MCB6937 Exam 2

*KNOW* S. aureus

Gram+ Epidemiology -part of HGM -30% of population is colonized -Ubiquitous in healthcare, environment, and agriculture -81,000 MRSA infections in US per year Disease -skin infections (impetigo), wound infections, osteomyelitis pneumonia, bacteremia, food poisoning -most common HAI -20-50% mortality rate from sepsis Resistance -in Thailand, over 60% of S. aureus isolates were MRSA -MRSA uses mecA/PBP2a -VRSA has change of Ala to Lac and vanHAX genes located on plasmid-residing transposon

*KNOW* K. pneumoniae

Gram- Epidemiology -part of the HGM -Ubiquitous in healthcare, environment, and agriculture Disease -highly pathogenic among immunocompromised people (HAI) -infection can be caused by endogenous or acquired pathogen -leading cause of CA and nosocomial pneumonia -up to 50% mortality rate Resistance -Carbapenemases: ~Metallo-B-lactamases (NDM-1) ~Carbapenem-hydrolyzing B-lactamases (KPC-1)

*KNOW* Tripartite transenvelope pumps in Gram- bacteria

Gram- bacteria need efflux pumps that span the inner and outer membranes, and the periplasmic space *E. coli* AcrAB-TolC efflux Pump -3 elements form channel transporting small molecules from cytoplasmic space into extracellular space -*AcrB* = inner membrane connecting AcrA and TolC -*AcrA* = periplasmic portion -*TolC* = outer membrane portion *P. aeruginosa* exhibits highly expressed constitutive homolog of AcrAB-TolC pump: -Constitutive = MexAB-OprM, MexXY-OprM -Inducible = MexCD-OprI, MexEF-OprN *Elevates MIC values by 2 to 8 fold*

What does it mean for a microbe to be resistant to antibiotics?

Human bodies are the limiting factor as to how much antibiotics/drug can be taken in order to kill bacteria -too much = toxic When dosage of antibiotic becomes *toxic*, *clinical resistance* to the antibiotic is developed

*KNOW* Anti-antibiotics are Natural Defense Mechanisms

Humans do NOT develop antibiotic resistance Bacteria naturally develop counter-attack mechanisms against other microbes that are trying to kill them High selective pressure (presence of antibiotics) accelerates bacteria to acquire resistance and lead to life-threatening infections

*KNOW* Positively charging cell envelope confers resistance to Lipopeptides

Lipopeptides are amphipathic molecules that are attracted to negatively charged phospholipids on cell membranes One of the most common antibiotic resistant mechanisms employed by bacteria is modification of membrane net charge -*Aminoarabinosylation* (glycosylation/ addition of sugar moiety) to Lipid A -*Lysinylation* (addition of amino acid, lysine) of membrane phospholipids -*Alanylation* (addition of amino acid, alanine) of anionic teichoic acids Result of ANY of the 3 modifications is *overall increase in positive charge* -leads to electrostatic repulsion of positively charged lipopeptide molecules

How resistance is acquired:

Low concentrations of antibiotics exposed to bacteria *naturally induce* resistance within the bacteria *LOW* concentrations of antibiotics *INDUCE* antibiotic resistance

Quiz 4: What does MIC stand for?

Minimum Inhibitory Concentration MIC = min concentration of an antibiotic required to completely inhibit bacterial growth

*KNOW* Antibiotic Target Protection

Molecules that bind to antibiotic targets and prevent antibiotic binding -Quinolone resistance protein -Tetracycline-resistant ribosomal protection protein -ATP-binding cassette F (ABC-F) ribosomal protection protein -Overproduction of peptidoglycan

*KNOW* 5 Classes of Membrane Efflux Pumps

Mostly within Gram- bacteria ALL of the efflux pump classes *require energy input* *Multidrug and Toxin Exclusion*/ *MATE* -powered by Sodium gradient *Major Facilitator Class*/ *MFC* -powered by Sodium gradient *Small Multidrug Resistance Class*/ *SMDR* -powered by proton gradient *Resistance-nodulation-division*/ *RND* -powered by proton gradient *ATP-binding Cassette*/ *ABC* -ABC transporter hydrolyze ATP on the cytoplasmic side of the membrane -drive either efflux or influx of small molecules

*KNOW* Bacteriophage Therapy against biofilm infections

OMKO1 Phage is capable of penetrating biofilms Once inside a biofilm, virus replicates and disrupts the biofilm matrix Antibiotics able to penetrate and kill exposed cells that were not killed by the virus

*KNOW* Structural Basis for the B-lactam resistance of PBP2a from MRSA

PBP binds to methicillin Structural work revealed resistance to B-lactams is *not* due to antibiotic's poor fit into active site - due to *inefficient formation of acyl-PBP intermediate* -intermediate formation between methicillin and PBP2a is 1,000x less efficient

*KNOW* Early Indication of Antibiotic Resistance

Penicillin became commercially available in 1942 2 years before commercialization, *Edward Abraham* and *Ernst Chain* made important discovery -identified an enzyme that could destroy penicillin -first official indication of antibiotic resistance mechanisms -did not have to crush the bacteria to get the enzyme, it appeared in the culture fluid -means enzyme was secreted 1940 paper in Nature: -took extract from E. coli and incubated with penicillin -extract inactivated penicillin and ability to kill S. aureus -di not know what this unknown enzyme was

*KNOW* Target Modification: Dihydropteroate Synthase (Sulfonamide Resistance)

Plasmid encoded *sul1* and *sul2* genes confer resistance to sulfonamide -highly effective at dissemination among bacteria sul1 and sul2 genes code for highly resistant variant of DHPS enzyme -catalyzes condensation reaction between PABA and dihydropterin pyrophosphate into dihydropteroate -DHPS variants do not bind sulfonamides, so bacteria that carry sul1 and sul2 genes are highly resistant to sulfa drugs

What came first, antibiotics or antibiotic resistance mechanisms?

