INTRODUCTION TO ENTEROBACTERIACEAE

Enterobactaeriacaea morphology characteristics:

• gram-negative bacilli

• They have flagella for motility

• Most have fimbriae or pili for attachment to other bacteria or host cells

• They have a complex cell wall

Outer membrane:

• Lipopolysaccharide portion which is responsible for the endotoxin and porin protein and lipoprotein.

Peptidoglycan

• gram-negative bacilli peptidoglycan is thicker compared to that of gram-positive microorganisms.

Capsular antigen (K antigen)

• found in the capsule

Somatic antigen (O antigen)

•  found in the cell wall

Flagellar antigen (H antigen)

which is found in the flagella

• Determinants of Pathogenicity: WHY THERE IS FEVER FOR A GI TRACT BACTERIA?

If an organism is engulfed by a macrophage, this organism will release endotoxin and will stimulate production of Interleukin-1 and this goes into the blood stream and into the Hypothalamus of the brain stimulating the pituitary gland to produce prostaglandins which will cause fever and this is why fever is present if you have a gram-negative infection.

Endotoxin

Integral part of the cell, Heat labile, toxoid cannot be produced, produced by gram - negative organisms only.

Subunit B (Enterotoxin)

attaches to the plasma membrane of epithelial cells lining the small intestine.

Subunit A (Enterotoxin)

• induces the formation of Cyclic AMP from ATP in the cytoplasm

• As a result, epithelial cells discharges large amounts of fluids and electrolytes such as chlorides, potassium, sodium, and bicarbonates. Normal contractions are disturbed leading to severe diarrhe

Subunit B (Shiga toxin)

• binds to the cellular receptor (GB3 or Globotriaosylceramide) found on the endothelial cells. It allows entry of Subunit A and modifies RNA component of the ribosome to inactivate it (inhibits protein synthesis).

Verotoxin/Shiga-like toxin

• similar to Shiga toxin

• Colonization Factors

• Capsule

• Vi Antigen

• Fimbriae

• O Antigen

• Intracellular survival

Capsule

• Inhibits phagocytosis

Vi Antigen

•  found in Salmonella, protects the organism from complement-mediated killing

Fimbriae

• attachment

O Antigen

• adherence of the organism to the host cell

Intracellular survival

• characteristics that makes them survive intracellularly

Mode of transmission

• From the feces (from humans or animals) containing pathogenic microbes or their toxins, they can be transmitted through the foods, fluids (water and milk), or fingers. The organisms/toxins are ingested by humans, it goes into the gut or the intestinal tract of humans. The organisms multiply in the gastrointestinal tract and toxins may be produced but infection remains localized in the gastrointestinal tract. If they are in the gastrointestinal tract, they produce diarrhea and will be excreted out also through the feces.

• Some organisms invade/the toxins are absorbed so they become disseminated they go to other parts of the body and causes symptoms of systemic infection like fever but can also be excreted out through the feces.

• Sites of Infection

• Central Nervous System

• Lower Respiratory tract

• Bloodstream

• Gastrointestinal tract

• Urinary tract

Central Nervous System

• Escherichia

Lower Respiratory tract

Klebsiella, Enterobacter, Escherichi

Bloodstream

Escherichia, Klebsiella, Enterobacter

Gastrointestinal tract

Salmonella, Shigella, Escherichia, Yersinia

Urinary tract

Escherichia, Proteus, Klebsiella, Morganella

other specimens used for diagnosis:

• Cerebrospinal fluid

• sputum

• blood

• stool

• urine

General specimen:

Generally, stool specimens are used for the diagnosis of the gram-negative bacilli because they are usually found in the gastrointestinal tract. 

