OPHTHALMIC ANTIMICROBIALS Alison Clode, DVM, DACVO Port City - - PowerPoint PPT Presentation

OPHTHALMIC ANTIMICROBIALS

Alison Clode, DVM, DACVO Port City Veterinary Referral Hospital Portsmouth, New Hampshire New England Equine Medical and Surgical Center Dover, New Hampshire

Overview

• Interpretation of efficacy
• Mechanisms of resistance
• Antibacterial agents
• Mechanism of action
• Applications in ophtho
• Antifungal agents
• Mechanism of action
• Applications in ophtho

Interpretation of Efficacy – in vitro

• 1. MIC = minimum inhibitory concentration
• Lowest concentration of antibiotic that inhibits growth of a specific
• rganism
• 2. MBC = minimum bactericidal concentration
• Lowest concentration of an antibiotic at which bacteria are killed
• 3. Breakpoint
• Antibiotic concentration dividing susceptible and resistant
• MIC < breakpoint à S
• MIC ≥ breakpoint à I, R

Interpretation of Efficacy – in vivo

• 4. PK/PD = pharmacokinetics (what the body does to the drug)

pharmacodynamics (what the drug does to the body)

• 5. Susceptible = bacteria inhibited by usually achievable concentrations
• f antibiotic when recommended dose used for particular site of infection
• 6. Intermediate = bacteria inhibited in sites were antibiotic is

physiologically concentrated or when higher-than-normal dosage can be used

• 7. Resistant = bacteria not inhibited by usually achievable concentrations
• f antibiotic with normal dosing schedules or when microbial resistance

mechanisms are likely

Interpretation of Efficacy

• Indices utilized:
• T > MIC = % time plasma

concentration is above MIC

• Cmax/MIC = max plasma

concentration relative to MIC

• AUC/MIC = plasma

concentration time curve (duration of drug exposure) relative to MIC

• Determined by various

animal models

www.rxkinetics.com

Time- versus Concentration-Dependent

www.slideshare.net

Mechanisms of Resistance

• Intrinsic to the bacteria
• Acquired by the bacteria

Acquired Mechanisms of Resistance

• 1. Modification of the antibiotic
• 2. Preventing antibiotic from reaching target
• 3. Modification of the target

Acquired Mechanisms of Resistance

• 1. Modification of the antibiotic
• Enzyme-induced damage to

antibiotic à inactive antibiotic

• Enzyme-induced acetylation,

adenylation, phosphorylation

• f antibiotic à alter affinity of

antibiotic for target

Acquired Mechanisms of Resistance

• 2. Prevent antibiotic from

reaching target

• Preventing intracellular drug

accumulation

• Alteration of porin channels à

reduced drug entry

• Production of active efflux

pumps à reduced drug retention

Acquired Mechanisms of Resistance

• 3. Modification of target by

altering:

• Binding proteins
• Ribosomes
• Chromosomes
• Cell physiology

Acquired Mechanisms of Resistance

• Vertical gene transfer = transfer
• f R-conferring gene to progeny
• Horizontal gene transfer =

sharing of R-conferring DNA among bacteria

• Same or different strains
• Transformation = DNA uptake

from environment

• Transduction = DNA transfer by

viruses

• Conjugation = plasmid exchange

via cell-to-cell contact

Antibacterial Agents

Antibacterial Agents

• Mechanisms of action = disruption of:
• 1. Cell wall synthesis
• 2. Cell membrane integrity
• 3. Protein synthesis
• 4. Folate metabolism
• 5. DNA synthesis

Bacterial Cell Wall

• Main component =

peptidoglycan

• PS + peptide crosslinks
• Formed by

transpeptidases (penicillin binding proteins)

• Gram positive:
• Thick cell wall with

greater peptidoglycan content and teichoic acid

• Cytoplasmic membrane

Bacterial Cell Wall

• Main component =

peptidoglycan

• PS + peptide crosslinks
• Formed by

transpeptidases (penicillin binding proteins)

• Gram negative:
• Outer membrane of LPS

and phospholipids

• Thinner cell wall with

lesser peptidoglycan content

• Cytoplasmic membrane

• 1. Cell Wall Synthesis Inhibitors
• Penicillins
• Cephalosporins
• Bacitracin
• Glycopeptides

