crosslink peptidoglycan

The majority of antibiotic classed were developed during the Golden Age of Antibiotics. Since then, very few new classes were introduced into clinical use. True/False

Trie

*KNOW* Antibiotics that inhibit folate synthesis in bacteria, which stops production of dTTP, which results in cellular arrest

Trimethoprim - target Dihydrofolate Reductase (DHFR) Sulfamethoxazole -targets Dihydropteroate Synthase (DHS) Abyssomicin C -targets Aminodeoxy chorismate

According to the WHO, antimicrobial resistance is one of the biggest threats to global health and security today. True/False

True

An infection manifests clinical resistance when the MIC of a specific antibiotic is toxic to humans and the infection can no longer be treated with that antibiotic. True/False

True

Carbapenems are effective against 98% of bacterial strains isolated from hospitals. There is a special caution in using this antibiotic to avoid development of resistance. True/False

True

Inhibiting DNA synthesis arrests the cell cycle but does not kill cells. Hence, trimethoprim is classified as a bacteriostatic antibiotic. True/False

True

Structural differences between eukaryotic and prokaryotic machinery involved in the Central Dogma of Molecular Biology (replication, transcription, translation) allow for the development of antibiotics that specifically target microbial cells.

True

*KNOW* The Scope of the Problem with AMR

-while AMR increases and spreads, development of novel antibiotics has completely ceased Failing Drug Efficacy - "Sisyphus goes to work" -Sisyphus had to push a boulder up a mountain, it falls, and he pushes it up again everyday -same with antibiotics, because AMR keeps making antibiotic obsolete

*KNOW* Community Acquired Infection

An infection that is acquired from a communal setting, such as gym, swimming pool, subway, etc. -MOST infections acquired from community

What are the reasons that make it difficult to eliminate antibiotics from their use in farm animals?

All of the above: -loss of profit to animal farming companies -loss of profit to pharmaceutical companies -easy access to antibiotics

*KNOW* Major Reservoirs of Antibiotic Resistance

Major Reservoirs = bacterial hosts that are exposed to selective pressures, that allows them to develop antibiotic resistance due to the exposure to antibiotics -food animals -humans (major reservoir) -pets Wild animals ate Soil also reservoirs (Natural and Acquired)

What is the problem associated with Citrus Greening Disease?

Massive application of clinically-important antibiotics on citrus crops

What does MIC stand for?

Minimum inhibitory concentration

*KNOW* Most Common Side Effects of Antibiotics

*Antibiotic resistance* *Gastrointestinal* (broadly caused by almost all antibiotics) -Diarrhea -Bloating and indigestion -Abdominal pain -Loss of appetite *Allergic reactions* (itchy skin and rashes) -usually from B-lactams *Photosensitivity* -Tetracyclines, Quinolones, Sulfonamides *Organ damage* -Kidney = Aminoglycosides -Liver = Carbapenems, Rifampicin, Isoniazid, Tetracyclines *Tooth discoloration* -Tetracyclines *Neurotoxicity* -Rifampicin, Isoniazid, Quinolones *Hearing Loss* -Aminoglycosides

Vancomycin causes kidney damage in 5-43% of patients. With such a high incidence of a serious side effect, vancomycin is still being used because:

-It's effective against multi-drug resistant bacteria -It's a last-resort antibiotic

*KNOW* Consequences of Antibiotic Use in Farm Animals on Human Health

(selective pressures due to imprudent antibiotic usage in animals) -Increase human morbidity (more zoonotic diseases that get people sick) -Increase human mortality -Reduced efficacy of related antibiotics used in humans -Increased human healthcare costs -Increased potential for carriage and dissemination -Facilitated emergence of resistance in human pathogens

Resistance to different Antimicrobials:

*Antibiotics* -kill bacteria or inhibit their growth -bacteria develop resistance to antibiotics and are able to replicate and survive in their presence (most common example is MRSA) -*KNOW* One (singular) = bacterium Two = bacteria Many (plural) = bacteria *Antiviral* -medications stop viral replication -ex) some therapeutics of prophylactics like vaccines, that lose their effectivity as virus mutates (why flu vaccines are constantly redeveloped) *Antifungal* -agents either inhibit growth or kill fungi -Most common fungus that lives on our bodies is Candida -Candida is an opportunistic pathogen (can attack human bodies when given opportunities to thrive) -according to CDC, Candida is leading cause of healthcare associated bloodstream infections in US hospitals *Antiparasitic* -drugs that target parasites like malaria -Plasmodium falciparum develops resistance to antimalarial drugs and poses serious threat to humans (takes over half-million lives annually) *Pesticides* -used to control invasive or disease carrying pests -ex) using pesticides to reduce incidence of malaria by killing mosquitoes -many mosquitoes have developed resistance to pesticides, making their control difficult *Herbicides* -agents used in agriculture to control growth of weeds that interfere with crop growth -Waterhemp is invasive weed that developed resistance to most common herbicidal agents Resistance in *Humans* -chemotherapy/chemotherapeutic agents that are used to control the uncontrollable cellular division characterized by cancer cells -*KNOW* Cancer Resistance -in many cases, cancer develops resistance to these drugs, which makes them difficult to treat

*KNOW* Adverse Reactions For:

*Chloramphenicol* -suppressed bone marrow activity -increases chances of developing aplastic anemia from 1:500K to 1:40k -physicians advised not to use this for infections where alternative can be used *Metronidazole* -ability to bind to human DNA covalently and irreversibly in dividing cells (placed on list of Human Carcinogens) -induces cancer in animals models, but acute exposure in humans was NOT linked to cancer -Rare instance, can cause Stevens-Johnson Syndrome (Severe cutaneous adverse rxn, also seen with sulfa antibiotics and penicillin) *Aminoglycosides* -use has been declining due to permanent hearing loss (even for topical application of Neomycin) -can also cause reversible kidney damage *Glycopeptides* -use of Vancomycin restricted due to high nephrotoxicity (5-43% patients) (preferable treatment for S. aureus was methicillin) -emergence of MRSA, vancomycin use was brought back as last-reserve antibiotic despite high toxicity

Match each ecosystem with its corresponding selection pressure:

*Clinical Ecosystem* = High Selectivity *Non-clinical Ecosystem* = Medium Selectivity *Environmental Ecosystem* = Low Selectivity

*KNOW* AMR Consequences

*Higher Medical Costs* - ABR infections cost ~$1.4k more per person compared to antibiotic sensitive infection ($2.2B to $20B in medical costs within US alone) -additional antibiotic prescriptions -prolonged hospitalization -additional diagnostic/lab tests to determine resistance -Infectious disease specialists have higher costs *Prolonged Hospital Stays* -estimated US household lost $35B in 2000 to ABR infections -can lead to additional health problems and family problems *Increased Mortality* -resistant strains have longer hospitalization when compared to sensitive strains (leads to higher mortality) -*REMEMBER*: ABR increases average hospital stay and increase mortality *Family Problems* -more health problems, like depression, anxiety, loss of jobs, etc.

