Week 10; Antibiotic Resistance, Methicillin‐resistant Staphylococcus aureus (MRSA) Lecture 22, 23, 24

DNA replication & transcription inhibitors

**Picture of 15-14*** Slide 10; Lecture 22 PP

Antibiotics and their mechanisms of action

**Picture of big table 15-2** Slide 8/9; Lecture 22 PP

How Antibiotic Resistance spreads

**Picture of graphic** Slide 17, Lecture 22 PP

β‐lactam resistance

**high level β‐lactamase expression (i.e. gene duplication) can overcome clavulanic acid supplementation (binding/sequestering ) **zinc β‐lactamases use a catalytic mechanism that does not involve active‐site serine residues to cleave β‐lactam ring; resistant to clavulanic acid **clavulanic acid only inhibits serine β‐lactamases -Cluvulanic acid: used in conjunction with antibiotic **Picture of Figure 16-1** Slide 10; Lecture 23 PP

Regulation of tetB (efflux pump) expression

**picture of Figure 16-6** slide 12; Lecture 23

Common disinfectants and their mode of killing

- Alcohols (ethanol, isopropanol): denature proteins - Alkylating agents (formaldehyde, ethylene oxide): form epoxide bridges that inactivate proteins - Halides (I-, Cl-, NaClO): oxidizing agents - Heavy metals (Hg2+, Ag+): Bind -SH groups, thus denaturing proteins - Phenols: Denature protein; disrupt cell membranes by intercalating in them - QACs: disrupt cell membranes by intercalating them - UV radiation: blocks DNA replication and transcription by damaging DNA

Regulation of blaZ (β‐lactamase) expression

- BlacR1 looks out for B-lactam - BlacR2 released into cytosol, cleave BlacI protein, prevents binding to promoter region of blaZ, allows blaZ expression/ secretion outside cell, cleaves antibiotic - blaZ only expressed in presence of antibiotic, actively repressed by BlacI in absence of antibiotic, DNA binding protein that binds the operator sequence and prevents blaZ expression, autoregulates its own expression **Picture of Figure 16-6** Slide 14; Lecture 23

Mechanisms of tetracycline resistance

- bacteriostatic; translation inhibitor 1. Tetracycline sensitive cell: diffuses into cell, accumulates, interacts with 16S RNA subunit, blocks protein synthesis 2. Tetracycline efflux pump (cytoplasmic membrane): Intracellular [tetracycline] remains too low; prevents binding to 16S rRNA 3. Ribosome modification/protection: TetM GTPase activity perturbs helix in 16S region bound by tetracycline; prevents binding to 16S rRNA -will accumulate but cannot bind to 16S subunit

Vancomycinresistant enterococci (VRE):

- genetically encoded resistance to vancomycin • Represents about 30% of all hospital‐acquired Enterococcus infections • Enterococcus sp. intrinsically resistant to various β‐lactams, fluoroquinolones, and aminoglycosides • VRE infections usually treated with newer antibiotics such as daptomycin • HORIZONTAL GENE TRANSFER A CONCERN (very good at exchanging DNA) - i.e. VRE MRSA = VRSA

Cell Wall Synthesis Inhibitors

- glycopeptides (vancmycin): block both transpeptidation and the transglycosylation step - all have four member ring, kill bacteria, inhibit last step in peptiodoglycan synthesis (cross-linking of side chains in backbone), bind and inhibit penicillin binding proteins, allows enzymes to become hyperactive and chew up and lyse the cell **Picture of 15-5*** Slide 12; Lecture 22 PP

Virulence factor expression is coordinately regulated by the agr quorum‐sensing system

- helps correlate which proteins are produced during which phase of growth - QS system; staph has two operons on its chromosome (RNA II is one). - two component system= AgrC and AgrA - AgrC is a membrane bound sensor kinase protein and AgrA is the response regulator which is the transcription factor - AgrD encodes a precursor peptide that is secreted through cell wall into external env. it is processed by AgrB which is a membrane bound protease; AgrB cleaves AgrD and the mature form of the peptide undergoes a cyclic event and is called AIP; AIP is the QS molecule - As the cell grows during exponential phase the system is expressed at very low levels (AIP); more dense culture causes AIP to accumulate and once it reaches threshold it binds AgrC, turns it on and causes phosphorlyation cascade from AgrC to AgrA and AgrA binds promoter turns on system at a high level by positive feedback, also turns on divergent transcript of RNA III which is an untranslated RNA molecule that acts as an effector of the system which coordinates the transition between colonization and invasive phenotypes **Picture of slide 10** Lecture 24 PP