Penicillins – Structure and function

• Side chain:
• Spectrum
• Susceptibility to destruction
• Pharmacokinetic properties
• β-lactam:
• Function
• Bind transpeptidase à inhibit

formation of peptide linkages between polysaccharides à inhibit formation of peptidoglycan side chain β-lactam thiazolidine ring

Penicillins – Resistance

• 1. β-lactamase production
• à hydrolysis of β-lactam

ring

• Occurs extracellularly in G+
• Occurs between cell

membrane and wall in G-

• Induced by drug binding to

bacterial cell wall or

• Constitutively produced by

bacteria

www.wiley.com

Penicillins – Resistance

• 2. Alter transpeptidases
• Penicillins unable to bind to

and inactivate transpeptidase

• ‘MRSA’

www.wiley.com

Penicillins – Classes

• Effective versus G+
• Resistant to penicillinase
• Extended spectrum
• Anti-pseudomonal

Penicillins

Effective versus G+ Penicillin G (parenteral) Penicillin V

• 1. Highly susceptible to β-lactamases à poor activity

versus Staph aureus and Staph epidermidis

• 2. Ineffective versus altered transpeptidases à poor

activity versus Streptococcus pneumoniae, viridans streptococci

Penicillins

Resistant to penicillinases Methicillin Oxacillin Cloxacillin Dicloxacillin Nafcillin

• 1. Structural modifications à increased efficacy versus β-

lactamase-producing Staph aureus, Staph epidermidis

• 2. Resistance now due to altered transpeptidases

Penicillins

Extended spectrum Ampicillin (+/- sulbactam) Amoxicillin (+/- clavulanate)

• 1. Penicillins inactivated by β-lactamases when not in combo
• 2. Irreversible inactivation of β-lactamases by sulbactam and

clavulanate

• 3. Ineffective versus altered transpeptidases

Penicillins

Anti-pseudomonal activity Carbenicillin Ticarcillin (+/- clavulanate) Piperacillin (+/- tazobactam) Mezlocillin Also effective versus Proteus and Enterobacter

Cephalosporins – Structure and Function

• Side chains:
• Spectrum/classification
• Susceptibility to destruction
• Pharmacokinetic properties
• β-lactam:
• Function
• Bind transpeptidase à inhibit

formation of peptide linkages between polysaccharides à inhibition of peptidoglycan formation

dihydrothiazine ring β-lactam side chain side chain

Cephalosporins – Resistance

• 1. Destruction by β-lactamases
• Cephalosporins less susceptible than penicillins
• S. aureus produces penicillinases
• G- bacteria produce β-lactamases
• Extended spectrum β-lactamases (E. coli,

Pseudomonas, etc.)

* Zapun A, et al., FEMS Microbiol Rev 2008

Cephalosporins – Resistance

• 2. Alteration of transpeptidases
• Cephalosporins unable to bind to and

inactivate enzyme

• Less common for cephalosporins than for

penicillins

• ‘MRSA’

* Zapun A, et al., FEMS Microbiol Rev 2008

Cephalosporins

First generation Second generation Third generation Fourth generation Drugs Cephalexin Cefazolin Cefadroxil Cephradine Cefuroxime Cefoxitin Cefaclor Cefprozil Cefotetan Ceftazidime Cefotaxime Ceftriaxone Cefixime Cefdinir Cefepime Other Good G+ activity Modest G- activity Increasing resistance of Streptococcus pneumoniae to cefazolin Good G+ activity Improved G- activity Modest G+ activity Improved enteric G- activity Ceftazidime has excellent activity versus Pseudomonas aeruginosa Good G+ activity Good G- activity

Penicillins and Cephalosporins in Ophtho

• No commercially available

topical ophthalmic preparations

• Systemic administration:
• Orbital disease
• Adnexal disease
• Limited use in ocular surface

disease

• Staph and Strep resistance

(penicillins)

