Topic Overview

🔶 ENTERIC GRAM-NEGATIVE RODS (ENTEROBACTERIACEAE)

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🔹 CLASSIFICATION & GENERAL CHARACTERISTICS

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🔸 Definition & Overview

• Family of Gram-negative, non-spore forming bacilli

• Primarily intestinal commensals, but many are important pathogens

• Facultative anaerobes

• Major cause of:

  • Gastrointestinal infections

  • Urinary tract infections

  • Septicemia

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🔸 Habitat

• Normal intestinal flora

  • E. coli (dominant)

  • Enterobacter, Klebsiella (less common)

• Environmental presence

  • Soil

  • Water

  • Food

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🔸 Morphology

• Gram-negative rods

• Non-spore forming

• Motility:

  • Motile → peritrichous flagella (E. coli, Salmonella, Proteus)

  • Non-motile → Klebsiella, Shigella

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🔸 Cultural Characteristics

• Grow on ordinary media

• MacConkey agar:

  • Lactose fermenters (LF) → pink colonies

  • Non-lactose fermenters (NLF) → pale colonies

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🔸 Cell Wall Structure (VERY HIGH-YIELD)

• Outer membrane contains LPS (Endotoxin)

Components:

• Lipid A

  • Toxic component → fever, shock

• Core polysaccharide

• O antigen

  • Serotyping basis

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🔸 Biochemical Properties

• Oxidase negative

• Catalase positive

• Ferment glucose

• Reduce nitrates to nitrites

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🔸 Antigenic Structure

Antigen	Location	Function	Example
O antigen	Cell wall	Serotyping	Salmonella
H antigen	Flagella	Motility	Salmonella
K antigen	Capsule	Virulence	Klebsiella
Vi antigen	Capsule	Anti-phagocytic	S. typhi

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🔸 Mechanism of Endotoxin (VERY IMPORTANT)

• LPS (Lipid A) released
  → Macrophage activation
  → Cytokines (TNF, IL-1)
  → Complement activation
  → Coagulation cascade
  → DIC + septic shock

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🔸 Virulence Factors

Factor	Function	Example
Capsule	Anti-phagocytic	Klebsiella
Fimbriae	Adhesion	E. coli (UTI)
Toxins	Tissue damage	ETEC toxins
Invasion factors	Cell entry	Shigella

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🔸 Plasmids & Resistance

• R plasmids → antibiotic resistance

• ESBL (Extended spectrum β-lactamase)

• Carbapenemases (NDM, KPC) → highly resistant strains

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🔸 Classification

Based on Lactose Fermentation

Lactose Fermenters	Non-Lactose Fermenters
E. coli	Salmonella
Klebsiella	Shigella
Enterobacter	Proteus
Serratia (late LF)

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Based on Motility

Motile	Non-motile
E. coli	Klebsiella
Salmonella	Shigella
Proteus

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Based on IMViC Reactions

Organism	Indole	MR	VP	Citrate
E. coli	+	+	–	–
Klebsiella	–	–	+	+
Enterobacter	–	–	+	+

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🔸 Important Genera

• Escherichia

• Klebsiella

• Proteus

• Enterobacter

• Citrobacter

• Serratia

• Shigella

• Salmonella

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🔬 SLIDES (EXAM FAVORITE)

Gram-negative bacilli

MacConkey agar (LF vs NLF)

Capsule demonstration (Klebsiella)

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🧠 DIAGRAMS / FLOWCHARTS

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🔹 Classification of Enterobacteriaceae

Gram-negative bacilli
↓
Enterobacteriaceae
↓
Lactose fermenters → E. coli, Klebsiella
Non-lactose fermenters → Salmonella, Shigella

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🔹 LPS Endotoxin Mechanism

LPS (Lipid A)
↓
Macrophage activation
↓
Cytokines (TNF, IL-1)
↓
Complement activation
↓
Coagulation cascade
↓
DIC → Shock

