Ocular Microbiology and Antimicrobial Agents

Introduction

Ocular microbiology is advancing with new technologies, improving the understanding and control of eye infections. While classical eye infections are decreasing, new ones are emerging. Essential knowledge includes eye anatomy, common microbial agents, infection transmission, and prevention methods.

Morphology and Classification of Bacteria

Microorganisms are divided into prokaryotes (bacteria, blue-green algae) and eukaryotes (fungi, algae, protozoa). Bacteria, measured in microns (\mum), are typically 0.2-1.5 mum in diameter and 3-5 mum in length.

Microscopy

• Light Microscopy: Simple (single lens) and compound (multiple lenses). Used for examining bacteria in living or stained states.
• Phase Contrast Microscopy: Enhances contrast by utilizing refractive index differences.
• Dark Field Microscopy: Uses reflected light to visualize slender organisms like spirochetes.
• Electron Microscopy: Uses electron beams for high magnification, requiring a vacuum.

Stained Preparations

• Simple Stains: Use dyes like methylene blue for color contrast.
• Negative Staining: Uses dyes like Indian ink to create a contrasting background for viewing slender bacteria.
• Impregnation Methods: Use silver to visualize thin cells and structures.
• Differential Stains:
  • Gram Stain: Differentiates bacteria based on cell wall structure (Gram-positive with thick peptidoglycan layer and teichoic acids, Gram-negative with lipid-rich outer membrane and thin peptidoglycan layer).
  • Acid-Fast Stain: Identifies bacteria resistant to decolorization by acids.

General Phenotypic Classification of Bacteria

Gram Positive Bacteria

Examples include Staphylococci, Streptococci, Pneumococci, Enterococci, Bacilli, Clostridia, Corynebacterium, Listeria, and Actinomyces. Each has distinct morphologies, oxygen requirements, reservoirs, and associated infections.

Gram Negative Bacteria

Examples include Enterobacteriaceae (E. coli, Klebsiella, Salmonella, Shigella), Bacteroides, Pseudomonas, Vibrio (cholera), Campylobacter, Legionella, Neisseria (N. meningitidis), Hemophilus, and Bartonella. Each has distinct morphologies, oxygen requirements, reservoirs, and associated infections.

Shape of the Bacteria

Bacteria are classified by shape into cocci (spherical), bacilli (rods), vibrios (comma-shaped), spirilla (rigid spirals), spirochetes (flexible spirals), actinomycetes (branching filaments), and mycoplasmas (cell wall deficient).

Growth and Multiplication of Bacteria

Bacteria multiply through binary fission. Growth is measured by total and viable counts. The bacterial growth curve includes:

• Lag phase
• Log phase (exponential growth)
• Stationary phase (equilibrium between cell division and death)
• Decline phase (cell death exceeds division)

Factors Affecting Bacterial Growth

Nutrition, oxygen, carbon dioxide, temperature (mesophilic, psychrophilic, thermophilic), moisture, light, pH, and osmotic effects influence bacterial growth.

Why Study Antimicrobials?

Antibiotics have significantly improved human health by controlling infections. However, antibiotic resistance is increasing due to overuse. Sensible and cautious antibiotic use is crucial. Common ocular pathogens include gram-positive (Staphylococcus, Streptococcus) and gram-negative (Haemophilus, Pseudomonas, Neisseria) organisms.

Common Ocular Pathogens

Lists various Gram-positive and Gram-negative organisms, including specific species of Staphylococci, Streptococci, Bacilli, Neisseria, Enterobacteriaceae, Haemophilus, Brucella, and Pseudomonas.

Sterilization and Antimicrobial Agents

Sterilization eliminates all microorganisms. Methods include:

• Radiation (UV, X-rays, gamma rays)
• Chemical Sterilization (Ethylene Oxide, Ozone, Glutaraldehyde, Formaldehyde, Hydrogen Peroxide)

Antimicrobials inhibit or kill microorganisms. Antibiotics are substances produced by microorganisms that act against other microorganisms.