Stool specimen is processed within 1 hour and no preservative is added

• rotavirus, direct wet mount, ELISA, and assays

Specimen cannot be processed within 1 hour then a transport medium is needed

• Carry Blair, Glycerol M Phosphate (Shigella), Stuarts medium (Shigella)

Enrichment medium

• Tetrathionate broth

• Bile salts- Salmonella and Shigella; Brilliant green- Salmonella

• Selenite F broth

• Hajna GN broth

• If growth, inoculate the plating medium. In some specimens like CSF, it can be inoculated directly to the plating medium

• Slightly selective

• allows growth of all gram (-) organisms; Eosin Methylene Blue and McConkey

Moderately selective

• allows growth of Salmonella and Shigella only; Salmonella-Shigella Agar, Hektoen Enteric Agar, and XLD (Xylose Lysine Deoxycholate)

Highly selective

• growth of Salmonella typhi and other salmonella; BGA (Brilliant Green Agar), BSA (Bismuth Sulfide Agar)

• Eosin Methylene Blue Agar 

• Sugar: Lactose and Sucrose

• Indicator: Eosin Y and Methylene Blue

• Lactose Fermenters: Dark-colored colonies, Escherichia coli- Greenish-metallic sheen

• Non-Lactose Fermenters: Colorless colonies

This medium contains aniline dyes which is inhibits other bacteria other than gram-negative bacill

• MacConkey Agar

• Sugar: Lactose

• Indicator: Neutral Red

• Lactose Fermenters: Pink Color

• Non-Lactose Fermenters: Colorless

• It inhibits gram-positive organisms because it contains bile salts and Crystal Violet.

• Salmonella Shigella Agar

• Sugar: Lactose

• Indicator: Neutral Red

• Lactose Fermenters: Pink Color

• Non-Lactose Fermenters: Colorless

• High concentrations of bile salts and sodium citrate to only allow growth of Salmonella and Shigella.

Hektoen Enteric Agar

• Sugar: Lactose, Sucrose, and Salicin

• Indicator: Bromothymol Blue

• Lactose Fermenters: Bright Orange to Salmon colonies

• Non-Lactose Fermenters: Green-Colored colonies

• If you have an organism which produces H₂S, you will have Blue green colonies with black centers.

• Xylose Lysine Desoxycholate

• Sugar: Lactose, Sucrose, and Xylose

• Indicator: Phenol Red

• Lactose Fermenters: Yellow Colored colonies

• Non-Lactose Fermenters: Red Colored colonies or Pink

• Brilliant Green Agar

• Sugar: Lactose and Sucrose 

• Indicator: Phenol Red

• Lactose Fermenters: Yellow to Greenish Yellow colonies

• Non-Lactose Fermenters: Pink with an Intense Red background

• Bismuth Sulfite Agar (BSA)

• Sugar: Dextrose

• Indicator: Bismuth Sulphite

• If there is Salmonella Typhi, it produces jet-black colonies with metallic sheens.

• If there are other colonies Dull-Green, Brown or sometimes Colorless colonies 

• Once the specimen has been inoculate in the plated medium, usually it would produce a typical colonies in the plating medium.

• Confirm these with Biochemical Tests

Triple Sugar Ion

• Sugars: Lactose, Sucrose, and Fructose (Glucose or Dextrose 0.1%)

• Indicator: Phenol Red

• Acid Production: Yellow Color

• There is a Slant and a Butt 

• The lactose fermenter will utilize first Glucose, since only have 0.1%, this will be utilized immediately.

• The organism will also use Lactose

• As a result of the utilization of these sugars, it will have mixed acid production

• Since the indicator present is phenol red, when there is acid production, it will produce yellow color.

• Peptides can also be used to convert it to Amines, but this can only be achieved in the presence of Oxygen.

• Lactose Fermenter with an Acid slant and Acid butt

• If the organism produces Gas it would produce Cracks or Bubbles

• If you have Non-Lactose Fermenters the initial reaction would be to use Glucose. Since all of the Enterobacteriaceae utilizes Glucose.

• When glucose is consumed, lactose can no longer be used.

• As a result of glucose fermentation, it will also have acid production in the butt the organism will then utilize proteins or peptides, Peptide Degradation requires oxygen and will have only utilization of peptides in the slant, since this is an alkaline reaction, your slant would be alkaline (Red Color) since your indicator is phenol red.

• When all amines have been utilized, the reaction will go down, but it cannot go down deep into the butt.

• If gas is produced, bubbles and cracks would be noticed. 

• Non-Lactose Fermenter with Hydrogen Sulfide (H₂S)

• Blackening of medium due to the reaction of H₂S with Iron, producing Iron Sulfide or FeS producing a black color.

• Alkaline slant and Alkaline butt but contains H₂S.