Timeline of AMR

*KNOW* It takes 8 years on average from the introduction of an antibiotic to get clinical resistance Dark Ages -when antiseptic techniques were not used to control spread of infections (we may be entering Dark Ages again) Biochemical Period -increasing efficacy of antibiotics Disenchantment -no more new antibiotics

Antibiotic Discovery vs Synthesis

*Naturally Derived* -penicillin is naturally occurring antibiotic from mold -Egyptians used moldy bread 3.5Kya to treat wounds *Semi-Synthetic* -derived from naturally derived antibiotics -amoxicillin *Synthetic* -FULLY synthesized in a test tube -quinolone

*KNOW* Transmission of Antibiotic Resistance Through Environment: *Air*

-Aerosol/fluid droplets -Solid particles *KNOW* Bacteria can enter air from: -soil and leaves (organic matter) - Water -Humans and animals -Fecal matter -Wastewater -Composting facilities ex) Netherlands '02-'09 -Coxiella burnetti causes Q-fever -one of the most infectious bacteria known -Largest Q-fever outbreak got 4k people sick, killed 74 -Bacteria disseminated by wind from nearby goat farms ex) MRSA bacteria can also move through air

AMR is a worldwide problem

-CDC says 2 million infections a year, with 23,000 deaths in US alone -AMR is worse in Europe, with greater deaths because the bacteria are pan-resistant, even to last reserve antibiotics -AMR is a global problem that is only getting worse -resistance can travel from one country to another

*KNOW* Tetracycline

-Doxycycline -Lymecycline -Oxytetracycline Targets protein synthesis -specifically 30S ribosomal subunit Isolated form soil-dwelling bacteria Streptomyces -1945 by Benjamin Duggar -commercialized in 1953 Broad Spectrum effective against: -Gram-, Gram+, intracellular bacteria (ricketssia and chlamydias) -even wipes out good human microbiota, predisposing patients to fungal infections Not advised for children because causes teeth discoloration MOST WIDELY used antibiotic in agriculture *KNOW* Bind to 30S and inhibit translation -tetracycline binding site overlaps partially with aminoacyl-tRNA binding site -leads to tRNA dissociation from ribosomes -bocks translation

What are some of the difficulties associated with antibiotic development?

-Drug development takes between 10-20 years -Drug development can cost over $1B -90% of drugs fail clinical trials

*KNOW* Scientific discoveries

-If you think you have discovered something important, blow your whistle until people start noticing -Be cautious when and how your share your results -Many discoveries are made by mistake -Science is a race and you need to act quickly -You only get credit if you are the first one to publish

Evidence of AMR rising from animal farming

-Same ABR bacterial strains that colonize animals are found in infected humans -E. faecalis is normally found in gut of animals and humans (will cause infection outside the gut) -causative strain found through MLST -E. faecalis is the most common clinical isolate of UTIs, abdominal or pelvis wound infections, and bacteremia (90% of clinical isolates) -infection treated with combination of antibiotics, like gentamicin (gentamicin resistant strains found in Denmark)

*KNOW* Aminoglycosides

-Streptomycin -Neomycin -Kanamycin -Gentamicin ALL target protein synthesis -specifically in 30S subunit Family of antibiotics whose structure has amino sugars linked to glycosidic bonds Given in combination with B-lactams -gives synergistic effect on bacterial killing (bactericidal) Amino groups are + charge at neutral pH -MoA dictated by groups -allows them to interact with - charged phosphate backbone of target RNA and 30S subunit Binding of aminoglycoside to 30S leads to inhibition of tRNA translocation from A to P site -causes misreading of mRNA, leaving bacterium unable to synthesize essential proteins -aminoglycosides increased mistranslation to ever 1-100, from 1k-10k

*KNOW* Humans can be exposed to antibiotic resistance through:

-What we eat -What we do -What we drink -Where we live

Structure of B-lactam antibiotics resemble:

-a house (with an attached garage)

*KNOW* Using Antibiotics in Farm Animals Leads to:

-enhanced development of resistance to antibiotics (Osterberg et al. 2016) -transmission of resistant bacteria to humans (Larsen et al. 2010) -contamination of the environment by air particulates that carry antibiotics, resistant genes, and bacteria (McEachran et al. 2015) -emergence of novel antibiotic resistance mechanisms (Liu et al. 2016)

Major host reservoirs that are exposed to antibiotic selective pressure include the following:

-food animals -pets -humans

Check all of the reasons why antibiotics are used in food animals:

-growth promotion (although banned in 2017) -infection control

*KNOW* It's Not Easy to Eliminate Antibiotics From Farming

-loss of profit to farm animal companies uncontrolled access to antibiotics

*KNOW* Suffix of Aminoglycosides

-mycin = derived from Streptomyces -micin = drived from Micromonospora

*KNOW* AMR Key Facts

-one of the biggest threats to global health and food security today -can affect anyone, of any age, in any country -occurs naturally, BUT misuses of drugs in humans and animals is accelerating the process -leads to longer hospital stays, higher medical costs, and increases mortality

UDP-MurNAc-pentapeptide accumulation with use of B-lactam antibiotics

-treatment of bacteria with sublethal concentrations of penicillin lead to accumulation of *UDP-N-acteylmuramyl-pentapeptide* -Park and Strominger suggested UDP-MurNAc-pentapeptide must be upstream of the B-lactam blockage (they are correct) Accumulation results from penicillin-mediated inhibition of peptidoglycan biosynthesis through inactivation of PBP

Antibiotic resistance occurs naturally, but the ___ of drugs in humans and animals is accelerating the process.