• Strep resistance (cephalosporins)
• Limited use in endophthalmitis

Bacitracin

• Interrupts transporter molecule à

inhibits movement of peptidoglycan precursor from cytoplasm to cell wall

• G+
• Staphylococcus
• Streptococcus pyogenes
• Administered topically (ointment)
• Nephrotoxicity
• May be administered IM in very few

approved situations

• Poor transcorneal penetration
• “Allergen of the Year” 2003

www.ccbcmd.edu

Glycopeptides

• Bind D-Ala-D-Ala

terminal portion of peptidoglycan precursor à peptidoglycan precursor unavailable for cell wall formation à decreased cell wall growth + decreased cell wall rigidity

vancomycin

Glycopeptides

• Strong activity vs G+
• Drug of choice for

MRSA, penicillin- resistant Strep pneumoniae

• Most G- are resistant
• Vancomycin
• Teicoplanin

vancomycin

Glycopeptides – Resistance

• 1. Alterations of the antibiotic target
• VanA resistance:
• Reduced affinity via alteration of terminal amino acid residues of

peptidoglycan precursor (D-Ala-D-Ala à D-Ala-D-Lac)

• VanC resistance:
• Steric hindrance caused by substitution (D-Ala-D-Ala à D-Ala-D-

Ser)

Glycopeptides – Resistance

• 2. Altered antibiotic penetration
• Inability to penetrate bacterial membrane (G- organisms)
• Intrinsic resistance

Glycopeptides – Resistance

Glycopeptides – Resistance

Enterococcal spp that are resistant to vancomycin but require vancomycin presence to grow have been isolated… Vancomycin presence induces resistance mechanisms…. This is VERY BAD…

Vancomycin – Ocular Application

• Reaches therapeutic AH levels when applied topically (50 mg/ml)
• Effective versus corneal infections with MRSA and MRSE
• Associated with cystoid macular edema when used intracamerally during

cataract surgery

• Non-toxic to the retina at 1 mg dose
• Intravitreal injection in combination with amikacin or ceftazadime for

endophthalmitis

* Alster Y, et al., BJO 2000 ** Sotozono C, et al., Cornea 2002 *** Penha FM, et al., Ophthalmic Res 2010

Teicoplanin – Ocular Application

• Alternative therapy for MRSA

infections

• No vitreal penetration when

administered topically

• Poor vitreal penetration when

administered IV

• 2. Cell Membrane Disruptors
• Polymyxin B
• Gramicidin
• Similarities between bacterial and human cell membranes

limit use

Polymyxin B

• Detergent/surfactant
• Disrupts cell membrane phospholipids

à increased permeability à cell death

• Positively charged drug binds

negatively charged LPS layer

• Binds to and inactivates endotoxin
• G- activity good
• Effective versus Pseudomonas
• Not effective versus Proteus
• G+ activity poor
• Thick cell membrane
• Absence of LPS
• Neurotoxic, nephrotoxic

Gramicidin

• Functions as membrane

channel

• Alters permeability
• Selective movement of monovalent

cations and water

• Stable in solution
• Predominantly G+ activity
• Hemolysis when administered

systemically, therefore topical administration only

www.physics.usyd.edu.au

• 3. Protein Synthesis Disruptors
• Aminoglycosides
• Tetracyclines
• Macrolides
• Chloramphenicol
• Oxazolidinones

Protein Synthesis – Short Version

• Translation = mRNA à protein
• Ribosomes
• 50S subunit + 30S subunit =

70S prokaryotic ribosome

• 40S subunit + 60S subunit =

80S eukaryotic ribosome

• Peptidyl transferase
• Enzymatic function of

ribosome to create peptide bonds between adjacent amino acids

www.wikipedia.com

Protein Synthesis Disruptors

Target = 30S Amino- glycosides Tetracyclines

Protein Synthesis Disruptors

Target = 30S Amino- glycosides Tetracyclines

• G- aerobes (Pseudomonas, Proteus, Klebsiella, E. coli, Enterobacter)
• Some Staphylococcus spp.
• Neomycin generally not effective versus Pseudomonas

Aminoglycosides – MOA

• Positively charged
• Bind negatively-charged LPS
• f G- outer membrane
• Bind negatively-charged rRNA
• Hydrophilic
• Poor lipid membrane

penetration

• Different AG have variable

specificity for different regions, leading to different spectrum of activity

Aminoglycosides – MOA

• 1. Bind outer membrane

(electrostatic)

