LC 7: ENTEROBACTERIACEAE: ENTERIC GRAM NEGATIVE RODS

I. GENERALITIES

  • Characteristics:

    • Large heterogeneous group of Gram-negative rods.

    • Differ in terms of carbohydrate fermentation.

    • Natural habitat is the intestine.

    • Typical organisms include:

    • Escherichia coli/Shigella/Salmonella/Enterobacter/Klebsiella/Serratia/Proteus/Morganell/Providencia/Citrobacter

    • E. coli:

    • Part of normal flora of the large intestine.

    • Can cause disease in immunocompromised individuals (diabetics, patients on steroids, undergoing chemotherapy).

    • Salmonella/Shigella: Always pathogenic.

  • Oxygen Requirement:

    • Facultative anaerobes (can tolerate environments with or without oxygen).

  • Carbohydrate Fermentation:

    • Ability to ferment a wide range of carbohydrates.

  • Antigenic Structures:

    • Possess complex antigen structures, produce toxins, and virulence factors (ability to cause disease).

II. CLASSIFICATION

  • Overview:

    • Most common group of Gram-negative rods.

  • Motility:

    • Either motile with peritrichous flagella or non-motile.

  • Growth Characteristics:

    • Grow on peptone or meat extract without additional supplements.

    • Well on MacConkey agar.

    • Grows facultatively aerobically and anaerobically.

    • Ferment glucose rather than oxidizing it, often with gas production.

    • Catalase positive.

    • All Enterobacteriaceae are oxidase negative (except Pleisomonas).

    • Reduce nitrate to nitrite as a form of energy.

    • DNA content: 35-59% Guanine+Cytosine.

III. MORPHOLOGY & IDENTIFICATION

A. DIFFERENTIATION

  • Biochemical Test:

    • Use of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) for identification of Gram-negative bacteria (except Shigella).

    • Identification of Shigella is through biochemical tests.

    • MALDI-TOF MS process:

    • Bacterial colonies placed on a target plate with chemical matrix.

    • A laser ionizes proteins for mass-to-charge ratio separation in a time-of-flight analyzer; spectral fingerprint compared with a database for identification.

B. TYPICAL ORGANISMS

  • General Characteristics:

    • Short Gram-negative rods.

    • Some species like Klebsiella have well-defined capsules.

C. CULTURE OF GRAM-NEGATIVE RODS

  • Colony Appearance:

    • Most enteric bacteria form circular, convex, smooth colonies with distinct edges.

    • Enterobacter: mucoid colonies.

    • Klebsiella: large, very mucoid, often coalesce with prolonged incubation.

    • Salmonella: colonies resemble E. coli but are distinct.

    • Shigella: non-lactose fermenters; may be transparent with distinct edges.

D. GROWTH CHARACTERISTICS

  • Carbohydrate (CHO) Fermentation Patterns:

    • Determined through the activity of amino acids, carbohydrates, and specific enzymes.

    • Examples:

    1. Indole production from tryptophan (e.g., E. coli).

    2. Voges-Proskauer reaction: production of acetylmethylcarbinol from dextrose.

  • Cultural Media:

    • Differential media used include:

    • Eosin-methylene blue (EMB) agar.

    • MacConkey agar.

    • Deoxycholate medium to distinguish lactose fermenters from non-lactose fermenters.

  • Lactose Fermentation Types:

    • Rapid Lactose Fermenters:

    • E. coli: exhibits metallic green sheen on differential media.

    • Enterobacter aerogenes: raised colonies without sheen.

    • Klebsiella pneumoniae: very viscous, mucoid growth, non-motile.

    • Non-Lactose Fermenters:

    • Shigella: non-motile, no gas from dextrose.

    • Salmonella: motile, gas from dextrose.

    • Proteus: swarming on agar; urease activity.

    • Pseudomonas: soluble pigments, blue-green color.

    • Slow Lactose Fermenters:

    • Edwardsiella, Serratia, Citrobacter, Arizona, Providencia.

E. TSI AGAR TESTING

  • Purpose:

    • Differentiate Salmonella and Shigella from other Enterobacteriaceae.