-underuse -use -overuse

*KNOW* Other possible approaches

1) *Hemoperfusion Devices* -MoA = extracorporeal filters/pumps that directly clear blood pathogens by their physiochemical properties -Possible Benefit = quickly reduce blood concentration of selected bacteria by order of magnitude 2) *Quorum Sensing* -MoA = disrupt intercellular signaling between bacteria to block coordinated tissue invasion -Possible Benefit = blocks sensing of necessary concentrations of bacteria for optimal synthesis of virulence and invasion genes 3) *Lytic Bacteriophage* -MoA = bacteriolysis induced by selected lytic phage or phage cocktails -Possible Benefit = parasitic predators of bacteria can be used as highly specific, targeted, bactericidal agents 4) *Polyclonal/Monoclonal Antibodies*/ *Immune Adjuvants* -MoA = improved bacterial vaccines, transgenic cattle for polyclonal immunotherapy (designer monoclonal antibodies, immune stimulant therapy for sepsis) -Possible Benefit = action of passive immunity to opsonize bacteria or inhibit exotoxins and virulence factors (adjuvant to stimulate cellular immune function) 5) *Liposome-based Cytotoxin Inhibitors* -MoA = engineered to serve as cell membrane decoys to absorb bacterial cytotoxins -Possible Benefit = capture pore-forming cytotoxins and protect host cell membranes from cellular injury 6) *Non-immune Tolerating Approaches* -MoA = treatments allowing host to survive and compensate for pathogen presence or until immune clearance removes the pathogen -Possible Benefit = permits host to tolerate pathogen until cleared by immune or non-immune mechanisms (oral or IV fluids for cholera)

*KNOW* From Population Increase to Antibiotic Resistance

1) Increase in global population drives increase for food demand 2) Increased food demand drives animal farming 3) Increased animal farming enhances occurrences of transmittable diseases 4) Enhanced occurrence of transmittable diseases increases use of antibiotics 5) Increase in antibiotic use enhances antibiotic resistance 5.5) Human exposure to low concentration of antibiotics enhances antibiotic resistance

Protein Synthesis Steps

1) Initiation -components required for protein translation come together (ribosomal subunits, tRNA, mRNA) -translation involves 50S + 30S subunits -1st amino acid is start codon, attached to tRNA 2) Accommodation -on assembled ribosome, tRNA carrying `st amino acid (formyl methionine, UAC) paired with start codon on mRNA (AUG) *KNOW* -fMet is start codon on prokaryotes, NOT Met like in eukaryotes -fMet is removed post-translationally (presence in humans indicates foreign bodies) 3) Accommodation -place on the ribosome where 1st tRNA sits is called P site -in A site next to P, 2nd codon of mRNA pairs with tRNA 4) Peptidyl Transfer -1st amino acid joins 2nd by peptide bond -1st tRNA release from E site 5) Translocation -ribosome moves along mRNA until 2nd tRNA is in P site, and process continues 6) Elongation Cycle -ribosome continues to move along mRNA molecule -new amino acids added to growing polypeptide chain 7) Termination -when ribosome reaches a stop codon (UGA), polypeptide is released from ribosome 8) Recycling -last tRNA is released, ribosome comes apart -released polypeptide forms new protein

Development of Bacteria

1) Lots of bacteria, few are resistant 2) Antibiotics kill bacteria causing illness, as well as good bacteria protecting body from infection 3) Drug-resistant bacteria are now allowed to grow and takeover 4) Some bacteria give their drug resistance to other bacteria, causing more problems by raising new antibiotic resistance

Reduction of antibiotic use in food animal production must be addressed on global basis by:

1) enforcing global regulations to cap antimicrobial use 2) adherence to nutritional guidelines leading to reduced meat consumption 3) imposing a global user fee in veterinary antimicrobial use

*KNOW* Transmission of Antibiotic Resistance Through Environment: *Water*

10-90% of antibiotics are excreted in urine and feces, leading to constantly increasing concentration of antibiotics in aquatic environments -exact concentration of antibiotics depends on class of antibiotic and species -tetracyclines generally exceed 50% Process: -bacteria or antibiotics from humans and pets enter water systems through ground wastewater -excretion from animal farms enter wastewater and are used in manure -both sludge from wastewater and manure are used as fertilizer carrying antibiotics and ABR bacteria onto commercial crops -No system is in place that would monitor antibiotic concentration in water, soil, sewage, or manure Water is nearly the source of ALL contamination because it is a large reservoir that is continuously fed through runoff

*KNOW* Discovery of Antimicrobial Property of Penicillium mold by Ernst Duchesne

1897 - Ernst Duchesne, French Physician, published thesis in which he described antimicrobial property of mold Noted Penicillium mold had antibacterial properties and carried out animal studies to confirm his findings -Guinea pigs injected with live bacteria (Salmonella or E. coli) and media from Penicillium mold culture -all infected animals that were injected with mold media were saved Proposed to use culture media as a therapeutic against bacterial infections -discovery went unnoticed and forgotten

*KNOW* "Re-discovery" of Penicillin by Alexander Fleming

1928 - Fleming was physician at St. Mary's hospital in London, UK Unintentionally made discovery of penicillin -Plates had become contaminated by Penicillium mold (common mold ubiquitously found in environment) -Saw Staph, aureus he was growing did not grow close tot he contamination source -there was zone of inhibition around the mold (FIRST indication of a substance having an inhibitory/killing effect on bacteria) He tested many mold species -Penicillium mold is the only mold strain that produced inhibitory substance Early tests of Penicillin's antimicrobial property -embedded penicillium into culture media, and streaked out different bacteria -saw zone of inhibition of some bacteria -not all species of pathogenic bacteria has same inhibition response to penicillin Also did research on sweat and tears of his grad students -led to discovery of Lysozymes (antimicrobial enzymes that destroy peptidoglycan layer of cell walls)

*KNOW* Penicillin and Derivatives (both natural and semi-synthetically derived)

1928: *Penicillin G* (benzyl-penicillin) -first penicillin discovered from penicillium mold -naturally derived -broken down by stomach acids and leaves body quickly through urine -delivered by IV or intramuscularly -antibiotic still excreted from body in 6 hours after injection 1948: *Penicillin V* (phenoxy methyl penicillin) -stays longer in body, so taken orally -*G and V are both part of the Narrow Spectrum of penicillin* 1961: *Ampicillin* (aminopenicillin) -has NH2 -similar structure to amoxicillin 1972: *Amoxicillin* -has NH2 and OH -OH makes it more lipid soluble, leading to better efficacy in killing Gram- than ampicillin -*Ampicillin and Amoxicillin belong to 2nd generation Broad Spectrum penicillin* (presence of NH2/amino group in both allows better penetration of outer membrane in Gram-) 1960: *Methicillin* -also part of *Narrow Spectrum* -1st penicillin antibiotic that is RESISTANT to cleavage by penicillinases