Antibiotic resistance mechanisms *HAD* to be made before antibiotics -otherwise, cell that made the antibiotics would kill itself

*KNOW* Examples of Highest Priority Critically Important Antimicrobials

*ALL fit C1, C2, P1, P2, and P3* *Cephalosporins* -3rd, 4th, and 5th gen -B-lactam antibiotics that target bacterial cell wall *Glycopeptides* -Vancomycin -targets bacterial cell wall *Macrolides and Ketolides* -Macrolides target ribosome and inhibit protein synthesis *Polymyxins* -poke holes in bacterial cell membranes *Quinolones* -kill cells by inhibiting type II topoisomerases -attack gyrase and topoisomerase IV Each of these 5 classes are the *ONLY bacteria that provide treatment options against zoonotic bacteria that can easily gain resistance* -important to monitor high priority pathogens and the use of priority antimicrobials against them -is the best effort against development of AbR -but many countries do not adhere to antibiotic use guidelines

*KNOW* B-lactamase Classification: 4 Classes

*Class A* Serine-B-Lactamases -inhibitor susceptible Chromosomal Genes/ Intrinsic Resistance -Penicillinases -Klebsiella spp. and Citrobacter koseri Plasmid-borne Genes/ Acquired Resistance -Penicillinases = TEM and SHV (include inhibitor-resistant variants) -ESBL = TEM, SHV, and CTX-M -Carbapenemase= KPC *Class B* Metallo-B-lactamases Plasmid-borne Genes/ Acquired Resistance -Carbapenamse= VIM, IMP, and NDM *Class C* Cephalosporins Chromosomal Genes / Intrinsic Resistance -Non-inducible AmpC (E. coli) -Inducible AmpC (Enterobacter spp.) Chromosomal Gene/ Acquired Resistance -Derepressed AmpC Plasmid-borne Gene/ Acquired Resistance -Plasmidic AmpC = CMY-2 and other types *Class D* Oxacillinases Plasmid-born Genes/ Acquired Resistance -Narrow Spectrum OXA (inhibitor resistant) -ESBL-OXA (uncommon) -Carbapeneames = OXA-48 variants

*KNOW* Regulation of Efflux Pumps

*Constitutive* -constantly being expressed *Inducible* -energy efficient to induce expression when needed -presence of reactive N and O species -antimicrobial peptides -antibiotics -antibacterial agents (ex. Triclosan) -membrane damaging biocides

*KNOW* WHO Antibiotic Classification - Criterion

*Criterion 1*/ *C1* -antimicrobial class is the sole, or one limited therapies, to treat bacterial infections in humans *Criterion 2*/ *C2* -antimicrobial class used to treat infections in people caused by either: 1) bacteria that may be transmitted to humans from nonhuman sources 2) bacteria that may acquire resistance genes from nonhuman sources *Critically Important* -antimicrobial classes which meet both C1 *AND* C2 *Highly Important* -antimicrobial classes which meet either C1 *OR* C2 *Important* -antimicrobial classes which meet neither C1 *NOR* C2

*KNOW* Major Mechanisms of Antibiotic Resistance

*Directly Targeting Antibiotic* Antibiotic Inactivation -enzymatic inactivation of antibiotics confers drug resistance *Targeting Antibiotic Availability* Antibiotic Efflux -transport antibiotic outside of the cell Reduced Permeability to Antibiotics -generally through reduced production of mediation of porins Resistance by Absence -deletion of a gene, usually a porin *Target Modification* Antibiotic Target Modification -mutational alteration or enzymatic modification of antibiotic target Antibiotic Target Replacement -replacement or substitution of antibiotic action target Antibiotic Target Protection -protection of antibiotic action target from antibiotic binding

*KNOW* Small Molecule (Antibiotic) Transport

*Efflux Pumps* -pumps molecules *out of* cells -antibiotic specific or broad (led to MDR) -provide *intrinsic resistance against antibiotic producing bacteria* -all pumps require energy to move molecules across (*Active Transport*) *Porins* -allow molecules *into* cells -reduce concentration of antibiotics even before pumps get a chance to pump them out -*provide first line of defense against toxic compounds* -*intrinsic resistance of Gram-* (P. aeruginosa has intrinsic resistance to a wide range of antibiotics, due to porins)

*KNOW* Selection for Antibiotic Resistance: Microbial Ecosystems

*Environmental Ecosystem* -*low resistance gene selection* -*The Resistome* -natural ecosystem where microbes encounter low concentrations of antibiotics -"Resistome" = every single antibiotic resistant gene is present in the environment, but all are not selected for -there are over 13,000 genes for antibiotic resistance that exist *Non-Clinical Ecosystem* -*medium selection pressure* -presence of manmade antimicrobials raise selective pressure for antibiotic resistance -includes use of antibiotics in food animals -increased concentration of antibiotics in environment, like wastewater *Clinical Ecosystem* -*high selection pressure* -relative concentration of antibiotics is high -so high selective pressure -contributes to MOST advent of antimicrobial resistance *ALL 3 microbial ecosystems contribute to selection for antimicrobial resistance* -humans mainly enhances selective pressure in clinical and non-clinical environments -contribution to enhancing selective pressure in environmental ecosystem through agriculture