• 2. Diffuse into periplasmic space
• 3. Transport into cytoplasm

(oxygen-dependent)

• 4. Bind 16s rRNA of 30S subunit

(energy-dependent)

• 5. mRNA misreading à

missense, premature stop codons

• Also bind to and disrupt cell

membrane

Aminoglycosides – Resistance

• 1. Alteration of AG à decreased affinity for ribosome
• AME (aminoglycoside modifying enzymes)
• Mutational pressure induced by exposure of bacteria to AG à resistance

genes within normal bacterial enzymes à modification of AG

• Transferred by plasmids or transposons
• Methylation of binding site on AG à decreased binding to ribosome à

decreased function of AG

• Most common resistance method
• Results in high-level resistance
• Resistance not predictable among different AG due enzyme variability

* Shakil S, et al., J Biomed Sci 2008

Aminoglycosides – Resistance

• 2. Reduced intracellular drug concentration
• Bacterial efflux pumps
• Energy-dependent
• Constitutively expressed à low-level, intrinsic resistance
• Substrate-induced or mutation-induced overexpression à increased

resistance

• Altered outer membrane permeability
• Decreased inner membrane transport
• Drug trapping

* Shakil S, et al., J Biomed Sci 2008

Aminoglycosides – Resistance

• 3. Enzyme-induced alteration of ribosome
• Normal bacterial enzymes
• Alter shape of ribosome à alter contact of AG with ribosome à decreased

function of AG

* Shakil S, et al., J Biomed Sci 2008

AGs – Toxicity

• Positive charge increases toxicity
• Nephrotoxicity
• Concentration in proximal tubule epithelial cells à disrupt tubule fxn à

cation-wasting in urine

• Ototoxicity
• Localization in hair cells à cell death
• Localize in the cochlea, spiral ganglion neurons, organ of Corti
• Neuromuscular blockade
• Affinity for rRNA of prokaryotes is only ~10X greater than

for eukaryotes

Aminoglycosides in Ophthalmology

• Topical:
• Neomycin – not considered effective versus Pseudomonas
• Gentamicin
• Tobramycin
• Combination with β-lactam for improved G+ spectrum – must be applied

separately!

• As a class, noted to have deleterious effects on corneal wound healing
• Delayed reepithelialization, punctate epithelial erosions, corneal

ulceration, chemosis

• Intravitreal:
• Amikacin – less toxic than gentamicin, may be used in combination with

vancomycin (G+ spectrum) for endophthalmitis

Protein Synthesis Disruptors

Target = 30S Amino- glycosides Tetracyclines

Rickettsia spp Borrelia spp Chlamydophila spp Mycoplasma spp Moraxella spp Brucella spp Some Staphylococcus and Streptococcus spp. Generally not effective versus Pseudomonas

Tetracyclines – Mechanism

• 1. Enter bacterial cell
• Outer membrane porins (G-) à

passive diffusion through inner cell membrane

• Active transport across

cytoplasmic membrane (G+)

• 2. Inhibit binding of tRNA to

mRNA-ribosome complex

• 16S subunit of 30S ribosome
• Reversible
• Weak binding to eukaryotic

ribosomes minimizes toxicity

www.antibioticsinfo.org

Tetracyclines – Resistance

Speer et al., Clin Microbiol Rev, 1992

Acquisition of tet genes by bacteria

Tetracyclines – Resistance

• 1. Efflux pumps
• 2. Ribosomal protection proteins
• Block binding of TCN to ribosome
• Bind to and distort ribosome to still allow t-RNA binding
• Bind to ribosome and dislodge TCN
• 3. Enzymatic inactivation (rare)
• Addition of acetyl group to drug

Thaker M, et al., Cell Mol Life Sci 2010 D’Costa VM, et al., Science, 2006

Tetracyclines

Short acting (t½ 6-8 hours) Intermediate acting (t½ 12 hours) Long acting (t½ 16 hours) Tetracycline (1st gen) Demeclocycline Doxycycline (2nd gen) Oxytetracycline (1st gen) Minocycline (2nd gen)