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🔹 Lactose Fermentation Differentiation

MacConkey agar
↓
Pink colonies → Lactose fermenter
↓
E. coli / Klebsiella

Colorless colonies → Non-lactose fermenter
↓
Salmonella / Shigella

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🔹 LABORATORY DIAGNOSIS (VERY HIGH-YIELD CORE)

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🔸 Specimen Collection

• Stool

  • Acute diarrhea, dysentery

  • Fresh sample preferred

• Blood

  • Enteric fever (1st week best)

• Urine

  • UTI (E. coli, Klebsiella, Proteus)

• Pus / body fluids

  • Abscess, wound infection, septicemia

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🔸 Transport Media

• Cary-Blair medium

  • Preserves enteric pathogens

  • Prevents overgrowth of commensals

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🔸 Culture Media

• MacConkey agar

  • LF → Pink colonies

  • NLF → Pale colonies

• XLD agar (Xylose Lysine Deoxycholate)

  • Salmonella → Red colonies with black center (H₂S)

  • Shigella → Red colonies

• SS agar (Salmonella-Shigella agar)

  • Salmonella → Colorless with black center

  • Shigella → Colorless

• DCA agar (Deoxycholate citrate agar)

  • Selective for enteric pathogens

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🔸 Enrichment Media

• Selenite F broth

  • Enhances Salmonella growth

• Tetrathionate broth

  • Selective enrichment for Salmonella

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🔸 Colony Characteristics

• Size, shape, margin, elevation

• Pigmentation

• Hemolysis (if any)

• Swarming → Proteus

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🔸 Biochemical Identification

IMViC Tests

• Indole

• Methyl red

• Voges–Proskauer

• Citrate

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TSI (Triple Sugar Iron) Test

• Glucose, lactose, sucrose fermentation

• Gas production

• H₂S production

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Other Tests

• Urease test

  • Positive → Proteus, Klebsiella

• Citrate test

  • Positive → Klebsiella, Enterobacter

• Motility test

  • Motile → Salmonella, E. coli

  • Non-motile → Shigella, Klebsiella

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🔸 Automated Systems (Brief)

• VITEK

  • Rapid identification + antibiotic sensitivity

• MALDI-TOF

  • Protein-based rapid organism identification

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📊 TABLES (VERY HIGH-YIELD)

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🔹 Selective & Differential Media

Medium	Function	Organism Identified
MacConkey	LF vs NLF	E. coli, Salmonella
XLD	Enteric pathogens	Salmonella, Shigella
SS agar	Selective for pathogens	Salmonella, Shigella
DCA	Enteric pathogens	Salmonella

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🔹 IMViC Reactions (Detailed)

Organism	Indole	MR	VP	Citrate
E. coli	+	+	–	–
Klebsiella	–	–	+	+
Enterobacter	–	–	+	+

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🔹 TSI Reaction Patterns

Organism	Slant	Butt	Gas	H₂S
E. coli	A	A	+	–
Salmonella	K	A	±	+
Shigella	K	A	–	–
Proteus	K	A	+	+

(A = Acid, K = Alkaline)

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🔹 Biochemical Test Interpretation

Test	Positive Organism
Urease	Proteus, Klebsiella
Citrate	Klebsiella, Enterobacter
Motility	Salmonella, E. coli

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🔬 SLIDES (EXAM FAVORITE)

TSI agar slants

XLD agar colonies

SS agar colonies

Motility test

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🧠 DIAGRAMS / FLOWCHARTS

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🔹 Stepwise Lab Diagnosis Algorithm

Specimen
↓
Transport medium (Cary-Blair)
↓
Culture on selective media
↓
Colony morphology
↓
Biochemical tests (IMViC, TSI)
↓
Identification
↓
Antibiotic sensitivity

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🔹 TSI Reaction Interpretation

Glucose fermentation
↓
Acid butt (yellow)