*KNOW* Antibiotic Inactivation - Enzymatic Modification of Antibiotics

*Enzymatic Degradation* Hydrolysis -B-lactamases -most common and well understood mechanism of AbR Linearization -cutting *Enzymatic Modification* Nucleotidylation -addition of nucleotides Phosphorylation -phosphate group addition Glycosylation -attachment of a carbohydrate Acylation -addition of the acyl (RCO-) group Hydroxylation -addition of hydroxyl (-OH) group *Antibiotic Inactivation by Sequestration*

*KNOW* Biofilms are Ubiquitous and Common Across All Aquatic Environments

*Household* -Sink drains, etc. *Industry* -Most common source is Water Towers/ Collin Towers -Legionella pneumophilia is one of the most common bacteria associated with cooling towers, which spread quickly and can kill elderly *Environment* -Yellowstone geysers or streams running through Appalachians

*KNOW* 2 Major Types of Resistance

*Intrinsic* -naturally occurring resistance to antibiotics -outer membrane of Gram- makes them more resistant than Gram+ -bacteria that naturally produce antibiotics have an intrinsic ability to defend themselves -so they are not self-destructive *Acquired* -genetic mutations -acquisition of mobile elements carrying antibiotic resistance genes -this type of resistance poses *HIGHEST risk*

*KNOW* Porin-mediated Resistance

*Loss of Porin* -Upon presence of selective pressure, a cell can completely lose its ability to make porins -complete inhibition of antibiotic entry -Happens through acquisition of *Mutational Resistance* *Decrease in Expression* -antibiotics can lead to decreases leveled of porins -less porins at outer membranes = less antibiotics getting in *Change in Structure* -antibiotics select for mutations that change the structure of porin channel -renders the porins impenetrable to antibiotics -total of 3 mechanisms with porins for antibiotic resistance to occur ~loss of porin ~decreased expression of porin ~narrow porin channel *Porins and Efflux Pumps confer MDR* -*Intrinsic AbR with acquired resistance gives rise to MDR superbugs*

*KNOW* Main B-lactamases in Enterobacteriaceae (Ambler's Classification)

*Most dangerous class of B-lactamases* =ones that are encoded on plasmids -they can be easily transmitted between bacterial cells -plasmid-encoded B-lactamases are responsible for *acquired resistance* Prior to the introduction of antibiotics, all B-lactamases were encoded on *non-mobile genomic DNA* -somehow during introduction of antibiotics, AbR genes are able to jump onto plasmids that could *facilitate rapid HGT* *Extended Spectrum B-lactamases*/ *ESBL* -endemic in hospitals and around the world -one of the most worrying classes of B-lactamases -are the source of *most dangerous* clinically resistant bacteria -initially isolated in 1980s from Klebsiella spp. in Germany Newly emerging ESBL = *CTX-M* -emerging in people with no medical condition, previous antibiotic exposure, or any previous contact with healthcare setting -5-10% of population in SE Asia and Eastern Mediterranean countries are infected by bacteria carrying CTX-M *Carbapenems are last-resort* antibiotics for drugs resistant infections *Most dangerous B-lactamases are Carbapenemases* -KPC1 -VIM -IMP -NDM1 -OXA-48 variants

*KNOW* Antibiotic Target Protection: Quinolone Binding Protein

*Mycobacterium fluoroquinolone resistance protein A* (*Mfpa*) expression in M. tuberculosis leads to fluoroquinolone resistance -mimics B-form DNA -quinolones bind to and inhibit type II topoisomerases -bacterial gyrase is a type II topoisomerase Mfpa binds to DNA gyrase and inhibits its activity when not bound to DNA Mfpa binding protects DNA gyrase from quinolone binding whenever they are *not* bound to bacterial DNA

*KNOW* Antibiotic Target Protection: ATP-binding Cassette F (ABC-F) ribosomal protection protein

*Peptidyl-transfer center* (*PTC*) targeting antibiotics: -Chloramphenicol -Streptogramin A -Lincosamides *Nascent-peptide exit tunnel* (*NPET*) targeting antibiotics: -Macrolides -Streptogramin B *ATP-binding Cassette F* (*ABC-F*) family proteins bind to the ribosome and displace PTC or NPET-bound antibiotics -ABC-F proteins found on Gram+ bacteria: ~Streptomyces ~Staphylococcus ~Streptococci ~Enterococci

*KNOW* Ribosomal Alteration, conferring AbR: Ribosomal Alteration - 50S

*Post-translational modification* -Macrolide -Lincosamide -Streptogramin B = MLSB -MLSB binds to 50S subunit of bacterial ribosome *MLSB Resistance* -post translational modification/methylation of 23S rRNA confers broad resistance to 23S targeting antibiotics -cross resistance by changing the binding site, inhibiting binding of antibiotic to bacteria Erythromycin ribosome methylation (erm) genes code for adenine-specific N-methyltransferases that catalyze ribosomal methylation -erm genes are encoded on plasmids across Gram+ and Gram- -troubling because erm genes are encoded on mobile element that can easily migrate among and between species of Gram+ and Gram- *Mutations* -Mutations in 23S rRNA is linked to decreases antibiotic affinity