Bonus Properties of TCNs

• Anticollagenase activity
• Inhibit MMPs
• Bind zinc and calcium ions within enzyme catalytic domain
• Likely irreversible
• May (or may not) modulate MMP expression
• Inhibit IL-1 synthesis
• Inhibit activated B cell function
• Inhibit NO synthesis via LPS activation

Golub LM, et al., J Dent Res 1987 Smith VA, et al., Br J Ophthalmol 2004 Solomon A, et al., Invest Ophthalmol Vis Sci 2000 Kuzin II, et al., Int Immunol 2001 D’Agostino P, et al., Eur J Pharmacol 1998

Tetracyclines in Ophthalmology

Federici, Pharmacological Research, 2011

Protein Synthesis Disruptors

Target = 50S Macrolides CHPC Oxazolidinones

Protein Synthesis Disruptors

Target = 50S Macrolides CHPC Oxazolidinones G+ cocci Chlamydophila spp, Mycoplasma spp, Borrelia spp Increased G- spectrum (azithromycin) Enterococci resistant Streptococcus spp developing resistance Erythromycin Clindamycin Azithromycin Etc..

Macrolides – Mechanisms

• 1. Prevent formation of peptide

bond between adjacent amino acids

• 2. Premature dissociation of

peptidyl-tRNA complex from ribosome

• 3. Inhibit ribosomal translocation
• 4. May also affect ribosome

assembly

• Macrolide binding to 50S

ribosome is reversible

• Accumulate within leukocytes

faculty.ccbcmd.edu

Macrolides – Resistance

• Resistance:
• Acquired ribosomal

alterations

• Drug-inactivating enzymes

(rare)

• Efflux pumps (rare)

Protein Synthesis Disruptors

Target = 50S Macrolides CHPC Oxazolidinones G+ G- Rickettsia spp, Chlamydophila spp, Mycoplasma spp Spirochetes Pseudomonas spp are resistant

Chloramphenicol – Mechanism

• 1. High lipid solubility à diffusion across cell

membrane

• 2. Inhibition of peptidyl transferase à inhibition of

protein elongation

Chloramphenicol – Resistance

• 1. Reduced membrane permeability
• Low-level resistance
• Most common
• 2. Enzymatic inactivation
• High-level resistance
• Prevents binding to ribosome
• 3. Mutation of 50S ribosomal subunit
• Rare

Chloramphenicol – Side Effects

• Dose-related bone marrow

suppression

• Due to inhibition of

mitochondrial synthesis

• Reversible with discontinuation
• Does not predict development
• f aplastic anemia
• Idiosyncratic aplastic anemia
• Not dose-related
• Weeks to months after

discontinuation of therapy

• Irreversible

Dose-Related Bone Marrow Suppression

• Inhibition of mitochondrial protein synthesis à mild BM

hypocellularity, anemia, neutropenia, thrombocytopenia

• 0.5% ophthalmic solution
• Four times daily x 7 days à total dose < 19 mg
• Four times daily x 14 days à total dose < 33 mg
• No measurable serum levels in either group (< 1 mg)
• Estimated total dosage for toxicity = 30 mg
• Estimated total exposure duration for toxicity = 18 days

Walker et al., Eye, 1998

Aplastic Anemia

CHPC intestinal bacteria dehydro-CHPC systemic absorption DNA damage in bone marrow cells nitroso-derivatives of CHPC

20X increased cytotoxicity

Aplastic Anemia

CHPC intestinal bacteria dehydro-CHPC systemic absorption DNA damage in bone marrow cells nitroso-derivatives of CHPC

20X increased cytotoxicity

Genetic susceptibility?

• 1. Increased ability to form nitroso-derivatives
• 2. Increased sensitivity of bone marrow cell DNA
• 3. Decreased ability to bone marrow cell DNA to repair itself

Aplastic Anemia

Lam et al, Hong Kong Med J, 2002

Aplastic Anemia

Possible association between ocular CHPC and aplastic anemia – the absolute risk is very low (Br J Clin Pharmacol 1998) 145 patients with aplastic anemia 1226 age- and sex-matched controls 3 affected and 5 controls used ocular CHPC preparation

Aplastic Anemia

BMJ, 1998

426 patients with aplastic anemia 3118 age- and sex-matched controls 0 affected and 7 controls used CHPC eye drops Possible association between ocular CHPC and aplastic anemia – the absolute risk is very low (Br J Clin Pharmacol 1998) 145 patients with aplastic anemia 1226 age- and sex-matched controls 3 affected and 5 controls used ocular CHPC preparation