*KNOW* Antibiotics - A Formal Definition

1942 - "A microbial product that kills or inhibits the growth of other microbes" *KNOW* Selman Waksman -pioneer in the field of antibiotic discovery -known as "Father of all known antibiotics" (discovered around 15 antibiotics) -saved the greatest number of people a scientist has ever saved Antibiotics are NOT developed -they are DISCOVERED -"microbial product" -can be misleading because we now have fully synthetic antibiotics that are not derived from microbes

*KNOW* Penicillin becomes the "miracle drug"

1943 - penicillin became commercially available (without prescription) 1945 - Nobel Prize to Fleming, Florey, and Chain Fleming's Important Lecture: -danger than a person can easily underdose themselves and expose their microbes to non-lethal quantities of drugs and make them resistant

*KNOW* The structure of Penicillin

1945 - structure solved by *Dorothy Crowfoot Hodgkin* -used x-ray crystallography -Nobel Prize for chemistry in 1964 B-Lactam -Penicillins characterized by B-lactam rings -B-lactam = 4 member ring, containing N attached to beta O -ALL B-lactam antibiotics target cell walls Structure -looks like house with attached garage -MoA mediated through inhibition of bacterial cell wall

*KNOW* Key facts about Antibiotic Use in Farm Animals

1951 -FDA approved 1st antibiotics for use in poultry, pork, an beef growth promotion -industrial farms using antibiotics since then ~70% of all antibiotics are used in animals 2016 -UN General Assembly announced use of antibiotics in animals is leading cause of antibiotic resistance crisis ABR can migrate from farms to our tables -antibiotics in animals selects for resistance -cross contamination leads to human exposure to AMR Antibiotic use in pigs enhances ABR

Timeline of Antibiotics in Farming

1951 - FDA approves 1st antibiotics are growth promoters 1969 - UK says antibiotic use in agriculture contributes to AMR 1970 - US recommends some antibiotics be banned from animal use 1977 - FDA proposes ban on penicillin/tetracycline use (opposed by drugmakers) 1980 - FDA concludes not enough data to support hazard caused by antibiotic use in animals 1997 - WHO recommends antibiotics used in animals should not be used as growth promoters 1999 - EU issues ban on common human antibiotics in animals 2003 - US institute of Medicine issues report on increased prevalence of "superbugs", and recommends ban on use of antibiotic growth promoters in animals 2006 - EU bans use of antibiotics as growth promoters 2012 - FDA orders limits on cephalosporin use in animals 2013 - FDA asks pharma companies to voluntarily label their products "not for growth promotion" 2017 - FDA bans use of antibiotics as growth promoters (implementing change on national level where you have 2 opposing sides is difficult and time consuming)

*KNOW* Sulfa-drugs

1st synthetic antimicrobial agents that preceded discovery of penicillin -discovered in 1932 by Gerhard Domagk NOT antibiotics -are antimicrobial agents Not true antibiotics because they are not of microbial origin Prodrug Prontosil: - 1st commercially available antimicrobial Prodrug = metabolized by intestinal enzymes into active form -not active in its original form Sulfa-drugs remained largely unknown until Franklin Roosevelt Jr., President's son -he was treated for septic sore throat -treatment was a success

*KNOW* Conversion of Dihydrofolate BACK to Tetrahydrofolate

2 step rxn catalyzed by 2 enzymes: -Dihydrofolate Reductase (DHFR) = converts dihydrofolate back into tetrahydrofolate (essential in prokaryotes and eukaryotes) -Serine Transhydroxymethylase = converts tetrahydrofolate to methylene tetrahydrofolate

On average, how long does it take for the development of clinical resistance to an antibiotic?

8 years

B-lactam antibiotics are characterized by:

A 4-member ring containing a nitrogen (is the attached garage on the house)

*KNOW* p-aminobenzoate (PABA) Synthesis

ADC (Aminodeoxy-chorismate) Synthase and Lyase facilitate synthesis of PABA -ADC Synthase converts chorismate compound to aminodeoxy-chorismate -ACD Lyase converts aminodeoxy-chorismate into PABA

*KNOW* 5 Classes of B-lactam Antibiotics

ALL have B-lactam rings and scaffold structure similar to D-Ala-D-Ala 1) *Penicillins* -naturally occurring from mold -characterized by 5 member ring attached to B-lactam ring 2) *Cephalosporins* -naturally occurring from mold -isolated in 1948 by *Giuseppe Brotzu* from sewage -both penicillins and cephalosporins target the same class of PBP (Cephalosporins are MORE effective because they are less receptive/more resistant to penicillinases) -6 member ring attached to B-lactam ring -over 70 different Cephalosporin antibiotics (5 gens of cephalosporins) 3) *Carbapenems* -naturally occurring from bacteria -2 differences from penicillins ~S to C substitution on thiazolidine ring ~double bond between C2 and C3 of 5 member ring -extremely broad spectrum antibiotics (effective against 98% of bacteria isolated from hospitals) -reserved for high risk or severe resistant infections (but still has widespread resistance) -Meropenem, has high affinity to P. aeruginosa -not easily inactivated by B-lactamases 4) *Monobactams* -naturally occurring from bacteria -single 4 member B-lactam ring -specifically targets Gram- bacteria (E. coli, Pseudomonas, etc.) 5) *Clavams* -really weak antibiotics -don't kills, sued to combat antibiotic resistance -naturally occurring from bacteria -typical B-lactam structure, but is not potent -MIMIC B-lactam antibiotics -SATURATE B-lactamases by tricking them that they are antibiotics -Clavams used to ENHANCE penicillin effectiveness

*KNOW* Factors contributing to AMR in Healthcare

Antibiotic usage -Underuse -Use -Overuse -lack of antibiotics -Poor sanitation and personal hygiene -Use of antibiotics in agriculture -Inadequate infection controls in healthcare facilities

*KNOW* Properties of antibiotics (or any other dugs) can be modified by generating semi-synthetic derivatives (usually by slightly modifying sidechains)

Antibiotics and other drugs are chemically modified in lab to achieve different properties of the molecule to do what's needed -ensure they reach infection site, are stable, or are not toxic

*KNOW* Antibiotic Safety (according to CDC)

Antibiotics responsible for 1 out of 5 emergency department visits for Adverse Drug Events Antibiotics are MOST COMMON cause of emergency department visits -for adverse drug events in children under 18 years of age