Biofilm infections

- multicellular mode of growth where bacteria adhere to a surface, protective matrix of bacterial community allows antibiotic tolerance, can cause infection at almost every body site -Biofilms are inherently tolerant to antibiotic treatment (various mechanisms) - Antibiotic tolerance: growth of bacteria stops in the presence of antibiotic, but cells are not killed 1. Failure to penetrate the biofilm; impairment of antibiotic in areas of waste accumulation of altered environment (pH, pCO2, pO2): not universal 2. Trapped/destroyed by matrix elements 3. Altered growth rate; slow growing= more resistant 4. Biofilm-specific resistance genes 5. Stress response to hostile environmental environmental conditions; expression of enzymes

Metabolic control of S. aureus virulence

- past focus on characterizing traditional virulence factors but Staph metabolism is important for virulence - two-component system w complete TCA cycle and ETC; sense changes in env and metabolic conditions to change metabolism to be suitable - TCA cycle, redox state of cell (NAD to NADH ratio), production of reactive O2 species from ETC, and two component systems all important influence of virulence factor production **Picture of slide 16** Lecture 24

S. aureus "arsenal" of virulence factors

- so many diseases because so many different virulence factors; expressed on growth phase dependent manor - growth sigmoidal in shape; early exponential phase has upregulated surface proteins, turned down in stationary phase and secreted proteins are more highly expressed - lipotechoic acid (endotoxin) secreted during cell lysis **Picture of Figure** slide 9; lecture 24 PP

S. aureus intracellular lifestyle

- staph can be taken up by nonphagocytic cells and hides from immune system -Small‐colony variants (SCVs): • stable or unstable (reversible) • Stable phenotype from genetic defects in electron transport chain co‐factors or thymidine biosynthesis • reduced virulence • increased antibiotic tolerance • avoidance of immune system - Non‐phagocytic internalization: Bacterial cell‐surface fibronectin‐binding protein Host fibronectin host cell integrin receptor - creates bridge between bacteria and host cell and induces internalization **Picture of slide 15** Lecture 24 PP

Staphylococcal biofilm development:

- staph detaches from mature biofilm in sub populations and finds new site to populate • Biofilms are inherently more recalcitrant to antibiotic treatment and the immune system • Need for a better understanding of biofilm physiology and development in order to develop novel therapies - each phase of growth is mediated by a specific series of genes and events that are highly regulated **Picture slide 13** Lecture 24

Translation inhibitors

- usually inhibits an aspect of the ribosome; different drugs have different targets **Picture of 15-9*** Slide 11; Lecture 22 PP

Persister cell hypothesis

-(subpopulation of dormant/non‐growing cells that Toxin‐antitoxin modules, survive antibiotic treatment) -ppGpp induced in subpopulation inhibit macromolecular synthesis processes dormancy - persisters reactivate growth and repopulate over time, why biofilms are chronic, reoccurring infections that are very resistant to treatment

Mechanism of vancomycin resistance

-Vancomycin binds to the C‐terminal acyl‐D‐Ala‐D‐Ala of the undecaprenol‐diphosphate MurNAc‐pentapeptide intermediate and inhibits transglycosylation and transpeptidation reactions in cell wall peptidoglycan polymerization and cross‐linking **There have been 11 VRSA (Vancomycin‐resistant S. aureus) strains isolated in the USA; HGT of VanA/B, VanX from VRE via a transposon **Gram -ve bacteria naturally‐resistant to Vancomycin (bulky cannot diffuse through outer membrane porins **Picture of Figure 16-4** Slide 7; Lecture 23 PP

