Enteric Gram-Negative Rods PDF

Enteric Gram-Negative Rods (Enterobacteriaceae and related organisms) Medical Microbiology Section 5  Characteristics of the family Enterobacteriaceae The Enterobacteriaceae are a large, heterogeneous group of gram-negative rods. Natural habitat is the in...

Enteric Gram-Negative Rods (Enterobacteriaceae and related organisms) Medical Microbiology Section 5  Characteristics of the family Enterobacteriaceae The Enterobacteriaceae are a large, heterogeneous group of gram-negative rods. Natural habitat is the intestinal tract of humans and animals. The family includes ;(Escherichia, Shigella, Salmonella, Klebsiella, and others). Some organisms, such as Escherichia coli, are part of the normal microbiota and incidentally cause disease, but others, the salmonellae and shigellae, are regularly pathogenic for humans. Although the members of Enterobacteriaceae are classified together, they cause a variety of diseases features common to all members of this heterogeneous family are 1- their anatomic location (colon) 2-and the following four metabolic processes : 1- they are all facultative anaerobic 2-they all ferment glucose (fermentation of other sugars varies) 3- don't have cytochrome oxidase (oxidase negative) 4-they reduce nitrates to nitrites as part of their energy-generting process These four reactions can be used to distinguish the Enterobacteriaceae from non-enterobacteraceae (pseudomonas aeruginosa)  MacConkey Agar MacConkey agar is a selective and differential media. Used for the isolation and differentiation of non-fastidious gram-negative rods. Used for the isolation and differentiation of the family Enterobacteriaceae and the genus Pseudomonas. Selectivity : Crystal violet dye and bile salts stop the growth of gram-positive bacteria. Only allows gram-negative species, which have a relatively bile-resistant outer membrane. Differentiation : Gram-negative bacteria that grow on MacConkey plate are differentiated by their ability to ferment lactose. Result Interpretation on MacConkey Agar A. Lactose fermenting strains: Grow as red or pink and may be surrounded by a zone of acid precipitated bile. The red color is due to production of acid from lactose. subsequent color change of the neutral red indicator when the pH of medium falls below 6.8 B. Lactose non-fermenting strains: Colorless and transparent and typically do not alter appearance of the medium. Eosin Methylene Blue (EMB) agar a selective and differential medium used for the isolation of gram-negative bacilli. Selectivity : contains two dyes, Eosin and methylene blue Slightly inhibits the growth of Gram-positive bacteria. Differentiation : Provides a color indicator distinguishing between organisms that ferment lactose and those that do not Principle of Eosin Methylene Blue (EMB) agar a. Lactose-fermenting gram-negative bacteria acidify the medium. 1. Under acidic conditions the dyes produce a deep purple complex usually associated with a green metallic sheen. Metallic green sheen is an indicator of the vigorous lactose and/or sucrose fermentation. 2. A smaller amount of acid production, which is the result of slow fermentation (by slow-fermenting lactose organisms) results in a brown-pink coloration of growth. b. Lactose non-fermenters Clear, colorless or amber colonies that are easily distinguished from coliforms. Notes: The distinctive metallic sheen produced on this medium is due to acid production resulting in an amide bond between eosin and methylene blue. Other coliforms not producing enough acid to cause this reaction. IMViC Test A series of four different biochemical tests it is mainly used for identifying Gram-negative bacteria.  It is the key to identifying and differentiating members of the Enterobacteriaceae family.  IMViC series contains the following biochemical tests: 1. “I” = Indole Test 2. “M” = Methyl Red (MR) Test 3. “V” = Voges – Proskauer (VP) Test 4. “C” = Citrate Utilization Test (simply Citrate Test) The letter “i” after ‘V’ is only for the rhyming purpose, it does not indicate any test. Purpose of IMViC The IMViC test is based on the variations in the metabolic requirements and properties of different genera and species of bacteria. The ‘indole test’ and ‘citrate utilization test’ in the series detect the ability of bacteria to produce specific enzymes and utilize specific nutrients. On the other hand, the ‘MR test’ and ‘VP test’ in the series detect the final metabolic products produced by the bacteria utilizing specific nutrients. What is Indole Test ? Detects the ability of organisms (bacteria) to produce indole as a metabolic product utilizing tryptophan in the medium Principle Some bacteria can produce an enzyme called ‘tryptophanase’ which helps them to metabolize the amino acid ‘tryptophan’ into ‘indole, pyruvic acid, and ammonia’. Procedure: 1- Incubate tryptone broth media with the tested organism for 24 – 48 hours. 