If lactose/sucrose ferment → Entire tube yellow (A/A)

If not → Slant red, butt yellow (K/A)

H₂S production → Black precipitate

Gas production → Cracks / bubbles

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🔹 DISEASES CAUSED BY ENTEROBACTERIACEAE (EXCLUDING SALMONELLA & SHIGELLA)

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🔸 ESCHERICHIA COLI

Types (Pathotypes)

• ETEC (Enterotoxigenic) → Traveler’s diarrhea

• EPEC (Enteropathogenic) → Infantile diarrhea

• EHEC (Enterohemorrhagic) → Hemorrhagic colitis, HUS

• EIEC (Enteroinvasive) → Dysentery-like illness

• EAEC (Enteroaggregative) → Persistent diarrhea

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Virulence Factors

• LT toxin (Heat-labile)

  • ↑ cAMP → secretory diarrhea

• ST toxin (Heat-stable)

  • ↑ cGMP → secretory diarrhea

• Verotoxin (Shiga-like toxin)

  • Inhibits protein synthesis → endothelial damage

• Adhesins (fimbriae)

  • Attachment to intestinal mucosa

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Mechanisms of Diarrhea

• Secretory → ETEC (LT, ST)

• Invasive → EIEC

• Hemorrhagic → EHEC

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Clinical Features

• Traveler’s diarrhea

• Hemorrhagic colitis

• Watery diarrhea (children)

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Other Important Syndromes

• Uropathogenic E. coli (UPEC)

  • Ascending UTI

• Neonatal meningitis

  • K1 antigen (capsule)

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🔸 KLEBSIELLA

Key Features

• Prominent capsule → major virulence

• Hypermucoviscosity (string test positive)

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Diseases

• Pneumonia

  • Thick “currant jelly” sputum

• UTI

• Rhinoscleroma

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Resistance

• ESBL-producing strains

• MDR hospital pathogens

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🔸 PROTEUS

Key Features

• Swarming motility

• Urease positive

• H₂S production

• Dienes phenomenon (strain differentiation)

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Clinical Importance

• UTI

• Renal stone formation

  • Urease → ammonia → alkaline urine → stones

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🔸 ENTEROBACTER, CITROBACTER, SERRATIA

Features

• Opportunistic pathogens

• Cause nosocomial infections

• High drug resistance

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Clinical Importance

• UTI

• Pneumonia

• Septicemia

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📊 TABLES (VERY HIGH-YIELD)

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🔹 Types of E. coli and Diseases

Type	Disease	Mechanism
ETEC	Traveler’s diarrhea	Secretory (LT, ST)
EPEC	Infantile diarrhea	Adherence
EHEC	Hemorrhagic colitis	Verotoxin
EIEC	Dysentery	Invasion
EAEC	Persistent diarrhea	Aggregation

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🔹 E. coli Toxin Comparison

Toxin	Mechanism	Effect
LT	↑ cAMP	Watery diarrhea
ST	↑ cGMP	Watery diarrhea
Verotoxin	Ribosomal inhibition	Bloody diarrhea

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🔹 Enterobacteriaceae Causing UTI

Organism	Feature
E. coli	Most common
Klebsiella	Capsule
Proteus	Stones (urease)

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🔹 Nosocomial Pathogens Comparison

Organism	Key Feature
Enterobacter	Opportunistic
Serratia	Red pigment (sometimes)
Klebsiella	MDR, ESBL

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🔬 SLIDES (EXAM FAVORITE)

E. coli colonies

Klebsiella mucoid colonies

Proteus swarming

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🧠 DIAGRAMS / FLOWCHARTS

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🔹 E. coli Diarrheal Mechanism

Adhesion
↓
Toxin production / invasion
↓
Electrolyte imbalance
↓
Diarrhea

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🔹 UTI Pathogenesis (Ascending)

Perineal flora (E. coli)
↓
Urethra
↓
Bladder
↓
Kidney (pyelonephritis)

