(2) Antibacterials II

CFU (Colony Forming Unit)

CONCEPT

• 1 live bacterial cell → forms 1 colony

• ~1 colony = ~1 million bacteria

Limit of Visible Growth

CONCEPT

• ~10⁷ CFU/mL (10 million bacteria per mL)

• Below this → growth not visible

MIC (Minimum Inhibitory Concentration)

CONCEPT

• Lowest drug concentration that inhibits visible growth

• ⚠ IMPORTANT: bacteria may still be alive

MBC (Minimum Bactericidal Concentration)

CONCEPT

• Lowest drug concentration that kills bacteria

• Defined as:

  • ≥ 3-log kill (99.9%)

Resistant Mutants Frequency

CONCEPT

• Occur spontaneously (NOT drug-caused)  in bacteria

• Drug only selects them

• Frequency:

  • 1 in 10⁵ to 10⁹ cells

 BREAKPOINTS

CONCEPT

• Used to interpret susceptibility testing results (MIC)

• Determines: SIR

  • Susceptible

  • Intermediate

  • Resistant

 BREAKPOINTS

Purpose

• Lab → reports to:

  • Physician

  • ID pharmacist

• YOU use it to choose antibiotic

BREAKPOINTS

Based on:

• Microbiology

• PK/PD

• Clinical outcomes

• Monte Carlo simulations (prof mentioned)

SUSCEPTIBLE ≠

 GUARANTEED SUCCESS

 SUSCEPTIBLE SUCCESS depends on…

• Drug penetration to infection site

• Immune status

• Comorbidities

• Need for surgery/debridement

RESISTANT

CONCEPT

• Strong prediction of failure

INTERMEDIATE

CONCEPT

1. MIC uncertainty

2. Works if higher dose used

3. Works if drug concentrates at site

• Example: UTI (urine concentration)

INTERMEDIATE

how treated?

• Clinically often treated like resistant

SDD (Susceptible Dose Dependent)

💡 CONCEPT

• Works only with specific higher dosing

SDD (Susceptible Dose Dependent)

Examples (she listed):

• Piperacillin/tazobactam

• Cefepime

• Ceftaroline

• Daptomycin

• Fluconazole

WHO SETS BREAKPOINTS

• FDA (initial)

• CLSI (US standard)

• EUCAST (Europe)

• EUCAST =

• CLSI =

• EUCAST = more strict

• CLSI = US standard used clinically

LIMITATIONS OF TESTING (antimicrobial susceptibility testing) - overview

1. 2-FOLD ERROR

2. INOCULUM EFFECT

3. TECHNICAL DIFFICULTIES

LIMITATIONS OF TESTING: 2-FOLD ERROR

CONCEPT

• MIC is not exact

LIMITATIONS OF TESTING: 2-FOLD ERROR

Example:

• MIC 2 → could be 1, 2, or 4

👉 Could change S ↔ R

LIMITATIONS OF TESTING: INOCULUM EFFECT

CONCEPT

• Lab bacteria count < real infection

• Real MIC may be higher

LIMITATIONS OF TESTING: TECHNICAL DIFFICULTIES

CONCEPT

• Mixed bacterial populations

• Fuzzy/trailing endpoints

• Technician judgment matters

She compared bacteria to humans → variation

QUANTITATIVE vs QUALITATIVE

Type	Meaning	Methods

Quantitative

Gives MIC

Macro, Micro, Agar, E-test

Qualitative

Yes/No only

Disk diffusion

MACROBROTH (GOLD STANDARD) ✅⭐

💡 CONCEPT

• Tubes with increasing drug concentration

• MIC = first tube with no visible growth

MACROBROTH (GOLD STANDARD) ✅⭐

MATH

• Inoculum: 5 × 10⁵ CFU/mL

• Volumes: 1–3 mL

MACROBROTH

MBC determination (STEP-BY-STEP)

1. Take MIC tube

2. Plate onto agar

3. If growth → NOT MBC

4. Next concentration with NO growth → MBC

MACROBROTH

Advantages

• Gold standard

• Can measure MBC

MACROBROTH

Disadvantages

• Time consuming

• Expensive

• NOT used clinically

MICROBROTH

CONCEPT

• Same idea as macro, but:

  • Smaller volume

  • Often automated

MICROBROTH (MOST USED)

MATH

• Inoculum: 5 × 10⁴ CFU/mL

• Volume: 0.1 mL

MICROBROTH (MOST USED)

IMPORTANT

Smaller sample → less accurate (miss mutants)

AGAR DILUTION

CONCEPT

• Antibiotic embedded in agar

• Multiple organisms tested at once

AGAR DILUTION

Advantages

Test many organisms at once

AGAR DILUTION

Disadvantages

• No MBC

• Time consuming/not common

E-TEST

CONCEPT

• Strip with antibiotic gradient

• Creates ellipse of inhibition

👉 MIC = where ellipse meets strip

E-TEST

Advantages

• Quantitative

• Easy

• Multiple drugs per plate (Can test ~6 drugs per plate)

E-TEST

Disadvantages

• Expensive

• Hard to interpret sometimes

DISK DIFFUSION

CONCEPT

Measure zone of inhibition

DISK DIFFUSION

 VERY IMPORTANT POINT

Zone size ≠ same for all drugs

→ Must compare to standards

DISK DIFFUSION: MIXED POPULATION

CONCEPT

• Growth inside zone = resistant mutants

👉 Clinical meaning:

• Infection may not resolve

 AUTOMATED TESTING

CONCEPT

Machines analyze growth

 AUTOMATED TESTING

 LIMITATION

• Algorithm-based

• New drugs → may not work properly

RAPID TESTING (CLINICAL IMPACT)

WHY IMPORTANT

• “Point of irreversibility” ≈ 3 days

• After that → treatment may not help

 RAPID TESTING

 IMPORTANT DISEASES:

• Pneumonia

• Sepsis

• Meningitis

• Neutropenia

 RAPID TESTING

 BENEFITS

• ↓ Mortality

• Faster switch:

  • Empiric → Targeted

• ↓ ICU time

• ↓ procedures

• ↓ cost

SOCIETAL IMPACT

CONCEPT

• More antibiotics → more resistance

• Reducing use → reduces resistance

SOCIETAL IMPACT'

Examples she mentioned:

• Neomycin → resistance

• Cephalosporins → Enterobacter

• Vancomycin → Enterococci

 MDR / XDR / PDR

Type	Meaning
MDR	≥ 3 drug classes
XDR	Most classes
PDR	All drugs