*KNOW* WHO Antibiotic Prioritization

*Prioritization Criteria 1*/ *P1* -high absolute # of people, or high proportion of use in patients in clinical settings where *the antimicrobial class is the sole/one of the few alternatives* to treat serious infections *Prioritization Criterion 2*/ *P2* -high frequency antimicrobial class *use for any indication in human medicine*, or high proportion of *use for serious infections* in clinical setting -*use favors selection of resistance in both settings* *Prioritization Criterion 3*/ *P3* -antimicrobial class used to treat infections in people for which there is *evidence of transmission of resistant bacteria or resistance genes from non-human sources* *Highest Priority* -3/3 of Prioritization Criteria -P1, P2, and P3 *High Priority* -2/3 Prioritization Criteria

*KNOW* Antibiotic Target Modification

*Restructuring of bacterial cell wall, conferring AbR* -Pentapeptide modification (Vancomycin) *Ribosomal alteration, conferring AbR* -23S rRNA mutations of 50S (MLSB) -16S rRNA mutations of 30S (Aminoglycoside and Tetracyclines) *Charge Alteration* -Positively charging cell envelope (Lipopeptides) *Point Mutations/ Deletions/ Insertions* -Dihydrofolate reductase/ DHFR (trimethoprim) -Dihydropteroate Synthase/ DHPS (Sulfonamide) -DNA gyrase and topoisomerase IV (quinolone) -RNA polymerase (rifampin)

*KNOW* Antibiotic Target Protection: Ribosomal Protection Protein

*Tetracycline-resistant ribosomal protection protein* (*tet(O)*) is a translational GTPase tet(O) is structurally similar to EF-G and can therefore bind to bacterial 70S ribosome -tet(O) binds to tetracycline-blocked ribosome, leading to conformation changes that release tetracycline -tet(O) leaves its binding site while ribosome remains in conformation that does not favor tetracycline binding

*KNOW* Tetracycline-dependent regulation of efflux pumps: Active Efflux of Tetracycline out of Cell

*tetA* = codes for transporter protein that removes tetracycline from the cell *tetR* = codes for repressor protein TetR that binds promoter regions to block own expression of tetR and expression of tetA -promoter binding represses transcription of both tetR and tetA Upon cellular entry, tetracycline binds to and releases tetR from the promoter regions -tetR and tetA genes are turned on -presence of tetracycline induces efflux of tetracycline out of cell -tetracycline binds to TetR, inhibiting its binding to promoter -more TetR is made because tetR is open to transcription -TetR binds promoter again when tetracycline is gone TetA begins to transport tetracycline out of the cell -tigtly regulating the inducible expression of tetracycline efflux system, which allows the cell to conserve energy

*KNOW* Two-Component Regulatory System (TCRS): VR/ Vancomycin Resistance by Target Modification

*vanS* =membrane associated sensory kinase *vanR* =transcriptional activator of vanHAX operon *vanH* =dehydrogenase that reduces pyruvate to D-Lac *vanA* =ligase that catalyzes ester bond formation between D-Ala and D-Lac *vanX* =dipeptidase that hydrolyzes/breaks D-Ala-D-Ala

Quiz 5: Which of the following antibiotics can be inactivated by B-lactamases?

-Ampicillin -Penicillin -Cephalosporin (They are all B-lactam antibiotics)

*KNOW* E. faecium

-Gram+ Epidemiology -Part of the HGM/ Human Gut Microbiome -66,000 infections in the US per year -30% are Van resistant Disease -UTI, abdominal or pelvic wound infections, bacteremia -3rd most frequent cause of nosocomial BloodStream Infections (BSI) -up to 70% mortality rate (VRE/ Van Resistant Enterococcus) Resistance -emerged in late 1980s -by 2002, over 60% VRE -D-Ala-D-Ala to D-Ala-D-Lac -vanHAX genes located on plasmid-residing protein -expression of PBP5 = low affinity PBP

Quiz 5: Which of the following is the mechanism of vancomycin resistance by target modification?

-alanine to lactate substitution

Quiz 5: What is the functional consequence of drugs that inhibit the generation of muropepetides?

-an increase in efficiencies of B-lactam antibiotics

Quiz 4: 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

Quiz 5: E. coli efflux pumps ___ MIC of antibiotics.

-increase Higher MIC = greater chance for antibiotic resistance Higher MIC = need to take more antibiotics to treat infection

Quiz 5: What is the purpose of aminoarabinosylation of lipid A, lysinylation of membrane phospholipids, or alanylation of anionic teichoic acids?

-increase membrane positive charge

Quiz 4: 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 MDR bacteria -it's a last-resort antibiotic

Quiz 4: Polymyxins and daptomycin are examples of:

-lipopeptides

Quiz 6: Canada has a much lower prevalence of MRSA compared to other countries. What is/are the main reason(s) for this low prevalence?

-low antibiotic prescription rate -MRSA patients are isolated upon hospital admission

Quiz 5: Bacteria gain resistance to trimethoprim mainly by which of the following mechanisms?

-mutations in DHFR

Quiz 5: S. aureus resists vancomycin by substituting its alanine to lactate in the termini pentapeptide of lipid II. What is another way S. aureus employs to resist vancomycin?

-overproduction of peptidoglycan This decreases crosslinking -deploys fake antibiotic binding molecules -less D-Ala-D-Ala for vancomycin to bind

Quiz 5: Which of the following statements best describes porins?

-provide the first line of defense against toxic compounds

Quiz 6: Biofilms are ___. Check all that apply.