    • Composition:

    • 0.1% glucose (butt), 1% sucrose (mid), 1% lactose (slant); typical ratio: 1:10:10.

  • Procedures:

    • Single stab into the butt for glucose fermentation; zigzag streak for lactose/sucrose fermentation.

  • Reactions:

    • pH Indicators: Phenol red.

    • Results:

    • Both slant and butt turn yellow (A/A) indicate glucose and lactose/sucrose fermentation.

    • H₂S detection via ferrous sulfate.

    • Common Outcomes:

      • Salmonella & Shigella yield alkaline slant and acid butt (K/A).

      • Proteus can indicate rapid red coloration in Christensen’s urea medium.

IV. SPECIFIC ORGANISMS ATTRIBUTIONS

A. ESCHERICHIA

  • Characteristics:

    • Positive results for indole, lysine decarboxylase, and mannitol fermentation.

    • Gas production from glucose and hemolytic activity on blood agar, characterized by iridescent sheen on EMB.

    • Negative oxidase test.

    • Figure: TSI test result representation showing yellow slant and butt, gas bubbles, and absence of H₂S.

  • Metabolic Activities:

    • Amino acids variably metabolized; enzymatic actions on amino acid decarboxylation.

B. KLEBSIELLA-ENTEROBACTER-SERRATIA

  • Klebsiella:

    • Mucoid growth, large polysaccharide capsule, non-motile, and positive for lysine decarboxylase and citrate.

  • Enterobacter:

    • Slight capsule, motile, positive tests for motility, citrate, and ornithine decarboxylase.

  • Serratia:

    • Positive for DNAse, lipase, gelatinase; usually positive for Voges-Proskauer reaction.

C. PROTEUS-MORGANELLA-PROVIDENCIA

  • Proteus:

    • Displays rapid urease activity and motility via peritrichous flagella; results in swarming on agar.

  • Morganella/Providencia:

    • Known for opportunistic infections, especially urinary tract infections.

  • Urease Test:

    • Positive results indicate breakdown of urea, making the medium alkaline and changing color due to phenol red.

D. CITROBACTER

  • Citrate Utilization:

    • Positive test results in a blue color change indicating citrate as a carbon source.

E. SHIGELLA

  • Characteristics:

    • Non-motile, non-lactose fermenters, and induce acid without gas from CHO.

    • Pathogenicity tied to infection and inflammation in the intestinal tract.

F. SALMONELLA

  • General Characteristics:

    • Motile, rapid growth in simple media, non-lactose & non-sucrose fermenters; produces H₂S.

    • Major serotype is Salmonella enterica, with multiple subspecies causing human illness.

V. DISEASES CAUSED BY ENTEROBACTERIACEAE (EXCLUDING SALMONELLA & SHIGELLA)

A. CAUSATIVE ORGANISMS

  • Common members causing infections include E. coli, Klebsiella, Enterobacter, Proteus, Morganella, Providencia, Citrobacter, and Serratia.

  • Typically non-pathogenic until reaching tissues outside their normal habitats, implicated in urinary tract infections and other systemic infections.

  • Organisms capable of causing bacteremia, peritonitis, and opportunistic infections in immunocompromised hosts.

B. ESCHERICHIA COLI

  1. Urinary Tract Infections (UTIs):

    • Caused in 90% of cases by E. coli. Symptoms include dysuria, hematuria, pyuria, flank pain.

    • Laboratory tests reveal RBCs and WBCs.

    • Uropathogenic strains elaborate virulence, such as hemolysins, and may express K antigens that facilitate adhesion.

    • Specific clones (e.g., E. coli 025b/ST1) are resistant to common antibiotic treatments.

  2. Sepsis:

    • Due to inadequate host defenses; especially risky in newborns lacking IgM antibodies.

  3. Meningitis:

    • Prominent cause in infants attributed to K1 antigen.

C. E. COLI ASSOCIATED DIARRHEAL DISEASES

  • EPEC (Enteropathogenic E. coli):

    • Affects infants, causing severe watery diarrhea through mucosal cell adherence and effacement, resulting in loss of microvilli.