Classification of Antibacterial Agents

Classified by:

• Spectrum of activity (broad, intermediate, narrow)
• Effect on bacteria (bactericidal, bacteriostatic)
• Mode of action

Disinfectants

Antimicrobial agents applied to non-living objects. Instruments contacting ocular surfaces should be disinfected or sterilized. Examples include cleaning tonometer probes with mild soap and hydrogen peroxide, and disinfecting gonioscopy lenses with glutaraldehyde or sodium hypochlorite.

Prevention of Hospital-Acquired Ocular Infection

Hand washing, disposable gloves, and proper disinfection of equipment are essential. Single-use eye drops are preferred. Autoclaving is recommended for sterilizing surgical equipment.

Contact Lens Care Systems

Methods include hydrogen peroxide systems, enzymatic cleaners, UV/vibration/ultrasonic devices, saline solutions, daily cleaners, and multipurpose solutions (MPS). MPS disinfectants include Polyhexamethylene biguanide (PHMB) and Polyquad. Biocompatibility involves EDTA, surfactants, and buffers.

Isolation of Bacteria

Steps include specimen collection, preservation and transport, microscopic examination (Gram stain), and culture methods (solid, liquid media, automated systems). Optimal conditions and growth requirements must be considered. High-risk samples should be labelled.

Normal Microbiota of the Human Eye

Normal flora consists of microorganisms inhabiting skin and mucous membranes. Tears contain lysozyme, inhibiting colonization. Common organisms include Staphylococcus aureus, Staphylococcus epidermidis, and diphtheroids. The flora can be transient or resident.

Infections of the Eye

Common infections include conjunctivitis, blepharitis, keratitis, keratoconjunctivitis, and dacryocystitis. Causes vary by age and condition.

Laboratory Diagnosis of Conjunctivitis

Requires careful specimen collection and appropriate media inoculation.

Standards for Microbiology Investigations

Aseptic technique, correct transport medium, and timely processing are crucial. Procedures should be performed in a microbiological safety cabinet.

Trachoma

Caused by Chlamydia trachomatis, leading to blindness from repeated infections. Transmission occurs via eye-to-eye contact, flies, and fomites. The WHO recommends the SAFE strategy: Surgery, Antibiotics, Facial cleanliness, and Environmental improvements.

Antimicrobial Therapy

Chemotherapy uses chemicals against invading organisms. Antibiotics are produced by microorganisms to harm other microbes. Selective toxicity targets cells without harming others. Drugs are classified by susceptible organisms and mechanism of action.

Acquired Resistance

Mechanisms include drug-metabolizing enzymes, reduced uptake, altered receptors, and antagonistic compounds.

Antibiotic Combinations

Responses can be additive, potentiative, or antagonistic. Disadvantages include increased toxicity, antagonism, suprainfection, resistance, and cost.

Specific Antibiotics

• Chloramphenicol carries a risk of fatal aplastic anemia.
• Penicillins weaken cell walls; penicillinases can inactivate them.
• Cephalosporins disrupt cell wall synthesis.
• Tetracyclines inhibit protein synthesis.
• Erythromycin inhibits protein synthesis.
• Aminoglycosides disrupt protein synthesis.
• Ciprofloxacin inhibits DNA replication.

Antifungal Agents

Polyene antifungals (Amphotericin B, Nystatin, Natamycin) disrupt cell membranes. Azoles (Clotrimazole, Miconazole, Econazole, Ketoconazole, Fluconazole, Voriconazole) inhibit ergosterol synthesis. Terbinafine and Tolnaftate target squalene epoxidase.

Bacterial Resistance

Results from enzymatic degradation, altered bacterial proteins, and changes in membrane permeability. Overcome this by producing new antibiotics and co-administering beta-lactamase inhibitors. Cell wall/membrane alterations, enzyme production, and ribosomal alterations are major mechanisms.

Actions to Fight Resistance

Prevent infections, improve antibiotic prescribing (stewardship), and develop new drugs and diagnostic tests. Hand washing, proper sanitation, and education are key.