• Does not use any of the sugar, only uses Proteins thus, have an Alkaline reaction.

• Different reactions

• Alkaline Slant and Acid Butt

• Acid Slant and Acid Butt

• Acid Slant and Acid Butt with H₂S (Fermenters with H₂S)

• Alkaline Slant and Acid Butt with H₂S (Fermenters with H₂S)

• Alkaline Slant and Alkaline Butt (Non-Fermenters)

• Alkaline Slant and Acid Butt (Non-Lactose Fermenters)

• Acid Slant and Acid Butt (Lactose Fermenters)

• Alkaline Slant and Acid Butt (H₂S)

Indole Test

• Tryptophan, the bacteria will ferment it to tryptophanase to Indole + Pyruvic acid + Ammonia 

• With the addition of Aldehyde Indicator (Kovac’s or Ehrich’s reagent), it will result into a purple ring

• Negative will be yellowish or greenish ring

• Methyl Red Test & Voges-Proskauer Test

• Taken together since they use the same medium

• Methyl Red Test

• Glucose is converted into pyruvic acid (mixed acid fermentation) with pH of less than 4.4. If methyl red is added, produces a red color (negative: yellow)

Voges-Proskauer Test

• Follows a different pathway of Glucose utilization.

• Glucose is broken down to acetoin. When KOH is added and exposed to air, it will be converted to diacetyl.

• When α-naphthol is added, it will produce a pink-red complex (negative: yellow)

• Citrate Utilization Test

• Determines whether the organism utilizes citrate as a source of Carbon

• Indicator: Bromothymol Blue

• Positive: Blue

• Negative: Green or retains its original color

• Indole, Methyl Red, Voges-Proskauer, and Citrate Utilization are generally called as IMViC tests

• Urease Test

• A rapid identification test for Proteus

• Converts urea to NH₃ and CO₂, NH₃ is alkaline 

• Indicator: Phenol Red

• Positive if Alkaline: Dark pink color

• Negative: Yellowish or retains its color.

• Nitrate Reduction Test

• Reduced Nitrate to Nitrite using the enzyme, Nitrate reductase. Adding sulfanilic acid will produce diazonium salt

• When α-naphthylamine is added to diazonium salt, it produces a red water-soluble azo dye

• Positive: Red Color (but will not stay long)

• Negative: Orange

• Sulfite Indole Motility Test

• Medium is Semi Solid and contains Iron so that if the organism produces H₂S, you have blackening of the medium

• Non-motile organisms usually grows only in the line of stabbing.

• If it is a motile organism, it causes haziness of the medium, which is conclusive that the growth is beyond the line of stabbing.

• Phenylalanine Deaminase Test

• If the organis has this certain enzyme, it is converted into phenylpyruvic acid

• Adding 10% FeCl₃ produces green color

• Identifies Proteus group

• Orthonitrophenyl-β-D-galactopyranoside (ONPG)

• Broken down by β-galactosidase into orthonitrophenol which is yellow in color.

• Determines whether if the organism is a Late or Slow Lactose Fermenter

• If the organism is slow or a late lactose fermenter, they have beta-galactosidase.

Positive: Yellow Color

• Carbohydrate Fermentation Test

• Some organism can be identified only from the other species by using carbohydrate

• Indicator: Phenol Red

• Positive: Yellow; Negative: Red

Rapid Diagnostic Tests

• For faster diagnosis (API)

• Enterotube test (same principle with API)

• If done in biochemical test, some organisms still can’t be differentiated with biochemical tests, so we need to use Serologic Tests  like in aSalmonell.

• With a Serologic Test, we can now differentiate different species.

Treatment

• Penicillin Derivatives, since gram negative bacilli are resistant to Penicillin

• Ampicillin

• Carbenicillin

• Mezclocillin

• Piperacillin

•  Ticarcillin

• Cephalosporins

• Aminoglycosides

• Amikacin

• Gentamicin

• Kanamycin

• Tobramycin

• Netilmicin

• Polymyxin

• Colistin

• Polymyxin B

• Quinolones

• Sulfonamides

• Tetracyclines

• Other antimicrobials

• Chloramphenicol

• Nalidixic Acid

• Nitrofurantoin

• Trimethoprim-sulfamethoxazole

• Imipenem