*KNOW* Quinolone Antibiotics

Antimicrobial activity of Nalidixic Acid was discovered in 1962 by George Lesher Characterized by 4-quinolone bicyclic ring Type II topoisomerase inhibitor: -Gyrase -Topoisomerase IV *KNOW* Targets: 1) DNA Gyrase (Type II Topoisomerase) -enzyme responsible for introduction of negative supercoils into DNA -Heterotetrameric (2 subunits) ~GyrA = mediates enzyme-catalyzed DNA breakage reunion ~GyrB = ATPase activity and facilitates strand passing -Quinolones BIND to GyrA subunits (means GyrA unable to reunite broken DNA, so DNA remains broken) 2) Topoisomerase IV -paralogue of DNA gyrase -facilitates DNA strand passing -relaxes positively supercoiled DNA -Heterotetrameric (2 subunits) ~ParC = breakage-reunion -Quinolones BIND ParC (means ParC unable to reunite broken DNA)

*KNOW* Cotrimoxazole

Antimicrobial that COMBINES -Sulfa-drugs (DHPS - dihydropteroate synthase inhibitor) -Trimethoprim (DHFR - dihydrofolate reductase inhibitor) Combination reduces chances of developing resistance *KNOW* Synergistic Effect -combined potency of a combination of drugs is much higher than sum of their individual potencies ALONE, Trimethoprim and Sulfamethoxazole are bacteriostatic -TOGETHER, become bactericidal

The use of antibiotics in farm animals has been ___ in 1951.

Approved

*KNOW* B-lactam containing compounds bind to and INHIBIT transpeptidases

B-lactam antibiotics bind transpeptidases much stronger than D-Ala-D-Ala Binding is covalent and IRREVERSIBLE -results in inhibition of PBP, cell wall synthesis, and consequently death by cell lysis MoA: -B-lactam binds to transpeptidase site (instead of D-Ala-D-Ala) -B-lactam has longer half-life than PBP, which is why binding is much stronger -PBP unable to bind to D-Ala-D-Ala -PBP unable to crosslink sugar glycan chains, disrupting cell wall synthesis

*KNOW* 2 Functions of Antibiotics

Bactericidal -killing/ kills bacteria Bacteriostatic -stationary/ inhibits bacteria

*KNOW* Ernst Chain

Chain = German born British biochemist Instrumental in purification of penicillin from Penicillium 1940 - Chain and Florey published work on "Penicillin as a chemotherapeutic agent" Fun Fact -tried to find penicillium mold strain that would be most effective in making penicillin -got it from a cantaloupe

Quinolone toxicity is well known, yet it's one of the most commonly prescribed antibiotics

Common warning for Fluoroquinolone: "Serious adverse reactions include tendinitis, tendon rupture, peripheral neuropathy, CNS effects, and exacerbation of myasthenia gravis" Fluoroquinolones include: -Cipro (most commonly used/prescribed) -Levaquin -Avelox

Johnny developed a MRSA infection when he got injured during his baseball practice. Johnny got a ___ acquired infection.

Community

What is the function of dihydrofolate reductase (DHFR)?

Converts dihydrofolate to tetrahydrofolate

What is this antibiotics? Figure: has NH and NH2

Cycloserine

Florida's Citrus Greening Disease / Huanglongbing (HLB) Yellow Root Disease

Diaphorina citri Kuwayama = Asian citrus psyllid (bacteria not culturable in lab setting) Pysllid = vector for Candidatus Liberbacter asiaticus Disease leads to overall declining health in tree and ceases fruit production -has yellowed trees and roots compared to normal Disease poses large threat to Florida economy and future of citrus industry Relation to ABR: -currently no treatment options -problem is that we don't want to put a lot of clinically important antibiotics on them to kill bacteria, because ABR will increase *MAY BE ON EXAM* -think about consequences of using antibiotics in fight against CGD (spread of ABR)

*KNOW* How different are bacteria from humans?

Differences in molecular level between bacteria and humans allows us to identify new antibiotic targets -can't have same targets in human as in bacteria *Bacteria* -Have peptidoglycan/cell wall -Structure of 50S/30S ribosome -BOTH have RNA pol (antibiotics tend to be specific towards RNA pol) -BOTH have isoluecyl-tRNA synthetase -metabolism synthesizes folates *Humans* -NO peptidoglycan/cell wall -Structure 60S/40S ribosome -BOTH have RNA pol -BOTH have isoluecyl-tRNA synthetase -CANNOT synthesize folate *PROBLEM* Despite humans being different from bacteria, there are not too many bacterial targets

*KNOW* Sulfanilamide

Drug that mimics PABA Sulfa-drugs are competitive inhibitors of dihydropteroate synthase -means instead of PABA binding to Dihydropteroate Synthase (enzyme) -Sulfa-drug binds to enzyme, stopping synthesis of dihydropteroate which is needed to make Dihydrofolate (DHF)

Penicillinases are:

Enzymes that deactivate penicillin

Folate Synthesis

Eukaryote/humans CANNOT synthesize folate/ Vitamin B9 -humans rely on diet, get it as a supplement -bacteria make their own folate

All strains of mold produce penicillin. True/False

False

Only some antibiotics affects human microbiota. True/False

False (generally, all antibiotics affect human microbiota)

Quinolone antibiotics target DNA gyrase and topoisomerase IV. These type II topoisomerases are also found within human proteome. True/False

False (humans and prokaryotes are different, which is why antibiotics work)

Trimethoprim binds to human DHFR. True/False

False (inhibits bacterial DHFR)

Sulfanilamide is an antibiotic. True/False

False (sulfa drugs are prodrugs)

Clavams are very potent antibiotics. True/False

False (they are the weakest)

*KNOW* Howard Florey

Florey = Australian pharmacologist and pathologist at University of Oxford, who got interested in penicillin Was aware of Paine's successful clinical work on penicillin -his team was successful at actually purifying penicillin and demonstrating its potential in a clinical setting

*KNOW* Cecil George Paine

First Clinical Use of Penicillin Paine = medical student at St. Mary's Hospital Medical School Obtained culture of Penicillium from Fleming and used it to successfully treat his patients using crude extracts -used on babies with gonorrheal infections, etc.