-resistant to antiseptics -resistant to antibiotics

Quiz 6: What are planktonic bacteria?

-single cells floating in liquid

*KNOW* Antibiotic Resistance Mechanisms of biofilms

1) *Physical Barrier* -results in restricted penetration/ slow penetration of antibiotics -thick DNA, protein, exopolysaccharide matrix -outer cells absorb damage -protecting cells deeper in biofilm 2) *Antibiotic Degradation* -acquired antibiotic resistance 3) *Presence of Persister Cells* -cells within biofilms are not as metabolically active -such non-growing (persister) cells are resistant to antibiotics 4) *Biofilm-specific Efflux Pumps* -acquired antibiotic resistance 5) *Stress Responses* -acquired antibiotic resistance 6) *Changing the microenvironment of the biofilm* *KNOW* Altered Microenvironment = unfavorable conditions -low O2, pH, and hydration -contributes *MOST* to antibiotic resistance

*KNOW* Strategies to treat biofilm infections

1) *Substances that destroy biofilm matrix* -destroy exopolysaccharides 2) *Molecules that target persister cells* -some things can go through outside layer 3) *Quorum-quenching enzymes* 4) *Substances that cause biofilm self-destruction* 5) *Strategies that boost antimicrobial agent action* -ex) electrical current 6) *Physical disruption of biofilms* -oral hygiene

*KNOW* Diversity of Biofilm-Associated Infections

1) Sinusitis 2) CNA 3) Keratitis 4) Otitis 5) Cochlear implant 6) Burn infections 7) IV catheter infections 8)Prosthetic valves 9) Pacemaker infections 10) Endocarditis 11) Biliary stent infection 12) Peritoneal dialysis catheter 13) Prosthetic join infection 14) UTI 15) IV stent infection 16) Cystic fibrosis 17) Ventilator-associated pneumonia 18) Breast implant infection

*KNOW* Target Replacement: Methicillin Resistance

1986: *Beck et al.* published study that described a novel DNA material encoding something that is responsible for Methicillin resistance Methicillin resistance was *not* dependent on typical B-lactamases -was encoded by *mecA* *mecA* =gene coding for PBP2a -gene located on mobile element called *Staphylococcal cassette chromosome mec* (*SCCmec*) -*intra* and *inter*species mobilization can be facilitated through HGT *PBP2a* =PBP homolog that exhibits low affinity for all clinically used B-lactam antibiotics *source of resistance and mechanism of acquisition are unknown*

*KNOW* Nosocomial Infections Associated with Biofilms

65% of all bacterial infections are thought to be associated with biofilms Common biofilm-harboring devices include: -Heart associated implants (ventricular-associated, shunts, pacemakers, defibrillators, valves) -mechanical ventilators and endotracheal intubation -catheters -breast implants -join prosthetics bacteria that grow on biofilms are 1,000x more resistant to antibiotics than other planktonic counterparts *KNOW* Planktonic Bacteria = floating as single cells in liquid

*KNOW* Enzymatic Modification of Antibiotics

= biochemical modification of antibiotics that increases steric hindrance -antibiotic loses its ability to recognize targets and MIC increases *Modifications* Nucleotidylation = addition of nucleotides Phosphorylation = phosphate group addition Glycosylation =attachment of a carbohydrate or sugar Acylation = addition of the acyl (RCO-) group Hydroxylation = addition of hydroxyl (-OH) group

*KNOW* Minimum Inhibitory Concentration (MIC)

=min concentration of an antibiotic required to completely inhibit bacterial growth Infection susceptible to treatment if antibiotic MIC is non-toxic to humans Manifests *clinical resistance* when antibiotic MIC is toxic to humans -can no longer be treated with antibiotic *KNOW* Most effective treatment of bacterial infections is when the antibiotic MIC is non-toxic to humans -administration of toxic concentration depends on benefits and risks -reserved for extreme cases where bacteria are pan-resistant

*KNOW* Clinical Resistance

=multiple factors such as: -type of bacteria -infection site -antibiotic pharmacokinetics -immune response All affect clinical outcomes of antibiotic treatment -deem resistant because antibiotic concentration needed for treatment is unachievable

*KNOW* B-lactamases are Enzymes that *Hydrolyze* B-lactam Antibiotics

Abraham, Chain, and Kirby discovered an enzyme that *hydrolyzes B-lactam rings* found in penicillins *KNOW* Penicillinase/ B-lactamase =enzyme that facilitates hydrolysis of B-lactams -renders them ineffective at killing bacteria

*KNOW* The Unknown Enzyme - Penicillinase

After series of additional experiments, *Abraham* and *Chain* concluded the excreted enzyme is responsible for deactivating penicillin Thought it was an enzyme because it was deactivated by heat -heated to 90*C for 5 minutes, inactivating it -inactivated with protease Papain, which digests enzymes -enzyme itself was non-dialysable through cellophane membrane -activity lost through alcohol precipitation -enzyme loses ability at lower pH *Named the enzyme Penicillinase* Not until 1940 when *William Kirby* extracted penicillinase from clinical isolates of Staphylococcal strains -2 years after penicillin introduced to market -few samples were already found to be completely resistant to penicillin

*KNOW* Charge Alteration: Alanylation

Alanylation = addition of alanine Alanylation of anionic teichoic acids at Gram+ cell wall changes negative charge of cell to net positive charge -call wall usually has negative charge due to phospholipids, attracting cationic molecules Positively charged membrane electrostatically repels cationic compounds/ cationic antimicrobial peptides -positively charged amine group pf each alanine adds net positive charge