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🔹 Proteus Stone Formation

Urease
↓
Urea → Ammonia
↓
Alkaline urine
↓
Struvite stones

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🔹 THE SHIGELLAE

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🔸 Classification

• S. dysenteriae

• S. flexneri

• S. boydii

• S. sonnei

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🔸 Morphology

• Gram-negative bacilli

• Non-motile

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🔸 Cultural & Biochemical Features

• Non-lactose fermenter

• No H₂S production

• Oxidase negative

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🔸 Antigenic Structure

• Only O antigen present

• No H antigen

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🔸 Infective Dose

• Very low (10–100 organisms)

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🔸 Pathogenesis

• Entry via contaminated food/water

• M cell invasion

• Intracellular multiplication

• Spread to adjacent cells

• Shiga toxin → inhibits 60S ribosome

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🔸 Clinical Features

• Bacillary dysentery

  • Blood + mucus in stool

  • Tenesmus

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🔸 Complications

• Hemolytic uremic syndrome (HUS)

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🔸 Laboratory Diagnosis

• Stool microscopy → RBC, pus cells

• Culture → NLF colonies

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🔸 Treatment & Resistance

• Antibiotics (based on sensitivity)

• Increasing resistance trends

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📊 TABLES (VERY HIGH-YIELD)

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🔹 Shigella Species Comparison

Species	Severity
S. dysenteriae	Severe
S. flexneri	Common
S. sonnei	Mild

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🔹 Shigella vs EIEC

Feature	Shigella	EIEC
Motility	Non-motile	Motile
Toxin	Present	Absent
Severity	Severe	Mild

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🔹 Biochemical Reactions

Test	Result
Lactose	–
H₂S	–
Motility	–

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🔬 SLIDES (EXAM FAVORITE)

Shigella colonies

Stool microscopy

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🧠 DIAGRAMS / FLOWCHARTS

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🔹 Pathogenesis of Shigella

Ingestion
↓
M cell invasion
↓
Intracellular multiplication
↓
Cell destruction
↓
Dysentery

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🔹 Shiga Toxin Mechanism

Toxin
↓
60S ribosome inhibition
↓
Protein synthesis blocked
↓
Cell death

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🔹 THE SALMONELLA–ARIZONA GROUP

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🔸 Classification

• Kauffmann–White classification

  • Based on O (somatic) and H (flagellar) antigens

• Major groups:

  • Salmonella typhi

  • Salmonella paratyphi (A, B, C)

  • Non-typhoidal Salmonella (NTS) → S. typhimurium, S. enteritidis

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🔸 Morphology

• Gram-negative bacilli

• Motile (peritrichous flagella)

• Non-spore forming

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🔸 Cultural Characteristics

• Non-lactose fermenter

• MacConkey → pale colonies

• XLD → red colonies with black center (H₂S)

• SS agar → colorless colonies with black center

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🔸 Biochemical Reactions

• Glucose fermentation → positive

• Lactose → negative

• H₂S production → positive

• Urease → negative

• Citrate → positive (most species)

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🔸 Antigenic Structure

Antigen	Feature	Significance
O antigen	Somatic	Early immune response
H antigen	Flagellar	Phase variation (Phase 1 & 2)
Vi antigen	Capsule (S. typhi)	Anti-phagocytic, carrier state

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🔸 Pathogenesis

• Ingestion via contaminated food/water
  ↓

• Survives gastric acid
  ↓

• Invades intestinal mucosa (Peyer’s patches)
  ↓

• Multiplies in macrophages
  ↓

• Primary bacteremia
  ↓

• Dissemination to liver, spleen, bone marrow
  ↓

• Secondary bacteremia → systemic disease (enteric fever)

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🔸 Clinical Features

Enteric Fever

• Step-ladder fever

• Relative bradycardia

• Hepatosplenomegaly

• Rose spots

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Food Poisoning (NTS)