2- an indole reagent is added following the incubation to read the result. The presence of indole can be detected through the use of Kovac’s reagent (para dimethy aminobenzaldehyde in amyl alcohol). Kovac's reagent, which is yellow, reacts with indole and produces a red color on the surface of the test tube Result of indole Test positive result: is indicated by the formation of red color at the surface of the test tube negative result: is indicated by no color change or a slight yellowish color ring at the top. What is Methyl Red (MR) Test? Detects the ability of bacteria to produce stable mixed acids as metabolic end products of glucose metabolism. Principle Some species of bacteria use the mixed acid fermentation pathway as their glucose metabolism process. Following this metabolic pathway, they convert pyruvate into stable mixed acids. When such acid fermenters bacteria are grown in a medium containing glucose they will release the acids; decreasing the pH of the medium to 4.4 or lower. methyl red indicator is added in a medium containing such acid fermenters, it will turn the medium red.” Result of Methyl Red (MR) Test positive result : is indicated by the development of red color negative result : is indicated by the development of yellowish color. What is Voges-Proskauer (VP) Test? Detects the ability of organisms (bacteria) to metabolize the pyruvate into a neutral intermediate product called ‘acetylmethylcarbinol’ or ‘acetoin’. principle Pyruvate can be metabolized into a neutral intermediate product called ‘acetyl methyl carbinol’, commonly called the ‘acetoin’ during the butanediol pathway of 2,3-butanediol production. Procedure Following the 48-hour aerobic incubation on broth , VP reagents I and II are added and the color change is observed within 30 minutes. The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B (potassium hydroxide). When these reagents are added to a broth in which acetoin is present; they produce a red color (a positive VP test). This color may take 20 to 30 minutes to develop. Result of VP Test VP Positive result : is indicated by the development of pink – red color at the top of the broth immediately or within 30 minutes but not more than 1 hour. VP Negative result :No change in color What is Citrate Utilization Test? Detects the ability of organisms (bacteria) to utilize citrate as the only source of carbon. Principle : Such bacteria produce citrase enzymes which will break the citrate into (organic acids ) Oxaloacetic acid will then be decarboxylated to produce pyruvate and CO2. Released CO2 will 1- combine with H2O and excess sodium from sodium citrate to produce alkaline ‘sodium carbonate’ that will increase the pH of the medium. CO2 + H2O + excess sodium from sodium citrate → Na2CO3 (alkaline) 2-trigger the metabolism of ammonium salts. Ammonium salt → Ammonium hydroxide (alkaline) The combined effect of ammonium hydroxide and sodium carbonate will increase the pH of the media above 7.6 which will turn the pH indicator bromothymol blue in the medium Result of Citrate Utilization Test Following the incubation of 24 – 48 hours (up to 4 days for some), bacterial growth and color change in the slant portion is observed. positive result : is indicated by growth and change in color of slant from green to intense blue. negative result : is indicated by no change in the color of the slant. (green due to the green color of bromothymol at neutral PH)  Gelatin Hydrolysis Test This test is used to determine the ability of an organism to produce extracellular proteolytic enzymes (gelatinases) that liquefy or hydrolyze gelatin, a component of vertebrate connective tissue. When an organism produces gelatinase, the enzyme liquefies the growth medium by hydrolyzing gelatin present in the medium. The gelatin hydrolysis is indicated by loss in ability to solidify even after refrigeration. Expected Results Positive: Partial or total liquefaction of the inoculated tube at 4°C within 14 days. On plates, gelatin hydrolysis is indicated by clear zones around gelatinase-positive colonies. Negative: Complete solidification of the tube at 4°C. On plates, no clear zones around colonies are observed. Urease production test: To test the ability of the organism to produce urease enzyme which split urea in urea media to form ammonia and CO2. Principle of Urease Test Urea medium, whether a broth or agar, contains urea (acidic ) and the phenol red as a pH indicator. Many organisms, especially those that cause urinary tract infections, produce the urease enzyme, which catalyzes the splitting of urea in the presence of water to release two molecules of ammonia and carbon dioxide. The accumulation of the ammonia will produce alkaline PH which turns the color of the indicator (phenol red) into pink. Expected Result positive: pink color Negative : yellow color ( the color of phenol red at acidic ph due to the presence of urea )