*KNOW* Antibiotic Target Replacement

Alternate proteins that have the *same functions* as other antibiotic target proteins, but are *structurally different* -replacement activity of other antibiotic-sensitive proteins in presence of antibiotics -ex) Methicillin-resistant Penicillin Binding Protein (PBP2a)

*KNOW* Target Modification: DNA Gyrase and topoisomerase IV (Quinolone Resistance)

Amino acid substitutions in DNA gyrase and topoisomerase IV result in decrease of quinolone binding affinity Most common amino acid substitutions are localized to GyrA and ParC of gyrase and topoisomerase IV, which can bind quinolones and responsible for DNA breakage-reunion

*KNOW* Ribosomal Alteration, conferring AbR: Ribosomal Alteration - 30S

Aminoglycosides and tetracyclines bind to 30S subunit of the bacterial ribosome -Aminoglycosides inhibit tRNA translocation from A to P site, causing misreading of mRNA -Tetracycline binding site overlaps partially with aminoacyl-tRNA binding site, leading to tRNA dissociation from ribosome, consequently blocking translation Nucleotide substitution mutation in 16S rRNA has ben linked to *resistance* of bacteria to both aminoglycoside and tetracycline antibiotics

*KNOW* S. aureus tightly regulates mecA

B-lactams activate MecR1 -*MecR1* = rapidly induces expression of *mecA-mecR1-mecI-mecR2 operon* Anti-repressor activity of *MecR2* is essential to sustain *mecA* induction -promotes inactivation of *mecI* inhibition by proteolytic cleavage In absence of B-lactams, MecR1 is *not* activated -MecI dimers inhibit mecA promoter -Residual MecR1 remain at the cell membrane

*KNOW* Antibiotic Inactivation by Sequestration

Bacteria have developed antibiotic sequestration mechanisms Klebsiella pneumonia, Streptococcus pneumoniae, or Pseudomonas aeruginosa -release *capsular polysaccharides* (both By Gram + and Gram-) -bind to polymyxins and inhibit their membrane destroying property Presence of antibiotics, like Vancomycin, increases *secretion of DNA* by both Gram+ and Gram- -DNA binds to Vancomycin and prevents its cellular entry Bacteria also secrete *proteins including lipocalins* and other *small molecules like monomeric phosphatidylglycerol* -membranes and molecules bind and sequester antibiotics, including polymyxins, rifampicin, and daptomycin Gram- have evolved mechanism by which it secretes *membrane vesicles* which sequester membrane disrupting antibiotics -antibiotics = colistin or polymyxin -prevents actual antibiotic from getting its target at the cell membrane

*KNOW* B-lactamase Classification

Based on amino acid sequence in their active sites, B-lactamases are divided into molecular classes: -A, C, and D class enzymes utilize *Serine in the active site* for B-lactam hydrolysis -Class B lactamases are *metalloenzymes* that require divalent zinc ions for substrate hydrolysis

Quiz 4: Match each ecosystem with its corresponding selection pressure.

Clinical Ecosystem = high selectivity Non-clinical Ecosystem = medium selectivity Environmental Ecosystem = low selectivity

*KNOW* Bulky side groups protect the B-lactam ring from B-lactamases

Each generation of Penicillin and Cephalosporin gets an increasingly bulky side group/ R group -is attached to B-lactam ring Bulky side chains *sterically protect* access of B-lactamases to the B-lactam ring -form a shield around B-lactam ring -decreasing ability of B-lactamases to inhibit the B-lactam ring Continually changing the sidechains to be increasingly bulky also stimulates the evolution of additional B-lactamases

*KNOW* Charger Alteration: Lysinylation

Daptomycin has an overall + charge Lysine residue added to a phospholipid by a multi peptide resistance factor (MprF) enzyme -shifts net charge from dianionic (2 negatively charged phosphate) to monocationic (2 positively charged amines) -negative charge of phosphate is neutralized by 2 amines *Opposites attract, like repels* -Resulting positive net charge repels daptomycin away from cell wall

*KNOW* Efflux Pump Inhibitor Strategies

Efflux pumps provide potential new targets against antibiotic resistance: 1) Suppress efflux pump expression 2) Alter antibiotic structure 3) Disrupt pump assemble 4) Disrupt AcrZ IMP/ Inner Membrane Protein (AcrB) interaction -AcrZ is recently discovered domain for efflux pump of small molecules 5) Inhibition of IMP/Inner Membrane Protein (AcrB) 6) Block OMP/ Outer Membrane Protein 7) Disrupt proton motif force with ionophores ex. Verapamil -potently decreases MIC of antibiotics such as bedaquiline and clofazimine in M. tuberculosis treatment by 8 to 16 fold -FDA approved drug that is used in treatment of hypertension -recently repurposed as successful efflux pump inhibitor

*KNOW* Efflux Pumps provide protection against own antibiotics

Employed to avoid toxicity from own antibiotics Streptomyces export *glucosyl oleandomycin (inactive macrolide)* via *OleB efflux pumps* -inactive antibiotic is transported out of the cell via OleB Streptomyces can also make and secrete *beta glucosidase* that cleaves the sugar moiety from oleandomycin, turning it into active form -Beta glucosidases are secreted into the extracellular space Oleandomycin gets activated once outside of the cell and poses no threat to Streptomyces cells that produce it -when glucosidases and glucosyl oleandomycin meet in extracellular space, glucosidase cleaves glucosyl sugar and makes active form of oleandomycin outside of the cell -activated antibiotics is outside, no harm to original cell

Quiz 6: ESKAPE pathogens attack only immunocompromised individuals. True/False

FALSE

Quiz 5: Efflux Pumps permit diffusion of molecules across the plasma membrane. Hence, they do not require energy. True/False

FALSE Efflux Pumps require energy This question is describing "porins"

Quiz 4: Only some antibiotics affect human microbiota. True/False

FALSE Essentially all antibiotics have an effect on the human microbiota

Quiz 6: VRSA is as prevalent as HA-MRSA. True/False

FALSE There are very few cases of VRSA -is becoming more prevalent now

*KNOW* What is a biofilm?