• Acute gastroenteritis

• Vomiting + diarrhea

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Septicemia

• Immunocompromised patients

• High mortality

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🔸 Carrier State

Type	Site
Intestinal	Intestine
Biliary	Gall bladder (most important)
Urinary	Urinary tract

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🔸 Stage-wise Disease Progression

Week	Findings
1st	Fever, bacteremia
2nd	High fever, organ involvement
3rd	Complications
4th	Recovery

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🔸 Complications

• Intestinal perforation

• Hemorrhage

• Septic shock

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🔸 Laboratory Diagnosis

• Blood culture

  • Best in 1st week

• Stool culture

  • Later stages

• Widal test

  • Detects antibodies against O & H antigens

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🔸 Vaccines

• Vi polysaccharide vaccine

• Oral Ty21a vaccine

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📊 TABLES (VERY HIGH-YIELD)

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🔹 Typhoidal vs Non-typhoidal Salmonella

Feature	Typhoidal	Non-typhoidal
Example	S. typhi	S. typhimurium
Disease	Enteric fever	Gastroenteritis
Host	Humans only	Animals + humans

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🔹 Antigenic Structure Comparison

Antigen	Function
O	Somatic
H	Motility
Vi	Capsule

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🔹 Widal Test Interpretation

Antibody	Significance
O	Acute infection
H	Past infection

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🔹 Stage-wise Culture Positivity

Stage	Best Sample
Early	Blood
Late	Stool
Carrier	Stool/urine

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🔹 Typhoid vs Paratyphoid

Feature	Typhoid	Paratyphoid
Severity	Severe	Mild
Organism	S. typhi	S. paratyphi

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🔬 SLIDES (EXAM FAVORITE)

Salmonella colonies

Blood culture

Widal test

 

🧠 DIAGRAMS / FLOWCHARTS

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🔹 Pathogenesis of Enteric Fever

Ingestion
↓
Intestinal invasion
↓
Macrophage survival
↓
Primary bacteremia
↓
Reticuloendothelial system
↓
Secondary bacteremia
↓
Systemic disease

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🔹 Carrier State (Gall Bladder)

Salmonella persistence
↓
Gall bladder colonization
↓
Chronic shedding
↓
Carrier state

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🔹 Widal Reaction Principle

Antigen (O/H)
↓
Patient serum
↓
Antibody binding
↓
Agglutination

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🔹 Antigen Structure Diagram

Cell wall
↓
O antigen

Flagella
↓
H antigen

Capsule
↓
Vi antigen

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🔹 CHAPTER SUMMARY

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🔸 Key Characteristics

• Gram-negative bacilli

• Non-lactose fermenters

• H₂S production

• Antigenic variation

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🔸 Important Organisms

• Salmonella typhi

• Salmonella paratyphi

• Non-typhoidal Salmonella

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🔸 Diagnostic Approach

• Blood culture (early)

• Stool culture (late)

• Widal test

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🔸 Virulence Mechanisms

• Intracellular survival

• Endotoxin

• Vi antigen (immune evasion)

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🔸 High-Yield Differentiating Points

• H₂S production

• Motility

• Antigenic variation

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📊 TABLES (VERY HIGH-YIELD)

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🔹 Organism vs Disease

Organism	Disease
S. typhi	Enteric fever
S. paratyphi	Mild enteric fever
NTS	Gastroenteritis

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🔹 Quick Revision Table

Feature	Salmonella
Motility	+
Lactose	–
H₂S	+
Urease	–

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🧠 DIAGRAMS / FLOWCHARTS

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🔹 Organism → Disease Concept Map

Salmonella
↓
Typhi → Enteric fever
Paratyphi → Mild fever
NTS → Gastroenteritis

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🔹 Diagnostic Algorithm

Fever
↓
Blood culture
↓
Positive → Salmonella

If negative
↓
Stool culture / Widal test
↓
Confirmation

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