Identification of Unknown Bacteria — Comprehensive Study Notes

Importance of Species-Level Microbial Identification

  • Species-level ID is critical for effective treatment of infectious diseases.
    • Determines correct antimicrobial therapy; prevents empirical over-use of broad drugs.
  • Main application arenas
    • Healthcare – rapid, accurate diagnosis → targeted therapy, shorter hospital stays, reduced resistance pressure.
    • Epidemiology – tracks outbreaks, discovers novel or resistant strains (e.g. MRSA, VRE).
    • Pharmaceutical & Food Industries – environmental isolates threaten asepsis; ID is a Good Manufacturing Practice (GMP).
    • Additional scenarios – criminal forensics, bioterrorism detection, environmental remediation (bio-augmentation studies).

Disease Diagnosis Workflow

  • Clinical evidence
    • Signs – objective (fever, rash, WBC count).
    • Symptoms – subjective (pain, malaise).
  • Laboratory evidence
    • Cultures → colony & cellular morphology, biochemical panels.
    • Antibiotic susceptibility: classifies isolates as \text{Sensitive} or \text{Resistant}.
  • Typical flow (Gram (+) cocci example)
    1. Gram stain → Gram (+) cocci.
    2. Catalase test.
    3. Hemolysis pattern on blood agar.
    4. Selective/differential plates (e.g. MSA).
    5. Disks (bacitracin, optochin) or coagulase assay.

Traditional Identification Techniques

  • Colony morphology – size, edge, color, elevation.
  • Cellular morphology / staining – Gram, acid-fast, capsule, spore stains; note size, shape, arrangement.
  • Biochemical testing – Bergey’s Manual keys; carbohydrate use, enzyme activity, respiration type.

Bergey’s Manual Highlights (Genus Pseudomonas example)

  • Morphology: straight/slightly curved rods, 0.5$–$1.0 \mu m \times 1.5$–$5.0 \mu m.
  • Features
    • Strictly respiratory (aerobic), some reduce \text{NO}_3^- anaerobically.
    • Motility via 1+ polar flagella (occasionally lateral).
    • Gram (−), oxidase variable, catalase (+).
    • Accumulate poly-\beta-hydroxybutyrate inclusions; hydrolyse it extracellularly in some spp.
    • Fail to grow at \text{pH} < 4.5; most free of growth-factor requirements.
    • Type species: P. aeruginosa.
  • Differentiation matrix (excerpt)
    • Flagella number, fluorescence, temperature growth at 41\,^{\circ}\text{C} or 4\,^{\circ}\text{C}, denitrification, gelatin/starch hydrolysis, specific carbon utilization.

Proper Specimen Collection

  • Sample type & device must suit infection site
    • E.g. nasal or throat swabs, sputum, midstream urine, stool.
  • Representation & purity
    • Avoid contamination by operator flora; maintain true pathogen ratio.
  • Storage
    • Protect heat-/cold-labile microbes; inhibit overgrowth of commensals.
    • Transport media, temperature control boxes.
  • Aseptic technique protects patient, collector, and culture integrity.

Healthcare-Associated Infection (HAI) Landscape

  • Majority of HAIs stem from a limited group (CDC NHSN 2009-2010):
    • Coagulase-negative staphylococci (CoNS), S. aureus, enterococci, Enterobacteriaceae, Pseudomonas, yeast.
    • Linked syndromes: SSI, CLA-BSI, CA-UTI, VAP.

Major Pathogenic Genera

1. Staphylococci

  • Key species: S. aureus (≈30\% nasal carriage).
    • Opportunistic: bacteremia/sepsis, ventilator pneumonia, endocarditis, osteomyelitis, skin/wound infections.
    • Notorious MRSA & VRSA.
    • ID markers: coagulase (+), ferments mannitol on MSA (yellow halo).
  • S. epidermidis
    • Normal skin flora; opportunistic similar to S. aureus.
    • Coagulase (−), mannitol (−); part of CoNS grouping.

2. Streptococci & Enterococci

  • Gram (+) cocci chains, catalase (−), often fastidious (blood, \text{CO}_2).
  • Hemolysins
    • \alpha: partial, green.
    • \beta: complete, clear.
    • \gamma: none.
  • Examples
    • Group A (GAS): S. pyogenes – \beta hemolytic, bacitracin sensitive; strep throat, scarlet fever, impetigo, rheumatic fever.
    • Viridans/Group D
    • S. pneumoniae: \alpha hemolytic, optochin sensitive; pneumonia, meningitis; prefers candle jar (high \text{CO}_2).
    • Enterococcus faecalis: usually \alpha hemolytic; UTI, wound, endocarditis; intrinsic resistance profile.
    • Group N: S. lactis (dairy starter), \gamma hemolytic.

3. Family Enterobacteriaceae

  • Gram (−), facultative anaerobic rods; ubiquitous — commensals, pathogens, plant parasites.
  • Economic & clinical impact: diarrheal disease, UTIs, sepsis.
  • Primary differentiator – lactose fermentation on EMB/MacConkey.
  • Lactose (+): E. coli, Klebsiella, Enterobacter, Serratia.
  • Lactose (−): Salmonella, Shigella, Yersinia, Proteus.