First described by *Arthur Henrich* in 1933 -"its is quite evident that for most part water bacteria are not free-floating organisms, but grow upon submerged surfaces" "Structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface." -Pozo & Patel, 2007 = *formal definition for biofilms* A diverse community with complex cell-cell chemical signaling (*Quorum Sensing*) to produce a protection shield from outside environment

*KNOW* Mechanism of B-Lactamase Induction: AmpG-AmpR-AmpC Pathway

For the most part, is conserved, bit variations exist throughout Gram- species *Mechanism* -Upon B-lactam antibiotic treatment, there is inhibition of cell wall synthesis -*lytic Transglycosylase (LT)* cleaves and releases fragments of peptidoglycan known as *"Muropeptides"* -Muropeptides = Lipid II -are major triggers for the AmpGRC Pathway -Muropeptides are transported into the cytoplasm by *AmpG transporter* -in cytoplasm, *NagZ enzyme* removes Nag sugar from Lipid II -remaining *NAG-oligopeptide* interacts with *AmpR* located upstream of AmpC (B-lactamase gene) -AmpR = transcriptional regulator -also has other functions -in P. aeruginosa, found to induce expression of other defense mechanisms, like protease quorum sensing, and other virulence factors -*AmpR* activates transcription of *AmpC* -B-lactamases are then secreted from the cell and degrade the B-lactam antibiotics, even BEFORE they have a chance to pass through the outer membrane

*KNOW* Porins allow transport of antibiotic into the cel

Porins allow transport of small molecules (B-lactams and tetracyclines) across the outer membrane Predominantly in Gram- bacteria -like efflux pumps *Porins* = transmembrane channels, usually B-barrels, that allow molecules to enter the cell Porins DO NOT require energy in order to transport molecules across membranes

*KNOW* Charger Alteration: Aminoarabinosylation

Represents a mechanism by which bacteria add amino-sugar moiety to Lipid A Such modification results in overall membrane charge increase -results in *electrostatic repulsion of cationic microbial peptides* -ex: Polymyxins Modification mainly seen in LPS/ lipopolysaccharides of Gram-

*KNOW* ESKAPE Pathogens

Represents the most debilitating HA infections -Enterococcus faecium -Staphylococcus aureus -Klebsiella pneumoniae -Acinetobacter baumannii -Pseudomonas aeruginosa -Enterobacter spp. All have in common: -found in both developed and developing countries -*most common bacteria found in all hospitals* -pathogenic -transmissible -highly resistant

*KNOW* TCRS control expression of mprf gene

Resistance regulated by a two-component histidine kinase regulatory system -Daptomycin is sensed by a sensory kinase, which in turn phosphorylates and activates the regulator that induces the expression of mprf gene Induction of mprf gene leads to elevated levels of mprf, adding lysines to phospholipids

*KNOW* Target Modification: Rna Polymerase (Rifampicin Resistance)

Rifampicin binds directly to RNA polymerase and inhibits its function -Bacteria introduce mutations in RNA polymerase -Majority of mutations are highly conserved on amino acids that maintain RNA pol functions, but abolished rifampicin binding *Amino Acid substitution* results in *high fitness cost* as a result of *reduced transcriptional efficiency* -Rifampicin resistance cripples bacteria a little, but allows them to stay alive

*KNOW* Enzymatic Linearization

Rifamycin Monooxygenase (ROX) =enzyme that linearizes rifampicin -this mechanism of resistance is common against *cyclic antibiotics* -lipopeptides -ansamycins -streptogramins

*KNOW* Antibiotic Target Protection: Overproduction of Peptidoglycan

S. aureus resists Vancomycin by overproducing peptidoglycan with decreased cross-linking -deploying fake antibiotic binding molecules -decreased cross-linking exposes more D-Ala-D-Ala peptides that bind Vancomycin -bound Vancomycin forms a barrier around the cell

*KNOW* Currently, there are over 1,000 B-lactamases identified

Since 1940 discovery of Penicillinase, MANY were found within 50 years Major contributor in growth of these enzymes is ability to *transfer genetic material* amongst strains and species Discovery of B-lactamases also triggered scientific community to pursue identification of compounds that would inhibit B-lactamases -Clavulanic acid (*Clavams*) is a B-lactam that does not posses any antibiotic property itself -but does bind and inhibit B-lactamases, preventing their destruction of other B-lactam antibiotics