Key Concepts in Antimicrobial Therapy

1. Bacteriostatic vs. Bacteriocidal Bacteriostatic - antimicrobial compounds that inhibit bacterial growth - Bacteria can frequently recover once treatment is removed; best used in patients with intact immune systems Bacteriocidal - antimicrobial compounds that kill bacteria - Best for patients with compromised immune systems **bacteriocidal vs. bacteriostatic can be blurred by properties of bacteria (i.e. fast vs. slow‐growing, biofilm growth)** Minimum Inhibitory Concentration (MIC) - Lowest [antibiotic] that will prevent bacterial growth - Dilution susceptibility assay Minimum Bacteriocidal Concentration (MBC) - Lowest [antibiotic] that will kill bacteria - rapidly growing bacteria more susceptible to bacteriocidal; if slow growing/metabolically inactive/biofilm bacteriocidal will act more like bacteriostatic 2. Pharmacokinetics (distribution of antimicrobial compound in host body): - Toxicity dictates external vs. internal use; something may be safe for external use but toxic if ingested at same [ ] - absorption, dosage, distribution/dissemination, route of administration, stability 3. Side Effects: - Antimicrobial agent specificity for bacterial target without affecting mammalian cells (differential toxicity) - "risk vs. reward" - some side effects are worth dealing with if infection is serious/life threatening

Overview of resistance mechanisms

1. Limiting access of the antibiotic • active efflux of antibiotic (antiporters, ABC transporters) - pumps pump antibiotic out of cell faster then it enters, low concentration of antibiotic ineffective 2. Enzymatic inactivation of the antibiotic • B‐lactamase (penicillin resistance), (chloramphenical/streptogramin acetyltransferases) - enzymes cleave/modify antibiotic to inactivate it 3. Modification or protection of target • PBPs (S. aureus methicillin resistance), glycopeptide resistance (VRE), tetracycline resistance (widespread), macrolide resistance (widespread) - penicillin binding proteins with modified structure so methicllin no longer recognizes it

How Antibiotic Resistance Happens

1. lots of germs, a few are drug resistant 2. antibiotics kill bacteria causing the illness, as well as good bacteria protecting the body from infection 3. the drug-resistant bacteria are now allowed to grow and take over 4. some bacteria give their drug resistance to other bacteria, causing more problems • Antibiotic‐resistant infections can happen anywhere. • Data show that most happen in the general community • However, most deaths related to antibiotic resistance happen in healthcare settings, such as hospitals and nursing homes.

Disinfectants vs. Antiseptics

Disinfectants - applied to non‐living objects or surfaces - i.e. bleach, alcohols, phenolics - primarily bacteriocidal, too toxic for use for external/internal treatment of patients Antiseptics - applied to living skin or tissue - Often contain diluted disinfectants • Often also active against viruses, fungi, protozoa • Often several targets in bacterial cell • Most effective against actively growing bacteria • In general, resistance mechanisms poorly understood relative to antibiotics - Membrane‐active agents less effective against Gram -ve bacteria - Membrane pumps (Quaternary Ammonium Cation resistance in staphylococci)

S. aureus agr two‐component system (TCS)

S. aureus agr two‐component system (TCS) • Coordinates the transition between colonization and invasive phenotypes (RNAIII) - Cell‐surface adhesins highly up‐regulated during exponential growth phase; secreted toxins and tissuedegrading enzymes up‐regulated during stationary phase - Transcriptional and post‐transcriptional mechanisms - Complex interplay between agr and many other transcriptional regulators (other TCS, sigma factors, etc) • Also an important regulator of biofilm development in S. aureus - Accumulation of AIP induces biofilm dispersal - Naturally‐occurring agr mutants have been isolated from biofilm infections (agr mutation = increased biofilm formation)

Tube dilution method for MIC determination

• A series of increasing concentrations of antibiotic is prepared in the culture medium. Each tube is inoculated, and incubation is allowed to proceed. • Growth (turbidity) occurs in those tubes with antibiotic concentrations below the MIC; can't be used to determine bacteriocidal concentration; would have to plate out onto agar plates and check viability for that - inoculate each tube with same concentration of bacteria amount

Antibiotic resistance in bacteria

• Although a trend for decades, public first began to really take notice of antibioticresistant bacteria in 1990s (hospital‐acquired MRSA**, MDR TB infections) **BOX 16‐1 read - Prohibitive treatment costs (insurance companies, HMOs) - Agricultural use of antibiotics in feed - Discovery of new antibiotics nearly ceased; not a big $$ maker for industry - 1980s research effort (mechanisms of resistance, regulation of resistance genes) came to be viewed as "old‐fashioned" de‐emphasis of work and funding • Nowadays, multi‐drug resistant (MDR) bacteria a huge concern, as treatment options dwindle and research/drug development catches up...