Aplastic Anemia

BMJ, 1998

426 patients with aplastic anemia 3118 age- and sex-matched controls 0 affected and 7 controls used CHPC eye drops Possible association between ocular CHPC and aplastic anemia – the absolute risk is very low (Br J Clin Pharmacol 1998) 145 patients with aplastic anemia 1226 age- and sex-matched controls 3 affected and 5 controls used ocular CHPC preparation

Relative risk = < 1: 1,000,000

Protein Synthesis Disruptors

Target = 50S Macrolides CHPC Oxazolidinones Predominantly G+ aerobes and anaerobes Including methicillin- and vancomycin-resistant strains Some G- activity (anaerobes) Poor activity versus G- aerobes

Oxazolidinones – MOA

• Bind 23S subunit of 50S

ribosome à non-functional initiation complex portion of 70S ribosome

• Inhibits translocation
• May also decrease bacterial

virulence factors and increase phagocytosis at sub-MIC concentrations

Oxazolidinones – Resistance

• Alteration of target ribosomal binding site
• Development low due to:
• Synthetic nature of compounds
• Unique method of inhibiting bacterial protein synthesis
• Multiple genes encode binding site to 23S ribosomal subunit
• Difficult to select for linezolid-resistant strains in vitro
• Risk factors for development of resistance:
• Lengthy or repeated course of therapy
• Use in presence of foreign bodies
• Reversal of resistance has been reported following discontinuation of

antibiotic

Oxazolidinones – Toxicity

• GI suppression
• Reversible thrombocytopenia and anemia
• Neuropathy

Oxazolidinones in Ophthalmology

• Topical administration
• 0.2% penetrates into rabbit anterior segment
• 0.2% successfully treated humans with vancomycin-resistant or

vancomycin-intolerant bacterial keratitis

• Intravitreal administration
• Effective in experimental model of S. aureus endophthalmitis in rabbits

(30 mg once)

• No retinal toxicity
• Oral administration
• Achieves levels >MIC for common organisms in human AH and VH

following single oral dose

Saleh et al., JCRS 2010 Tu et al., AJO 2013 Saleh et al., IOVS 2012 George et al., JOPT 2010

• 4. Folate Metabolism Disruptors
• Sulfonamides
• Trimethoprim
• Pyrimethamine

Folate Metabolism Disruptors

• Folate
• Necessary for DNA and RNA

synthesis and maintenance

• Mammals acquire folate (or

folic acid) in food

• Bacteria must synthesize folate
• MOA = enzyme inhibition
• Dihydropteroate synthetase
• Sulfonamides
• Dihydrofolate reductase
• Trimethoprim
• Pyrimethamine (primarily

antiprotozoal)

Folate Metabolism Disruptors

• Resistance:
• 1. Overproduction of PABA by

bacteria

• 2. Decreased enzyme affinity for

drug

• 3. Decreased bacterial

permeability of drug

• 4. Increased inactivation of drug

by bacteria

• If resistant to one

sulfonamide, resistant to all

Sulfonamide Toxicity – KCS

• Direct toxic effect on lacrimal acinar

cells

• N-containing pyridine and

pyrimidine rings

• Dose-dependent or idiosyncratic
• 15-25% incidence in dogs treated

with sulfas

• May develop months to years after

discontinuation

• May be reversible upon

discontinuation

• Other systemic toxicity signs

variable (i.e., hepatotoxicity)

Barnett K, et al., Human Toxicol 1987 Trepanier L. J Vet Pharm Therapeutics 2004 Trepanier L, et al. J Vet Intern Med 2003

• 5. DNA Synthesis Disruptors
• Fluoroquinolones

DNA Synthesis – Very Short Version

• Topoisomerase II (DNA

gyrase)

• gyrA + gyrB
• Relaxes positive supercoils

that accumulate ahead of DNA polymerase during DNA replication

• Bacterial enzyme only
• Topoisomerase IV
• ParC + ParE
• Unlinks newly formed DNA

strands

• Relaxes positive supercoils

that accumulate ahead of DNA polymerase during DNA replication

• Not present in all bacteria

Fluoroquinolones – MOA

• 1. Enter bacterial cell
• Porin- and LPS-mediated (G-)
• Lipophilicity (G+)
• 2. Bind to enzyme à interrupt

