2.3: Medical Microbiology & Infectious Pathogens

2.3 Medical Microbiology & Infectious Pathogens

Pharmacists play a key role in antimicrobial stewardship in Australia. The OPRA exam requires a strong understanding of bacterial structures, mechanisms of antibiotic resistance, pathogen-specific characteristics, and viral replication cycles to guide rational antimicrobial selection and dosing.

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1. Gram-Positive vs. Gram-Negative Bacteria

Bacteria are classified based on the structural properties of their cell wall, which dictates their staining characteristics and susceptibility to different classes of antibiotics.

Cell Wall Structural Differences

• Gram-Positive Bacteria:
  • Peptidoglycan Layer: Thick, multi-layered peptidoglycan mesh (20-80 nm) composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) glycan chains. These chains are cross-linked by peptide bridges synthesized by transpeptidases, also known as Penicillin-Binding Proteins (PBPs). In Gram-positive cells, this layer constitutes up to 90% of the cell wall.
  • Teichoic and Lipoteichoic Acids: Penetrate the peptidoglycan layer, contributing to cell wall rigidity, maintaining cell wall charge, and acting as antigenic determinants.
  • Outer Membrane: Absent.
• Gram-Negative Bacteria:
  • Peptidoglycan Layer: Thin, single-layered peptidoglycan (1-3 nm) located within the periplasmic space between the inner cytoplasmic membrane and the outer membrane. It constitutes only 10% of the cell wall.
  • Outer Membrane: Present. It is an asymmetric bilayer. The inner leaflet consists of phospholipids, while the outer leaflet is composed of Lipopolysaccharide (LPS). LPS consists of:
    1. Lipid A: The toxic component (endotoxin) that binds to toll-like receptor 4 (TLR4) on host immune cells, triggering a massive release of inflammatory cytokines (TNF-alpha, IL-1, IL-6) that can lead to vasodilation, systemic inflammatory response syndrome (SIRS), and septic shock.
    2. Core Polysaccharide: Provides structural stability.
    3. O-antigen: A highly variable polysaccharide chain used for serotyping.
  • Porin Channels: Transmembrane proteins in the outer membrane that form water-filled channels, allowing the passive diffusion of small hydrophilic molecules (including nutrients and many antibiotics like beta-lactams).

Gram Staining Procedure and Biochemical Rationale

The Gram stain differentiates bacteria based on their cell wall's ability to retain crystal violet dye.

1. Primary Stain (Crystal Violet): Crystal violet enters the cytoplasm of all cells, staining them purple.
2. Mordant (Gram's Iodine): Iodine binds to crystal violet, forming a large, water-insoluble crystal violet-iodine (CV-I) complex inside the cell.
3. Decolourisation (Alcohol or Acetone): This is the critical differentiating step.
   • In Gram-positive cells, alcohol dehydrates the thick peptidoglycan layer, causing it to shrink and close its pores, which traps the large CV-I complexes inside. The cells remain dark purple.
   • In Gram-negative cells, alcohol dissolves the lipid-rich outer membrane and breaches the thin peptidoglycan layer. This allows the CV-I complexes to wash out, leaving the cells colourless.
4. Counterstain (Safranin): Safranin enters the colourless Gram-negative cells, staining them pink/red. The Gram-positive cells remain purple, as the dark violet dye masks the lighter pink counterstain.

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2. Major Clinical Pathogens

Staphylococcus aureus (Gram-Positive Cocci)