*KNOW* Disruption of normal microbiota - antibiotics do not discriminate between commensal and pathogenic bacteria

Gastrointestinal problems (same as side effects) Secondary infections -Vaginitis/yeast infection and other fungal infections Generally all antibiotics effect human microbiota -effect is directly linked to gastrointestinal issues -disruption of gut bacteria indirectly linked to number of other side effects not related to GI tract

*KNOW* Vancomycin

Glycopeptide -isolated from actinobacteria in 1953 -introduced to clinics in 1958 -takes 10-20 years to introduce single antibiotic to market -in past, things happened quickly *Glycoprotein* -molecule that is composed from combination of amino acids and polypeptide fused with sugar moiety -synthesized by non-ribosomal peptide synthase in actinobacteria Large 1.4kDa Molecule -cannot pass through porins of Gram- (B-lactams are small to pass through) -why vancomycin is only effective in killing Gram+ Unique MoA Independent of B-lactams -effective against MRSA -used on Gram+ resistant to penicillin/methicillin *MoA* -Caps D-Ala-D-Ala and prevents its incorporation into peptidoglycan ad subsequent crosslinking Took 30 years for resistance to appear -vancomycin was considered "last resort"

*KNOW* Non-B-Lactam Cell Wall Synthesis Inhibitors

Glycopeptides: -Vancomycin -Bacitracin Cycloserine Fosfomycin

*KNOW* B-lactam antibiotics are more effective in Gram+ than Gram-

Gram- -peptidoglycan and PBP are less accessible -peptidoglycan is in periplasmic space between inner an outer membranes Gram+ -more accessible peptidoglycan and PBP -peptidoglycan more exposed on outer membrane

*KNOW* Why use antibiotics in farming?

Growth Promoters -antibiotics are known to increase animal weight Infection Control -cheaper alternative to hygiene maintenance Video: -80% of antibiotics in US are given to animals -gives rise to resistant superbugs - "USDA Process Verified" and "Organic" means no antibiotics in meat

*KNOW* Nosocomial/ Hospital Acquired Infections (HAIs)

Infections that are caught in healthcare setting, like hospital, clinic, nursing home, etc. -MOST deaths related to antibiotic resistance happen in healthcare setting

What is the function of sulfanilamide?

Inhibits dihydropteroate synthase

*KNOW* Streptogramins

Isolated from soil bacteria, Streptomyces, during "Golden Age" of Antibiotics (1950s) Mixture of 2 structurally distinct compounds produced by Streptomyces at 70:30 ratio 1) Streptogramin A -binds to peptidyl transferase catalytic center on 50S subunit -tRNA cannot bind to A nor P sites (prevents tRNA attachment) -leads to arrest of peptide bond formation and stops polypeptide elongation -Strep A binding leads to CONFORMATIONAL CHANGES in ribosome that ENHANCES binding of Strep B 2) Streptogramin B -Binds to 23S rRNA within 50S subunit exit tunnel -inhibits growth of nascent protein -leads to release of unfinished polypeptide fragments INDIVIDUALLY = Bacteriostatic COMBINED = Bactericidal -potency increases 100-fold -Cooperative Binding (Strep A enhanced ability of Strep B to bind)

*KNOW* Chloramphenicol

Isolated in 1947 from Streptomyces -much cheaper to synthesize in vitro -FIRST antibiotic synthesize in test tube Broad Spectrum, Bacteriostatic -inhibits replication -does not kill Binds to 23S of 50S subunit -inhibits peptidyl transferase (no facilitation of peptide bond formation between amino acids in P and A sites) Small size allows diffusion to body areas normally not accessible by antibiotics -used for bacterial meningitis SERIOUS side effects -similarity in mammalian mitochondrial ribosomes to bacterial ribosomes enhances cross reactivity

*KNOW* Macrolides / Macrocyclic Lactones

Isolated in 1952 by McGuire et al. from soil actinobacteria, Saccharapolyspora -natural products Characterized by macrocyclic lactone connected to sugar moiety via glycosidic bonds -Erythromycin -Clarithromycin -Azithromycin High affinity for 50S subunit -bind to 23S of 50S subunit (same as Lincosamides) -bind proximal end of peptide exit tunnel (nascent polypeptide chain grows here) -leads to peptidyl transferase inhibition (stop enzyme from adding additional amino acids)

*KNOW* Cycloserine

Isolated in 1954 from Streptomyces D-alanine analog -D cycloserin inhibits D-Alanyl-D-Alanine formation/synthesis Works upstream of B-lactams, Vancomycin, and bacitracin through distinct mechanism Small size -can penetrate Gram- bacteria -reach places in body not accessible by other antibiotics Broad Spectrum antibiotic -restricted as secondary choice for MDR-Tb and XDR-Tb Constituents of Lipid 2 -made of 2 sugar molecules NAM and NAG, and 5 amino acids -2 terminal amino acids on pentapeptide are alanines, coming from dipeptide D-Alanyl-D-Alanine -then added into polypeptide *MoA* 1) D-cycloserine inhibits alanine racemase (Alr) -Alr = enzyme responsible for racemization of Alanine into D-Alanine 2) D-cycloserine inhibits D-Alanine: D-Alanine Ligase (Ddl) -Ddl = enzyme that ligates 2 Alanine molecules to make D-Alanyl-D-Alanine -as a result, Lipid 2 molecule missing 2 terminal Alanines, which leads to disruption of bacterial cell well and leads to cell lysis

Lincosamides

Isolated in 1962 from Streptomyces lincolnensis -Clindamycin -Lincomycin Bind to 23S within the 50S subunit of bacterial ribosome -leads to bacteriostatic inhibition of protein synthesis

Which of the following statements is true about rifampicin?

It inhibits bacterial RNA polymerase

*KNOW* Nomenclature Rules for Bacteria

Italicize scientific names -Genus and species italicized -if you want to refer to all species, Genus is italicized and spp. left alone

Factors contributing to AMR in healthcare include (choose the best answer):

Lack of antibiotics, poor sanitation, inadequate infection controls

*KNOW* Other Factors Contributing to AMR

Limiting exposure to microbes dampens immune system -weak immune system increases chances of developing infection that will require antibiotic treatment Kids whose pacifier was licked by mom and given back had betters stimulation of immune system FDA banned use of antibiotic agents in personal hygiene products -exposure to bacterial environment is important for proper development of our immune system

Polymyxins and daptomycin are examples of:

Lipopeptides

*KNOW* Multilocus Sequence Typing (MLST)

Method for identifying bacterial isolates by directly measuring the DNA sequences variation in housekeeping genes -ex) E. faecalis ST16

*KNOW* Inhibition of DHFR

Methotrexate inhibits HUMAN DHFR -used in cancer treatment/immunosuppressant Trimethoprim inhibits BACTERIAL DHFR -antibiotic Structural similarities between DHF, methotrexate, and trimethoprim allow their binding to and inhibition of DHFR Similarity in structures is what allows binding and inhibition -Similar mechanism to B-lactam antibiotics that bind to PBP and stop function

*KNOW* Fosfomycin

Most upstream of all B-lactam and Non-B-Lactam Cell Wall Synthesis Inhibitors Isolated in 1969 from Streptomyces Highly reactive epoxide ring with *Broad Spectrum*, *Bactericidal* Properties Enters cell via hexose monophosphate or glycerophosphate transporter -IRREVERISBLY inhibits early steps of bacterial cell wall biosynthesis *MoA* -MurA = enzyme that catalyzes addition of enolpyruvate to UDP-N-acetylglucosamine -MurA involved in one of the first steps in peptidoglycan biosynthesis -There are antibiotics that also target other parts of peptidoglycan synthesis

Future looks promising, but it's not!