DNA stabilization à inhibit DNA synthesis

• DNA gyrase target for G-
• Topoisomerase IV target for G+
• Spectrum depends upon

substituents added to core structure

www.pharmainfo.net

Fluoroquinolones – Resistance

• Low-level resistance = in vitro determination that may not

correlate with clinical failure due to ability to achieve greater local concentrations

• Single-step mutants
• High-level resistance = in vitro determination that is more

likely to correlate with clinical failure due to inability to achieve appropriate local concentrations

• Multi-step mutants
• Due to repeated exposure to sub-lethal concentrations of FQN

Fluoroquinolones – Resistance

Chromosome-mediated (mutational)

• 1. Mutations in genes encoding protein targets of FQNs
• Mutation in gyrA à low-level resistance
• Mutation in parC à moderate-level resistance
• Second mutation in gyrA à high-level resistance
• Second mutation in parC à highest-level resistance
• 2. Mutations causing reduced drug accumulation
• Decreased uptake
• Decreased expression of porins (G-)
• Increased efflux
• Upregulation of efflux pumps

Fluoroquinolones – Resistance

Plasmid-mediated

• 1. Qnr
• Encodes protein that protects topoisomerases from antibiotic
• Results in low-level resistance
• 2. Enzymatic inactivation
• Alters antibiotic structure à inactivation of antibiotic
• In combination with Qnr à higher-level resistance
• 3. QepA
• Gene encoding for efflux pump

Fluoroquinolones – Avoiding Resistance

• Low-level resistance:
• Maintain drug concentrations above mutant prevention concentration
• Generally several-fold higher than minimum inhibitory concentration
• High-level resistance:
• Avoid repeated exposure to low levels of FQN
• Avoid intermittent FQN exposure
• Avoid tapering FQN
• Newer FQN:
• Structural features confer less resistance potential
• Structural features increase ocular tissue concentrations
• MICs generally lower

Fluoroquinolones in Ophthalmology

Generation First (quinolone) Second Third Fourth Drugs Nalidixic acid Norfloxacin Ciprofloxacin Ofloxacin Levofloxacin Gemifloxacin Sparfloxacin Moxifloxacin Gatifloxacin Besifloxacin Spectrum G- G- Some G+ G- Some G+ G- G+ Other Weak versus Pseudomonas Improved versus Pseudomonas Increasing resistance of Strep pneumoniae Efficacy versus MRSA, resistant Pseudomonas strains

4th Generation Fluoroquinolones

• Moxifloxacin
• Gatifloxacin
• Besifloxacin (formulated for ophthalmic use)
• Balanced inhibition of DNA gyrase and topoisomerase IV
• Strong Gram+ activity, strong anaerobic activity, retain Gram-

activity

• Labeled for bacterial conjunctivitis
• Clinical efficacy in bacterial keratitis, post-operative

endophthalmitis, etc.

• No difference in tolerability, adverse events

O’Brien, Adv Thera, 2012 Majmudar, Cornea, 2014 Garg Asia Pac J Ophth 2015 Etc…

Fluoroquinolones in Vet Ophthalmology

• [Ofloxacin] > [ciprofloxacin] in canine AH
• [Moxifloxacin] > [ciprofloxacin] in equine AH
• [Moxifloxacin] > [ciprofloxacin] in equine tears, cornea, and AH
• Increased resistance of β-hemolytic Streptococcus to

ciprofloxacin in dogs

Fluoroquinolones – Side Effects

• Corneal cytotoxicity in vitro
• Delayed wound healing in

vivo

• 1.5% levo > 0.5% moxi >

0.3% gati > 0.5% levo

• Corneal precipitates
• Endothelial damage with

intraocular injection

• Concentration-dependent

Summary

• Important to understand the mechanisms of bacterial

resistance

• Significantly increasing understanding of these

mechanisms via genomics

• Appropriate selection of antibiotic in ophthalmology

depends upon spectrum of action and toxicities