4. Pseudomonas

  • Non-enteric, non-fermentative Gram (−) bacilli.
  • P. aeruginosa
    • Multi-drug resistant baseline; accumulating further resistance.
    • Opportunistically infects burns, wounds, lungs (CF/VAP), GI, urinary tract.
    • Produces soluble pigment (pyocyanin/pyoverdin) → greenish media.
    • Oxidase (+) – key bench ID.

Selective & Differential Culture Media

Mannitol Salt Agar (MSA)

  • 7!\text{–}!10\% NaCl = selective for staphylococci.
  • Phenol red indicator.
    • Mannitol (+)/coagulase (+) → yellow colonies + yellow agar (S. aureus).
    • Mannitol (−)/coagulase (−) → pink/red colonies, medium unchanged (S. epidermidis).
  • Comparative plate results: 1 Sterile | 2 S. epidermidis | 3 S. aureus | 4 E. coli (no growth).

Blood Agar

  • 5\% sheep blood; detects hemolysis.
    • \beta – clear zone (S. pyogenes, S. agalactiae, S. aureus, P. aeruginosa, Listeria).
    • \alpha – green halo (S. pneumoniae, oral streptococci).
    • \gamma – none.
  • Result key: 1 Sterile | 2 \alpha | 3 \beta | 4 \gamma.

Eosin–Methylene Blue Agar (EMB)

  • Selective: bile salts + dyes inhibit Gram (+).
  • Differential: lactose fermentation rate.
    • Vigorous (++): green metallic sheen (E. coli).
    • Slow (+): pink-purple (Enterobacter aerogenes).
    • Non-fermenter: colorless (Proteus, Salmonella).

Simmon’s Citrate

  • Green slant contains citrate as sole C source + bromothymol blue.
    • Utilization → alkalinization → blue (positive).
    • Remains green if citrate (−).

Quick Reference Table

  • MSA: Mannitol (+) = S. aureus; (−) = S. epidermidis.
  • EMB: Lactose (+) = E. coli, Enterobacter; (−) = Proteus, Salmonella.
  • Blood agar: \alpha = S. pneumoniae; \beta = S. pyogenes; \gamma = Enterococcus.
  • Citrate (+) = Enterobacter, Proteus; (−) = E. coli.

Antibiotic Susceptibility Assays (Gram + Focus)

  • Bacitracin disk (0.04 U) on \beta-hemolytic streptococci
    • Sensitive = zone ≥10\,\text{mm} → Group A (S. pyogenes).
    • Resistant → other \beta-streps.
  • Optochin disk (ethylhydrocupreine)
    • Sensitive (≥14\,\text{mm}) → S. pneumoniae.
    • Resistant → other \alpha streps (Viridans group).
  • Methicillin (oxacillin) screen – detects MRSA among staphylococci.
  • Bacitracin further separates Micrococcus (susceptible) from staphylococci (resistant).

Representative Bench Flow Charts

Gram (+) Cocci

  • Catalase (+)
    • MSA: Mannitol (+)/Coagulase (+) → S. aureus.
    • Mannitol (−)/Coagulase (−) → S. epidermidis.
  • Catalase (−)
    • Blood agar \alpha + Optochin S → S. pneumoniae.
    • \beta + Bacitracin S → S. pyogenes.

Gram (−) Bacilli

  • EMB Lactose (+)
    • Metallic green, Indole (+), Citrate (−) → E. coli.
    • Mucoid pink/purple, Indole (−), Citrate (+) → Enterobacter aerogenes.
  • Lactose (−)
    • Colorless, H_2S (+), Pdase (+), Citrate (+) → Proteus mirabilis.

Constructing Your Own Identification Flow Chart

  • Start broad → narrow.
    • Exclude non-relevant groups rapidly (Gram stain, oxygen tolerance).
  • Identify the terminal test unique for each target; plan backward.
  • Combine tests to minimize media & time (e.g. triple sugar iron gives glucose/lactose/sucrose + H_2S data simultaneously).
  • Make parallel lists
    • All Gram (+) vs Gram (−) organisms under consideration.
    • Their distinctive biochemical/enzymatic traits.
  • Iterate forward to ensure logical progression.
  • Supplementary tutorial: https://www.youtube.com/watch?v=JIu1nL2_xE0

Beyond Classical Methods

  • Molecular
    • 16S rRNA sequencing, PCR, whole-genome sequencing provide precise ID & resistance genes.
    • Virtual lab simulation: https://media.hhmi.org/biointeractive/vlabs/bacterial_id/index.html.
  • Serology
    • Detects pathogen antigens or host antibodies; useful for non-cultivable or intracellular agents.
  • Ethical/Practical Considerations
    • Stewardship: only order targeted tests; avoid unnecessary broad empirics to curb resistance.
    • Biosafety: correct handling of potential bioterror organisms (B. anthracis, F. tularensis).
    • Data privacy when sequence data link to outbreaks.

Quick Equation & Value Recap

  • High-salt in MSA: 7\text{–}10\% NaCl.
  • Sheep blood in blood agar: 5\%.
  • Acidic growth cut-off for Pseudomonas: \text{pH} < 4.5.
  • Nasal carriage of S. aureus: \approx30\% of population.