Neither Teixobactin nor Odilirhabdin made it to clinical trials yet - even if they do, 90% of drugs fail Drug development takes time and money -10-20 years -$2B Pharmaceutical companies do not pursue antibiotic development due to profit loss

Teixobactin

Newly discovered cell wall synthesis inhibitor Isolated from "unculturable" soil bacteria Non-ribosomal peptide made of unusual amino acids Made by Gram-, and is EFFECTIVE against Gram+ Has not reached clinical trials yet Targets: -thought to bind highly conserved part of Lipid II (precursor of cell wall teichoic acid) -Lipid II = synthesized at cytoplasmic section of the cell -flipped by flippase across inner membrane into periplasmic space -multiple antibiotics like Bacitracin and Vancomycin inhibit here

Odilorhabdin

Newly discovered ribosomal inhibitor Discovered in 2018 by Alexander Mankin and Yury Polikanov Non-ribosomal peptide isolated from bacteria which was isolated from nematodes Binds specifically to small ribosomal subunit at completely novel site -when compared to other known translational inhibitors

*KNOW* Mupirocin

Non-ribosomal Binding protein synthesis inhibitor Isolated in 1971 from Pseudomonas fluoresencs -is a carboxylic acid Non-ribosomal translation inhibitor Binds to an inhibits Isoleucyl-tRNA Synthetase -enzyme responsible for charging tRNA with isoleucyl (messes up polypeptide chains without one amino acid) -enough differences between bacterial and eukaryotic tRNA synthetases that allows specificity of Mupirocin to target bacteria and NOT Eukaryotic tRNA synthetase Concentration dependent bactericidal antibiotic Rapidly metabolized into inactive monic acid and carboxylic acid -restricted to topical application only (Bactroban) -impetigo is common bacterial skin infection in kids that Bactroban is used for

*KNOW* Folate Synthesis Pathway in Bacteria

Opens new opportunities to develop antibiotics that target folate biosynthesis -consequently leads to inhibition of DNA synthesis, leading to cellular arrest Dihydrofolate (DHF) Synthesis requires -dihydropterin -PABA Steps: -GTP initially converted into dihydropterin pyrophosphate -requires rxn catalyzed by Dihydropteroate Synthase in presence of p-aminobenzoate (PABA) -converts molecules to dihydropteroate -Dihydropteroate has reaction catalyzed by Dihydrofolate Synthase to make Dihydrofolate (DHF) p-Aminobenzoate (PABA) is an important precursor in DHF synthesis

*KNOW* We have run out of antibiotic targets

Over 20 new classes of antibiotics discovered between 1940-1962 since 1962, only few classes of antibiotics were commercialized -We are now in Discovery Void Period We have nothing new/novel -only combination of antibiotics or structural derivatives of known

*KNOW* Penicillinases/B-lactamases

Penicillin antibiotics are prone to degradation by penicillinases -enzymes produced by bacteria that degrade B-lactam ring and render the antibiotic ineffective

Peptidoglycan in Gram-

Peptidoglycan = thick layer of inner cell membrane -polymer whose sidechains are crosslinked by amino acids, to form protective grid over cell membrane -gives cell shape and strength, counteracts internal osmotic pressure from cytoplasm (will rupture and die without it) Gram- -peptidoglycan layer is thinner -located in periplasmic space between inner plasma membrane and outer membrane

Structure below is D-ala-D-ala dipeptide. What is its function?

Peptidoglycan terminus

Lipopeptide

Poke holes in bacterial membranes -Polymyxins -Daptomycin Polymyxins first isolated in 1947 from Bacillus polymyxa Characterized by peptide ring attached to fatty acid sidechain -hydrophobic fatty acid chain allows penetration and disruption of lipid bilayer -Strong + charge of fatty acid in polymyxin interacts with - charged LPS (lipopolysaccharide chains) -specifically with lipid A portion of molecule at outer membrane of Gram - -Hydrophobic fatty acid chain responsible for bactericidal action -polymyxins disrupt outer membrane and allow access to periplasmic space and into inner membrane Polymyxins initially successful against Gram- -neurotoxic behavior and nephrotoxic effect -replaced by safer antibiotics Increase in prevalence of "superbugs" prompted return of polymyxin clinical use -Transmission Electron Microscope (TEM) reveals polymyxin-mediated disruption of membrane (blebbing on outer surface) Daptomycin is effective against Gram+ -has mechanism that is distinct from polymyxins that target Gram- -Exact mechanism is not understood -Believed that Daptomycin oligomerizes inside of the bacterial membrane, affecting membrane curvature -curvature leads to disruption in membrane integrity

Use of antibiotics on farms leads to emergence of new resistance

Polymyxins resistance only occurs through chromosomal mutation -dies not spread from bacteria to bacteria Instead, the mutated gene itself moves in the wind and gets to other bacteria, which in turn infects humans -how polymyxin resistance occurred in China -bad because polymyxins are a last reserve antibiotic

Isoniazid

Prodrug that is metabolized into active form by Mtb (mycobacteria) catalase peroxidase enzyme -facilitates isoniazid fusion with an NAD molecule Activated isonicotinic acyl-NAD binds to and INACTIVATES enoyl acyl carrier protein reductase -responsible for synthesis of mycolic acid, major component of Mtb cell wall MOST commonly used anti-tuberculosis drug -specifically inhibits synthesis of mycolic acid

Prokaryotic vs Eukaryotic Ribosomes

Prokaryotes: 70s ribosome -50S subunit -30S subunit Eukaryotes: 80S ribosome -60S subunit -40S subunit

Abyssomicin C

Recently discovered antibiotic that inhibits ADC synthase -inhibits synthesis of PABA -stops synthesis of DHF -does not allow dTTP to be made -results in cell arrest Isolated in 2004 from actinomyces strain, collected from 300m depth in Japanese sea Recently discovered, still not commercially available

