Identification of the Enterobacteriacae

THE ENTERIC BACILLI

Diseases: Enteric bacteriosis (hospital-acquired or nosocomial infections with various Gram-negative rods acquired by patients during hospitalization).

Etiologic Agents: Enterobacter, Escherichia, Klebsiella, Proteus, and Serratia.

Source: environmental sources, clinical sources, and normal flora

Pathogenesis: Bacteremia, pneumonia, urinary tract infections, wound infections, CNS infections, abscess formation in various organs, and colonization and infection of implants, prostheses, and catheters.

Laboratory Diagnosis: Bacteriologic isolation of etiologic agents from blood, other body fluids, wounds, and exudates. Antimicrobial susceptibilities tests characteristically reveal resistance to commonly used antibiotics. Molecular epidemiological analysis may identify resistance plasmids. Several companies manufacture diagnostic kits based on biochemical test properties of the organisms. The Enterotube method will be demonstrated.

THE LACTOSE FERMENTERS

E. coli INFECTIONS

Diseases: Intestinal infections (diarrhea, dysentery, hemorrhagic colitis). Urinary tract infections (asymptomatic bacteriuria, cystitis, pyelonephritis, prostatitis). Bacteremia. Neonatal meningitis.

Etiologic Agents: Intestinal infections - enterotoxigenic Escherichia coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), and enterohemorrhagic E. coli (EHEC). Urinary tract infections - E. coli, usually strains carrying uropathogenic determinants. Bacteremia - E. coli, usually strains belonging to the same serogroups associated with urinary tract infections. Neonatal meningitis - E. coli (usually strains with K1 Ag).

Source: Intestinal infections - human intestinal tract, cattle (EHEC). UTI, Bacteremia and Neonatal meningitis- human intestinal tract

Pathogenesis: Intestinal infections - interaction of the intestinal mucosa with colonization factors, heat-labile (LT) and heat-stable (ST) enterotoxins (ETEC); enterocyte invasiveness factors (EIEC); enteroadhesiveness factors. Urinary tract infections - interaction of urinary tract mucosa with adhesins (type 1 fimbriae, P-fimbriae, X-adhesins); possibly hemolysin, O polysaccharides; immature host immunity; shock associated with endotoxin. Neonatal meningitis-acidic polysaccharide capsule K1.

Laboratory Diagnosis: Isolation of E. coli from the appropriate clinical samples. Kits are available commercially. These are based on the biochemical tests that will be presented in this lab section.

Adapted from Laboratory Diagnosis of Infectious Diseases, ed. A. Balows

A SIMPLIFIED BRANCHING FLOW DIAGRAM FOR GRAM-NEGATIVE RODS

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ISOLATION OF ENTEROBACTERIACEAE

There are numerous plating media in use today, some selective, and others inhibitory. Most laboratories prefer to employ one selective medium such as SS agar, and one inhibitory medium, such as EMB or MacConkey agar for fecal specimens. For specimens other than feces or rectal swabs, a combination of MacConkey or EMB agar together with a BAP plate is usually sufficient.

Characteristics and Tests Used for the Species Identification of Enterics:

Identification of the Enterobacteriaceae is based on colony morphology on primary isolation media and on biochemical reactions. The following set of tests is a subset of those tests widely used in clinical laboratories:

• Utilization of carbohydrates (fermentation)
• The IMViC reactions:
  I = production of Indole from tryptophan
  M = Methyl Red Reaction
  Vi = Voges-Proskauer test (Production of acetyl-methyl carbinol)
  C = Utilization of Citrate
  
• Motility
• Production of Urease
• Production of H₂S gas

Carbohydrate fermentation pattern for an isolate can be determined using any of several commercially-available minaturized test configurations (Enterotube, API test strip). In the clinical lab these tests are usually performed using a Vitek automated analyzer and liquid culture isolates.

Automated Analysis System for Enteric Microorganisms

Hospital microbiology laboratories typically employ fully automated computerized methods for the identification and classification of enteric bacterial species. One example of such a system is the Automicrobic System (bioMerieux Vitek, Inc.) The system uses disposable polystyrene cards that include 30 reaction micro-wells. Each microwell contains biochemical substrates or dehydrated antibiotics. A filling module automatically fills each microwell with the test bacterium and seals the card. An incubation and reading module optically scans the cards on a 1-hour rotational basis as they are incubated. A computer module records the biochemical reactions and performs identification analyses. The final identification report can be available with as little as 6 hours of incubation. The total time required to culture and identify a suspect enteric pathogen, however, includes the initial primary culture for isolation, generally on MacConkey agar.

I) Primary isolation medium (Inhibitory).

Important Purpose and Differential Medium Ingredients

MacConkey agar

Uninoculated MAC Plate

MAC Agar Formula Bile salt (1.5g/l) & Crystal violet Purpose: Recover the Enterobacteriaceae & related Lactose-fermenting Gram-negative enteric bacilli. Inhibitor: Bile salt & Crystal Violet. Inhibit the growth of Gram-positive bacteria and some fastidious Gram-negative bacteria. Carbohydrate Source: Lactose Neutral Red, pH indicator

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Eosin-Methylene-Blue agar (EMB)
Eosin Y and Methylene-blue

Purpose: Isolation & detection of the Enterobacteriaceae or Lactose-related coliform bacilli from specimens with mixed bacteria.
Inhibitor: Eosin & methylene blue. Inhibits the growth of Gram-positive bacteria and some fastidious G- organisms
Carbohydrate Source: Lactose
pH Indicator: Eosin & methylene blue combine to form a precipitate at acidic pH.