• Morphology & Physiology: Gram-positive cocci arranged in clusters ("grapes"). They are catalase-positive (distinguishing them from streptococci) and coagulase-positive (distinguishing them from coagulase-negative staphylococci like S. epidermidis). They are beta-haemolytic on blood agar, forming golden-yellow colonies.
• Virulence Factors:
  • Protein A: Binds the Fc region of IgG antibodies, preventing opsonization and phagocytosis.
  • Coagulase: Converts fibrinogen to fibrin, coating the bacterial cell to shield it from immune cells.
  • Toxins: Includes Toxic Shock Syndrome Toxin-1 (TSST-1), a superantigen that activates T cells non-specifically, leading to massive cytokine release.
• Methicillin Resistance (MRSA): Methicillin resistance is mediated by the mecA gene, which is carried on a mobile genetic element (SCCmec). The mecA gene encodes Penicillin-Binding Protein 2a (PBP2a). Unlike normal PBPs, PBP2a has a very low binding affinity for almost all beta-lactam antibiotics (penicillins, cephalosporins, and carbapenems). PBP2a allows cell wall cross-linking to continue even in the presence of these drugs. Treatment in Australia typically relies on glycopeptides like vancomycin or teicoplanin, or linezolid. The only beta-lactam with MRSA activity is the 5th generation cephalosporin ceftaroline.

Escherichia coli (Gram-Negative Bacillus)

• Morphology & Physiology: Gram-negative bacillus (rod). It is a facultative anaerobe that ferments lactose, forming pink colonies on MacConkey agar, and is oxidase-negative.
• Virulence Factors:
  • Fimbriae (Pili): Enable adherence to the uroepithelium, preventing the bacteria from being washed out by urine flow. This makes E. coli the leading cause of urinary tract infections (UTIs) and pyelonephritis.
  • K1 Capsular Antigen: Prevents phagocytosis and is associated with neonatal meningitis.
• Clinical Presentation: Causes UTIs, neonatal meningitis, sepsis, and gastroenteritis. Strains like Enterohaemorrhagic E. coli (EHEC / STEC) produce Shiga-like toxins that cause hemorrhagic colitis and Haemolytic Uraemic Syndrome (HUS), characterized by microangiopathic haemolytic anaemia, thrombocytopenia, and acute renal failure.

Pseudomonas aeruginosa (Gram-Negative Bacillus)

• Morphology & Physiology: Gram-negative bacillus. It is an active aerobe, oxidase-positive, and does not ferment lactose (forming clear colonies on MacConkey agar). It produces blue-green pyocyanin and yellow-green pyoverdine pigments, and has a characteristic sweet, grape-like or tortilla-like odor.
• Virulence Factors:
  • Exotoxin A: ADP-ribosylates elongation factor 2 (EF-2), inhibiting host cell protein synthesis (identical mechanism to diphtheria toxin).
  • Alginate (Mucoid Exopolysaccharide): Forms a thick biofilm that physically shields the bacteria from antibiotics and immune cells, particularly in the lungs of cystic fibrosis patients.
• Clinical Presentation: An opportunistic pathogen causing hospital-acquired infections (ventilator-associated pneumonia, catheter-associated UTIs, burn wound infections, sepsis). It also causes otitis externa ("swimmer's ear") and hot tub folliculitis.
• Antipseudomonal Agents in Australia: Piperacillin-tazobactam, ceftazidime, cefepime, meropenem, ciprofloxacin, gentamicin, tobramycin, colistin.

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3. Mechanisms of Bacterial Resistance

Bacteria develop resistance through several biochemical pathways:

1. Enzymatic Inactivation (e.g., Beta-lactamases):
   • Simple Beta-lactamases: Cleave the beta-lactam ring, inactivating narrow-spectrum penicillins.
   • Extended-Spectrum Beta-Lactamases (ESBLs): Plasmid-encoded enzymes (common in E. coli and Klebsiella) that hydrolyze penicillins, narrow-spectrum cephalosporins, and third/fourth-generation cephalosporins (e.g., ceftriaxone, cefotaxime, ceftazidime), and monobactams (aztreonam). They are typically inhibited by beta-lactamase inhibitors (clavulanic acid, tazobactam) and do NOT hydrolyze carbapenems. The treatment of choice for ESBL-producing infections is a carbapenem (e.g., meropenem).
   • Carbapenemases: Hydrolyze carbapenems and most other beta-lactams. Examples include metallo-beta-lactamases (MBLs like NDM-1, IMP) which require zinc for activation and are resistant to beta-lactamase inhibitors. MBL-producing Enterobacteriaceae are a growing clinical concern in Australian hospitals.
2. Target Site Modification:
   • MRSA: Production of PBP2a (encoded by mecA).
   • Vancomycin-Resistant Enterococci (VRE): Plasmid-mediated vanA or vanB genes encode enzymes that alter the terminal D-alanyl-D-alanine of peptidoglycan precursors to D-alanyl-D-lactate or D-alanyl-D-serine. Vancomycin cannot form the hydrogen bonds necessary to bind D-Lac, resulting in resistance.
   • Macrolide Resistance: The erm gene encodes a methylase that methylates the 23S rRNA of the 50S ribosomal subunit, preventing macrolides (clarithromycin, erythromycin), lincosamides (clindamycin), and streptogramin B from binding.
3. Active Efflux Pumps: Transmembrane proteins that actively pump antibiotics out of the periplasm or cytoplasm. For example, the MexAB-OprM pump in Pseudomonas aeruginosa extrudes beta-lactams, fluoroquinolones, and tetracyclines.
4. Porin Loss or Mutation: Downregulation or structural alteration of outer membrane porins restricts antibiotic entry into Gram-negative cells. Loss of the OprD porin in Pseudomonas aeruginosa selectively prevents carbapenem entry, causing resistance.

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4. Viral Replication & Antiviral Pharmacology

Viruses are obligate intracellular parasites. Their replication cycle depends on the type of nucleic acid they possess:

DNA vs. RNA Viruses

• DNA Viruses: Typically replicate in the host cell nucleus (except Poxviruses) and use host DNA-dependent RNA polymerase for transcription. Examples include Herpes simplex virus, Varicella-zoster virus, and Adenovirus.
• RNA Viruses: Replicate in the cytoplasm (except Influenza and Retroviruses). Because host cells do not possess enzymes to copy RNA, RNA viruses must encode or carry their own RNA-Dependent RNA Polymerase (RdRP). Examples include Coronavirus, Hepatitis C virus, and Influenza.

The Retrovirus (HIV) Replication Cycle & Drug Targets

Retroviruses are RNA viruses that replicate through a DNA intermediate.

1. Attachment & Fusion: Viral gp120 binds to host CD4 receptors and CCR5 or CXCR4 co-receptors. The viral gp41 protein then mediates membrane fusion, releasing the viral core into the cytoplasm.
   • Drug Targets: Maraviroc (blocks CCR5 co-receptor) and Enfuvirtide (blocks gp41).
2. Reverse Transcription: Viral Reverse Transcriptase copies the single-stranded viral RNA into double-stranded DNA. This process is highly error-prone, leading to rapid mutation.
   • Drug Targets: NRTIs (e.g., tenofovir, emtricitabine) compete with host nucleotides and cause chain termination; NNRTIs (e.g., efavirenz) bind non-competitively to a pocket on the enzyme, inducing a conformational change.
3. Integration: Viral Integrase transports the double-stranded viral DNA into the nucleus and inserts it into the host genome.
   • Drug Targets: Integrase Strand Transfer Inhibitors (INSTIs) (e.g., dolutegravir, bictegravir) block the catalytic active site of the enzyme.
4. Transcription & Translation: The host cell's RNA polymerase transcribes the integrated viral DNA into viral mRNA, which is translated into long, non-functional polyproteins (Gag-Pol).
5. Assembly & Budding: The viral RNA and polyproteins assemble at the host cell membrane and bud off as an immature virion.
6. Maturation: The viral Protease enzyme cleaves the long polyproteins into functional structural and enzymatic proteins, maturing the virion so it can infect other cells.
   • Drug Targets: Protease Inhibitors (PIs) (e.g., darunavir, atazanavir) block this cleavage step.

Influenza Replication and Release

In influenza replication, the virus binds to sialic acid receptors on host cells and enters via endocytosis. After replication, the newly formed virions bud from the host cell membrane but remain tethered by sialic acid. The viral Neuraminidase enzyme cleaves sialic acid residues, releasing the virions.

• Drug Targets: Neuraminidase Inhibitors (e.g., oseltamivir, zanamivir) inhibit this enzyme, trapping the virions on the host cell surface and limiting viral spread.