Staphylococcus aureus

• Capable of infecting nearly every tissue and organ system in the human body - Boils, skin/soft tissue infection, abscess - Sepsis can spread to and grow in nearly all organ systems • Kidneys, heart, bone, spleen, liver, brain abscess, etc. - Also a major cause of biofilm infections • 20% persistent colonization; 30% transiently colonized - Primarily nares; also groin, GI tract - Colonization increases the risk for subsequent infection - Those with S. aureus infections are generally infected with their colonizing strain • Emergence of antibiotic resistant strains - MRSA: both community and hospital‐acquired strains - VRSA: not a serious problem...yet... - vancomycin last line of defense against staph, a few cases of vancomycin resistant staph but not many yet - yellow= keratanoid pigment that is an important virulence factor, more resistant to reactive O2 species and respiratory burst

Scary facts (2013 CDC report)

• Each year in the United States, at least 2 million people become infected with drug‐resistant microbes. • At least 23,000 people die each year as a direct result of these infections.

MRSA: Methicillin‐resistant S. aureus

• Emerged shortly after introduction of methicillin (2nd generation penicillin) in clinical setting (1960 1961) - developed due to Staph resistance to penicillin • Methicillin‐resistance encoded by the mecA gene alternative penicillin binding protein PBP2a resists β‐lactam antibiotics such as methicillin - Found on staphylococcal cassette chromosome (SCC) mec element: 50-60 kb extrachromosomal cassette that carries resistance, virulence, etc. - Often associated with genetic resistance to other classes of antibiotics: multi-drug resistant staph • In the USA, MRSA is among the leading causes of death by any single infectious agent - CDC estimates that 80,000 MRSA infections and 11,000 MRSA‐associated deaths occur in the US per year - recently categorized by the CDC as a "significant antibiotic‐resistant threat"

Antibiotics "agents against life"

• General characteristics: - Low‐medium MW compounds that kill or inhibit bacterial growth - Can be ingested or injected into humans or animals with minimal side effects - Generally interfere with a specific cellular process or enzyme • Characteristics of a "good" antibiotic: - Few or no side effects to the host "differential toxicity" - Broad spectrum of activity (but can have drawbacks...i.e. C. difficile) wipe out normal flora, can cause microbial shift diseases - Appropriate bioavailability (rate or amount of drug that reaches the infection site) and pharmokinetics to get to site(s) of infection - Pill/ingested form: eliminates need to keep patient in the hospital - Cost: limits for patient and medical system

Development of antibiotic resistance in bacteria

• Genomic plasticity of bacteria (core genome mutation acquisition/accumulation, intra‐/interspecies genetic exchange (HGT), mobile genetic elements(plasmids, transposons)) • 50% of antibiotic use in US estimated to be inappropriate - Antibiotics prescribed for viral or inappropriate bacteria - Use of antibiotics to enhance growth of livestock • Crowding, homelessness, poor nutrition/sanitation • International travel

Enterococcus faecalis

• Gram +ve facultative anaerobe • Causative agent of UTIs, abdominal/pelvic wound infections, bacteremia • Environment, GI tract • Opportunistic pathogen usually infections are hospital‐acquired

HA‐MRSA vs. CA‐MRSA

• Hospital‐acquired MRSA (HA‐MRSA) endemic in health‐care facilities in most developed countries - Highly‐transmissible "epidemic" strains; outbreaks in 1990s • 1st genuine cases of community‐acquired (CA)‐MRSA infection were reported among individuals in Western Australia in the early 1990s - Further outbreaks in late 1990s, early 2000s in patient groups that had not been hospitalized in recent past - USA400 strain: highly associated with necrotizing pneumonia or pulmonary abscesses and sepsis - USA300 strain: skin and soft‐tissue infections (outbreaks in prisons, locker rooms/sports teams, military); now the predominant strain - Increased virulence of CA‐MRSA is a subject of active research

In vivo biofilm studies - interplay with host immune system?