*KNOW* TCRS controlling B-lactamase synthesis

Staphylococcus resistance to B-lactams is mediated by B-lactamase encoded by *blaZ* -expresion of blaZ is negatively regulated by BlaI *BlaI* = transcriptional inhibitor that binds to the promoter region of blaZ -prevents activation of blaZ transcription *BlaR* sits on the membrane and is a transmembrane sensor that, upon exposure or presence of B-lactams, auto cleaves itself -cleaved fragment becomes protease, leads proteolytic cleavage of *BlaI* -*BlaI* leaved the promoter from blaZ and turns gene on *SUMMARY* Exposure to B-lactams: -*BlaR1* cleaves itself and becomes an active protease -*Protease *BlaR2* cleaves and inactivates BlaI repressor -leads to activation of expression

*KNOW* Mechanism of B-Lactamase Induction: Two Component Regulatory System (TCRS)

TCRS = signaling cascade that allows a cell to sense the outside environment/ extracellular signal such as antibiotic, and responds accordingly through a signaling cascade 2 components: 1) Sensory Kinase 2) Regulator Histidine Kinase TCRS, BlrAB, is not well understood -most evidence from its environment comes from genetic studies of Aeromonas *BlrAB* = BlrA sensory kinase + BlrB response regulator -overexpression of BlrA increases production of B-lactamase -BlrA knockout attenuated expression of B-lactamase -showed BlrA is most likely a sensory kinase -studies of promoter region revealed that deletion of the BlrB DNA binding site attenuated expression of B-lactamase -suggested BlrB is a response regulator Similar TCRS identified in other species: -E. coli -different Aeromonas spp. -P. aeruginosa -S. maltophilia Recent studies in Parahaemolyticus identified the *histidine kinase sensor to function directly as a B-lactam receptor* -translated the signal to a repsonse regulator that controls the expression of B-lactamase

*KNOW* TCRS are involved in sensing inactivation of many antibiotics

TCRS VanS and VanR are responsible for the detection of Vancomycin/ other glycopeptides *VanS* =sensory kinase that detects presence of extracellular glycopeptide -sends the signal to VanR *VanR* =receives signal from VanS and activates transcription of VanHAX genes

Quiz 4: The majority of antibiotic classes were developed during the Golden Age of antibiotics. Since the, very few classes were introduced into clinical use. True/False

TRUE

Quiz 6: It is estimated that ~30% of the world's population is colonized by S. aureus. True/False

TRUE

Quiz 6: Staphylococcus aureus is the cause of the most common hospital-acquired infections. True/False

TRUE

Quiz 5: Tetracycline-resistant ribosomal protection protein, tet(O), binds to the bacterial ribosome leading to conformational changes that prevent tetracycline binding to its target even after tet(O) dissociates. True/False

TRUE tet(O) = translational GTPase -structurally similar to EF-G -can therefore bind to bacterial 70S ribosome tet(O) binds to tetracycline-blocked ribosome -leads to conformational change to release tetracycline -tet(O) leaves binding site -ribosomes remains in changed conformation so tetracycline cannot bind when tet(O) is gone

Quiz 4: 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 MIC = higher amount of antibiotic needed to treat infection Higher MIC can be toxic, leading to clinical resistance

*KNOW* Pentapeptide Modification that Confers Vancomycin Resistance

Target Modification = Vancomycin Resistance Glycopeptides/ *Vancomycin* -Gram+ are now resistant tp penicillin/ methicillin -Van used to treat MRSA -"last resort antibiotic" -caps D-Ala-D-Ala and prevents its incorporation into peptidoglycan and subsequent crosslinking Vancomycin Resistance appeared shortly after its introduction -S. aureus (VRSA) is able to substitute lactate for alanine -peptide to ester bond -makes D-Ala-D-Lac, *NOT* D-Ala-D-Ala Ala to Lac change prevents Van binding -no effect on cell wall integrity -only affect against antibiotic Horizontal Gene Transfer (conjugation) of *vanA operon* that encodes components necessary for substitution -activation of vanA operon occurs in presence of low Van concentration

*KNOW* Point Mutations/ Deletions/ Insertions confer antibiotic resistance by modifying antibiotic targets

Target Modification: Dihydrofolate Reductase (Trimethoprim Resistance) Single amino acid substitutions in DHFR are enough to diminish trimethoprim binding Amino acid substitutions at the active site confer most resistance

Some bacteria can produce enzymes that inactivate antibiotics: Other than B-lactamases

Tetracycline destructase =enzymes that degrade tetracycline antibiotics

*KNOW* Biofilms and our bodies

Toothbrushing is *better than antibiotics* as it *mechanically removes* harmful biofilms Biofilms are *resistant to antibiotics and other antiseptics*

*KNOW* Mechanisms of B-lactamase Induction: Potential new targets to combat resistance

Understanding mechanisms of B-lactamase induction provides additional therapeutic targets to combat antibiotic resistance *Bulgecin A* =compound that selectively binds to and inhibits bacterial lytic transglycosylase -increases efficacy of B-lactam antibiotics -is a selective inhibitor *Bacterial Lytic Transglycosylase* =enzyme responsible for clipping and releasing peptidoglycan as muropeptides -inhibition of this enzyme inhibits degeneration of muropeptides -decreases production of B-lactamases through AmpGRC system -*Increases* efficacy of B-lactam antibiotics Identified additional small molecules that target: -LT -NagZ -AmpG -AmpR -shown to increase efficiency of B-lactam antibiotics by inhibiting levels of B-lactamases Inhibitors are currently used as drugs in combination with B-lactam antibiotics -treat infections caused by B-lactamase producing bacteria

Quiz 6: What does VRE stand for?

Vancomycin Resistant Enterococci