*KNOW* Oxazolidinones

Repurposed drugs -initially developed as monoamine oxidase inhibitors for treatment of depression Linezoid -approved for clinical use in 2000 (one of most recent antibiotics) -FIRST antibiotic with completely NOVEL structural class in 1980-2000 Binds to 50S subunit -inhibits formation of initiation complex (no formation of ternary complex between mRNA, ribosomal subunits, and tRNA charger with fMet) -binds to an earlier step, compared to rival similar inhibitors

*KNOW* Rifampicin

Rifamycin B first isolated from soil bacteria Nocardia mediterranei in 1965 by Dow-Lopetit Research Lab Rifamycin belongs to group of antibiotics called *Ansamycin* -characterized by aromatic ring bridged by aliphatic chain Broad Spectrum -effective against Mycobacteria, Gram+, Gram-, and most anaerobes Specifically BINDS to bacterial RNA polymerase and prevents RNA synthesis -depending on concentration, can be bactericidal or bacteriostatic *KNOW* RNAP Complex -crystal structure of RNA polymerase bound with rifamycin molecule -Rifamycin directly BINDS in the path of the elongating RNA molecules by simple steric block (DIRECTLY blocks the path) Only 3 years after discovery, antibiotic was introduced into clinical use in Italy and approved by FDA -now takes 10-20 years after discovery to get to clinical use -takes ~$2B in funding to release

*KNOW* Trimethoprim

SYNTHETIC antibiotic -synthesized in 1962 by Barbara Roth Arrests cell cycle -Bacteriostatic -does NOT kill 100,000-fold selectivity for bacterial DHFR -specific for bacteria over humans Broad Spectrum Antibiotic -commonly prescribed to treat MRSA ALWAYS in combination with other antibiotics, that also inhibit folate acid synthesis pathway

*KNOW* B-lactam containing compounds mimic D-Ala-D-Ala

Skeleton structure of B-lactam antibiotics mimics shape of D-Alanine-D-Alanine peptide -structures look almost identical when superimposed

*KNOW* Transmission of Antibiotic Resistance Through Environment: *Soil*

Soil has the highest density of bacteria -bacteria are not culturable under lab conditions -makes studying soil bacteria difficult Antibiotic Resistant Bacteria occur naturally in soil (almost all bacteria isolated form soil have ABR to various antibiotics) MRSA can also spread through soil from farms

*KNOW* Most Probable Number (MPN)

Statistical method to estimate number of bacteria in liquid sample based on number of dilutions required to detect no growth

*KNOW* Bacitracin

Strain of bacillus isolated from fracture wound of 7-year-old girl Margaret Tracy -exhibited strong antibacterial property *C-55-isoprenyl pyrophosphate* flippase shuffles Lipid 2 across membrane -flippase facilitates transport of Lipid 2 across bacterial membrane -flippase binds to Lipid 1/NAM -Lipid 1 modified with NAG, becomes Lipid 2 -Lipid 2 transported by flippase, by flipping across membrane -flippase release Lipid 2 in periplasmic space -Lipid 2 incorporated into growing peptidoglycan structure, crosslinked by PBP *MoA* -Bacitracin physically binds to C-55-isoprenyl pyrophosphate, inhibiting dephosphorylation -inhibition of dephosphorylation STOPS flippase from going back to cytoplasmic region -stops synthesis of peptidoglycan wall *KNOW* Bacitracin inhibits cell wall synthesis through mechanism that is DISTINCT from B-lactam and vancomycin *KNOW* Due to its kidney toxicity, is only available topically -also approved by FDA for use in agriculture/poultry

Use of antibiotics on farms enhances transmission of resistance into the environment

Study assess: 1) antibiotics 2) antibiotic resistance genes 3) bacteria via particulate matter from large-scale cattle farms Particulate matter collected downwind and upwind from cattle farm -large concentrations of antibiotics, bacteria, and resistance genes found in wind-spread particulate matter

*KNOW* Metronidazole

Synthetic derivative of parasitic drug produced by Streptomyces -repurposed in 1962 Prodrug -activated by anaerobic bacteria, leading to formation of DNA adducts -ONLY active against anaerobic bacteria, because need to be activated by them (C. difficile, H. pylori, bacteria vaginosa, etc.) Reduced, activated metronidazole covalently binds to DNA -forms DNA adducts -inhibits RNA synthesis and leads to bactericidal death Clinical study involved over 5k metronidazole users shows no link between cancer and acute metronidazole use -compounds that bind to DNA often known to be carcinogenic *KNOW* Metronidazole is known to cause DNA damage, not seen to be good to treat something in the body -activity comes from non-specific and irreversible covalent binding to DNA in rapidly dividing cells, and leads to cell death -belongs to class of drugs called *Ansamycin* -other drugs in this class, like Geldamycin, used in cancer treatment

*KNOW* Components of the Cell Wall are NOT present in Eukaryotes

Targeting cell wall synthesis is an attractive way to inhibit bacterial growth WITHOUT affecting the host

*KNOW* Colony Forming Unit (CFU)

Unit used to estimate the number of viable bacteria under the assumption that a single bacterium will form a single colony 1 bacterium = 1 colony

What is this antibiotics? Figure: antibiotic caps D-ala-D-ala

Vancomycin

Antimicrobial Resistance (AMR)

WHO: "ability of a microorganism to stop an antimicrobial from working against it. As a result, standard treatments become ineffective, infections persist and may spread to others." Microorganism = bacteria, viruses, and some parasites Antimicrobial = antibiotics antivirals, and antimalarials

Conversion of dihydrofolate back to tetrahydrofolate is essential to keep a constant supply of which nucleotide?

dTTP

Synthesis of deoxythymidine triphosphate (dTTP) *KNOW* Dihydrofolate

dTTP synthesized from dUMP (deoxy uracil monophosphate) Conversion of dUMP to dTMP (deoxy thymidine monophosphate) -requires enzymatic reaction catalyzed by Thymidine Synthase -Thymidine Synthase = enzyme that catalyzes dUMP to dTMP -biproduct made is 7,8-dihydrofolate -dTMP reduced to dTTP through Ribonucleotide Reductase -dTTP directly used as substrate for DNA synthesis *KNOW* Conversion of 7,8-dihydrofolate BACK into methylene tetrahydrofolate is essential to keep constant supply of dTTP molecules as building blocks of DNA