• In vivo catheter model (mouse) • S. aureus biofilms appear to be capable of attenuating traditional host proinflammatory responses, which may explain why biofilm infections persist in an immunocompetent host

Symptoms of MRSA skin infection

• Infected area resembles a spider bite (bump/infected area); unless a spider is actually seen, the irritation is likely not a spider bite: • Red, swollen, painful and warm to the touch • Full of pus or other drainage • Accompanied by a fever Treatment: infection lanced/drained by physician, and antibiotic prescribed; swab and test for MRSA to confirm **IMPORTANT not to ignore symptoms; if MRSA escapes the abscess, serious systemic infection can occur

Alternative Lifestyles of the Staphylococci

• Intracellular vs. Extracellular • Planktonic vs. Biofilm • Abiotic contamination vs. Biotic colonization

Extended Spectrum B‐lactamase (ESBL) producing Enterobacteriaceae

• Klebsiella pneumoniae - healthcare‐associated infections, including pneumonia, bloodstream infections, wound or surgical site infections, and meningitis. - Part of normal GI microbiota - opportunistic pathogen • E. coli - Variety of pathogenic strains and diseases (diarrhea, dysentery), UTIs, meningitis, respiratory tract, others) - Fecal‐oral, contaminated food, birth canal • In addition to penicillin, ESBLs can hydrolyze the B‐lactam ring of various cephalosporins - Usually associated with active site mutations of plasmid‐encoded β‐ lactamase genes - These plasmids often encode other antibiotic‐resistance genes (i.e. aminoglycosides), making treatment choices limited - Carbapenems usually used to treat these infections ■ 19% of healthcare‐associated Enterobacteriaceae infections are caused by ESBL‐producing Enterobacteriaceae. ■Patients with bloodstream infections caused by ESBL‐producing Enterobacteriaceae are about 57% more likely to die than those with bloodstream infections caused by a non ESBL‐producing strain.

Staphylococcus aureus

• May display hemolysis on blood agar plates • May display golden‐pigmented colonies •white colonies in some cases Catalase‐positive • Gram‐positive cocci "grape‐like clusters" • Coagulase positive: converts fibrinogen fibrin, results in clotting/agglutination • Tube coagulase test detects secreted coagulase • Slide coagulase test detects surfacebound coagulase • Thought to prevent phagocytosis - by coating bacteria in fibrin molecules

M. tuberculosis

• Multi‐drug resistant strains a BIG problem - A four‐drug regimen (incl. rifampin) is recommended for persons in US with slight to moderate risk for infection with multi‐drug TB. Examples of Genetic Resistance: (primarily modification of drug target) 1. Rifampin‐resistance: mutations in β subunit of RNA polymerase (transcription inhibitor) 2. Streptomycin‐resistance: mutations in genes encoding S12 protein and 16S rRNA (translation inhibitor) 3. Fluoroquinolone‐resistance: mutations in DNA gyrase gene (DNA replication inhibitor) - Other factors that makes TB difficult to treat and/or promotes drug resistance: ‐slow growth of organism:inherent tolerance to antibiotic treatment ‐intracellular: need antibiotics that can both penetrate the host cell membrane and get to the bacteria ‐patient compliance (treatment time can be from 2‐9 months, depending on resistance profile) - epidemiology: outbreaks in homeless shelters, etc. affect patient compliance also

In summary...

• S. aureus/MRSA continues to be a devastating cause of morbidity and mortality - On CDC's "one to watch" list • Treatment of genetically‐resistant strains (MRSA) complicated by alternative lifestyles (biofilm, intracellular growth) in vivo • A better understanding of virulence factor arsenal, metabolism, mechanisms of biofilm formation and intracellular survival will help in the development of desperately‐needed new antibiotic therapies

Mycobacterium tuberculosis

• Tuberculosis (TB) • "Acid‐fast" bacillus (Ziehl‐Neelsen stain of sputum) • identified from sputum culture • Chest X‐ray • TB skin test (indicates exposure; does not necessarily mean infection) • Aerosols from infected individuals (**Efficient!) alveoli internalized by macrophage inhibition of lysosomal fusion SLOW replication • Potential outcomes dictated by health status of infected individual: - Infecting microbes are killed/cleared - Infecting microbes remain viable but controlled in granuloma for many years - Growth, lung damage, dissemination and destruction of other organs - cell mediated immunity required to kill TB in the macrophage

CA‐MRSA transmission:

• direct contact with an infected wound • sharing personal items (towels, razors) that have touched infected/colonized skin - football teams/lockers rooms; quarantine area and bleach everything • MRSA infection risk increased when a person is in certain activities or places that involve crowding, skin‐to‐skin contact, and shared equipment or supplies - team sports