  • ETEC (Enterotoxigenic E. coli):

    • Major cause of traveler's diarrhea, with factors promoting adherence and localized production of heat-labile and stable toxins, leading to hypersecretion of fluids.

  • STEC (Shiga Toxin-Producing E. coli):

    • Known as EHEC or VTEC, linked to hemorrhagic colitis and hemolytic uremic syndrome. E. coli O157:H7 is most notable for clinical isolation.

  • EIEC (Enteroinvasive E. coli):

    • Resembles shigellosis, prevalent in developing countries.

  • EAEC (Enteroaggregative E. coli):

    • Causes prolonged diarrhea, notably in HIV patients.

D. KLEBSIELLA, ENTEROBACTER, SERRATIA, PROTEUS, AND CITROBACTER

  • Klebsiella pneumoniae:

    • Causes severe lung infections in debilitated patients, may produce extensive necrosis and bleeding in tissue.

  • Enterobacter: Generally opportunistic, can be resistant to multiple antibiotics due to intrinsic resistance mechanisms.

  • Proteus: Produces creating an alkaline urinary environment, promoting stone formation.

  • Providencia and Citrobacter: Known for urinary and some systemic infections, resistant to multiple treatment options.

E. DIAGNOSTIC & LABORATORY TESTS

  1. Specimen Types:

    • Blood, urine, spinal fluid, or culture as dictated by clinical scenarios.

  2. Culture Techniques:

    • Plating on blood agar and differential media assists in identification.

  3. Nucleic Acid Amplification Tests:

    • Rapid tests available for quick pathogen identification in various specimens including blood and stool.

F. IMMUNITY

  • Immunity may develop following infections; systemic infections typically elicit specific antibodies, but long-lasting immunity is uncertain.

G. TREATMENT

  • No definitive treatments available; management primarily revolves around surgical interventions where necessary and antibiotic therapy for infections.

H. EPIDEMIOLOGY, PREVENTION, AND CONTROL

  • Enteric bacteria are typically part of the normal flora established shortly post-birth; contamination through water and food remains critical in outbreak prevention.

  • Handwashing, sterilization, and aseptic techniques are crucial for preventing infections.

VI. SHIGELLA

A. TRANSMISSION

  • Primary transmission through feces, fingers, food, flies with low infectious dose making young children particularly vulnerable.

B. MORPHOLOGY & IDENTIFICATION

  • Slender Gram-negative rod; grows best aerobically and forms characteristic colonies on selective media.

C. GROWTH CHARACTERISTICS

  • Non-lactose fermenters that mainly ferment glucose producing acids.

D. ANTIGENIC STRUCTURE

  • Composed of LPS; infection leads to distinctive intestinal pathologies.

E. TOXINS

  • Release of endotoxin and specific exotoxins that influence intestinal and CNS functions leading to dysentery-like symptoms.

F. CLINICAL FINDINGS

  • Short incubation with symptoms including abdominal pain, fever, and water-to-bloody diarrhea progression.

G. DIAGNOSIS & LABORATORY TEST

  • Fresh stool samples typically analyzed for growth on selective media; serologic testing of limited use.

H. IMMUNITY & TREATMENT

  • Type-specific antibody responses observed but reinfection is possible. Treatment options include multiple antibiotics, avoiding opioids.

VII. SALMONELLA

A. MORPHOLOGY & IDENTIFICATION

  • Motile with characteristic flagella, rapidly grows in simple media and non-lactose fermenters.

B. CLASSIFICATION

  • Includes multiple subspecies with varying pathogenic potential, primarily sourced from human and animal infections.

C. PATHOGENESIS AND CLINICAL FINDINGS

  • Manifestations include typhoid fever, bacteremia, and enterocolitis from various serotypes, differing primarily by incubation periods and clinical presentations.

D. DIAGNOSTIC AND LABORATORY TEST

  • Blood cultures, stool testing, and upholding isolation measures are imperative for identifying Salmoanella infection.

E. TREATMENT

  • Severe infections managed with specific antibiotics, while many gastroenteritis cases typically self-resolve without treatment.

F. VACCINES

  • Different vaccines available for typhoid fever, including TCV conjugate vaccines which show promise in inducing immunity in susceptible populations.