Biochemical Study of E. Coli PDF

MICROBIOLOGY AND PARASITOLOGY BIOCHEMICAL STUDY OF E.COLI Dr. Olive Grace G. Tolentino, M.D. | Oct. 23, 2024 OUTLINE Sepsis I. Enterobacteria...

MICROBIOLOGY AND PARASITOLOGY BIOCHEMICAL STUDY OF E.COLI Dr. Olive Grace G. Tolentino, M.D. | Oct. 23, 2024 OUTLINE Sepsis I. Enterobacteriaceae group Blood: entry of E.coli will cause sepsis especially in A. Escherichia coli immunocompromised individuals like neonates II. Diseases caused by E.coli → often the major clone associated is E. coli O25b/ST131 III. E.coli associated diarrheal diseases IV. Culture medium: Meningitis MacConkey agar Meninges: caused by strains of E.coli having K1 antigen V. Biochemical testing (K) = capsular antigen I. Enterobacteriaceae group Most common group of Gram negative rods cultured in clinical laboratories III. E. coli associated diarrheal diseases Can reduce nitrate to nitrite Natural habitat: intestinal tract of humans and animals Enteropathogenic E. coli Severe water diarrhea in Motile/nonmotile: either motile (E.coli) with peritrichous (EPEC) infants flagella, or nonmotile (Shigella and Klebsiella) Growth in medium: grow well on Mac-Conkey agar Mechanism: Due to adherence Facultative anaerobes: ferment rather than oxidize of EPEC to the mucosa and glucose, often with gas production flattening the villi Catalase: positive Cytochrome oxidase: negative Complex antigenic structure Enterotoxigenic E. coli Travellers diarrhea Produce variety of toxins and virulence factors that (ETEC) can successfuly cause many diseases Mechanism: Due due ETEC Differentiated to species level by Biochemical tests producing two toxins: Examples: Heat labile toxin (LT) Escherichia, Shigella, Salmonella, Enterobacter, Proteus, Heat-stable toxin (ST) Yersinia etc. Enteroinvasive E. coli diseases similar to Shigellosis A. Escherichia coli (EIEC) Mechanism: Due to invasion of the lining Enteroaggregative E. coli Acute and Chronic diarrhea (EAEC) -persistent diarrhea in patients with HIV Chronic: more than 14 days Mechanism: Unknown Shiga toxin-producing E. coli Mild non-bloody diarrhea, (STEC) hemorrhagic colitis, hemolytic uremic syndrome Mechanism: Due to production Morphology: Gram negative bacilli of toxins: Motile due to presence of Peritrichous flagella Complex antigenic structure Shiga-like toxin 1 → Capsule (K) antigen Shiga-like toxin 2 → LPS (O) or Somatic (O) antigen → Fimbriae (F) antigen O157:H7 (most common strain) Produce toxins Diseases caused by E.coli (NOTE: “Maask ni siguro sa mga lecture exams” -DOC T) Manifestation of E.coli will depend on the site A. Urinary Tract Infection (UTI) Urinary tract: E.coli is the most common cause of UTI in 90% of women → Caused by certain O serotypes → strains have pili that help them adhere to specific receptors on the urinary tract epithelium Transcribed by: NMD 2027  Biochemical study of E.coli Lactose Fermenting Strains IV. Culture Medium: MacConkey agar → Pink → Production of acid from lactose: Neutral red ▪ = color change of the dye when the pH is below 6.8 Lactose Non-Fermenting Strains → Colorless and transparent MAC: Growth pattern of some Enterobacteriaceae Selective and Differential → Enterobacteriaceae group grow well in MacConkey agar because this medium is selective for gram negative organisms and most gram negative organisms’ morphology are rods (bacilli) Bile salts and Crystal violet → inhibit the growth of Gram positive organisms Lactose fermenter, Lactose fermenter, Non-Lactose fermenter Lactose → provides a source of fermentable carbohydrate, allowing for differentiation LACTOSE FERMENTATION → lactose will be added into the medium because it will provide a source of fermentable carbohydrates which Lactose fermenters some organisms can ferment and cannot ferment → B-galactoside permease - transport protein in the wall that will facilitate the fast entry of the lactose into the pH indicator: Neutral red cell. → turns pink at pH 6.8 Late or Delayed lactose fermenters → B-galactosidase only How to detect fermentation in Mac? “If the transport of lactose does not have the permease enzyme they can only absorb slowly. So that's why they are “carbohydrate fermentation in most of your bacteria, they slow or delayed lactose fermenter or late lactose fermenter” will produce acid and gas, your CO2 and hydrogen. So - DOC T carbohydrate fermentation releases acid, for example lactose fermentation - lactic acid, which will decrease the Non-lactose fermenters pH of the medium and the pH change will be detected by → Lack both enzymes the indicator dye added to the medium” -DOC T Differentiation between Lactose fermenters (LF) and Non lactose fermenters (NLF) by Growth on MacConkey agar MacConkey agar is selective and differential medium for Enterobacteriaceae How to differentiate lactose fermenters, Delayed and See image at the last page Non? Based on the image above MAC: Result interpretation Lactose fermenters (right side) → Pink colony with halo - due to more acid produced Late or Delayed lactose fermenters (middle) → Pink colony only - due to less acid produced Non-lactose fermenters (left) → Colorless - lack enzymes See image at the last page for additonal info 2 of 13  Biochemical study of E.coli BIOCHEMICAL TESTING Results Read both slant and butt. Observe the following Used for further differentiation of Enteric bacilli color changes: After growth in MacConkey agar, it will be transferred to Red color: Alkaline (K) biochemical tubes for inoculation Yellow color: Acidic(A) ○ If: Yellow butt: only glucose is fermented Yellow slant: glucose, lactose and/or sucrose are fermented Reading Results Note: Read both slant and butt. Observe the following color changes: - Yellow color: Acidic (A) - Red color: Alkaline (K) Triple Sugar Iron agar, Lysine Iron Agar, Citrate and Urease - solid tubes ○ Example 1: → Due to large percentage of agar alkaline slant / acid butt = red/yellow= K/A Sulfide Indole Motility - semi solid Only butt is yellow: glucose → Test for motility fermented → bacteria needs to be able to move in the medium Only glucose is fermented Slant ○ Example 2: acid slant/ acid butt = → Exposed to air yellow/yellow= A/A → Aerobic reactions Both slant and butt are yellow: → Inoculate by Simple streaking glucose, lactose and/or sucrose are fermented Butt All sugars are fermented → Anaerobic reactions → Inoculate by Stab Triple Sugar Iron Test Is this result possible? Composition ○ Protein sources: peptone, beef and yeast extract ○ Sugars: 1% lactose, 1% sucrose, 0.1 % glucose Indicator ○ Phenol red: pH indicator Alkaline: Red Acidic: Yellow ○ Ferrous sulfate/ Ferrous ammonium A/K? sulfate: hydrogen sulfide (H2S) production Acid/Alkaline Lactose fermentation only? indicator ANSWER: NO, If a microbe can ferment lactose it must be ○ Sodium thiosulfate (sulfate source) able to ferment glucose. H2S production: BLACK precipitate “Lactose is disaccharide, which means that sugars making up lactose is glucose and galactose. If bacteria will To inoculate: Stab, Streak breakdown lactose, it must automatically be able to break Test for carbohydrate fermentation: down your glucose to use it as carbohydrate” - DOC T ○ Carbohydrate fermentation causes acid production lowers pH, turns phenol red to yellow 3 of 13  Biochemical study of E.coli If only glucose is fermented: ○ Slant and butt initially turn yellow from the small amount of acid produced ○ After 12 hours: glucose is consumed, bacteria on the slant use up the peptones aerobically: alkaline reaction in slant: red color “If the peptone breaks down, it releases an alkaline product producing an alkaline slant, turning it into red” - DOC T ○ Fermentation of glucose (anaerobic) in Test for gas formation the butt: produces larger amounts of acid, overcomes the alkaline effects of peptone Bubble formation degradation: butt remains acidic (yellow) cracking or splitting of the agar upward displacement of the agar “Butt is still yellow because glucose fermentation is mainly pulling away of the medium from the walls of test tube anaerobic and it happens more in the butt and in the butt we have more concentration of acid. So the acidity here can still neutralize the alkaline of the peptone. So remaining in the butt is the yellowing of the glucose fermentation” - DOC T Test for H2S production Blackening of the butt indicates H2S positive If glucose + lactose or sucrose are fermented: H2S is produced only in acid enviroment ○ So much acid is produced that the slant and butt remain yellow (acid) Sample TSI tubes Sample Results Let’s combine the three tests (CHO, gas and H2S) and practice reading! 4 of 13  Biochemical study of E.coli More TSI tubes: Read the following TSI results: Last batch of TSI tubes: “Ang mag labas sa evals E.coli lang, memorize Let’s read the following: E.coli only” - DOC T Lysine Iron Agar Test It has lysine in the tube which the bacterial enzymes lysine deaminase or lysine decarboxylase will act about Composition: ➔ Peptones and yeast extract, glucose, lysine Indicators: ○ Bromcresol purple: pH indicator REMEMBER! Below pH 5.2 (Acidic): Yellow Above pH 6.8 (Alkaline): Puple Timing is critical in reading TSI test results: ○ Sodium thiosulfate/ Ferric ammonium early reading could reveal yellow throughout the citrate: H2S production indicator medium H2S production: BLACK late reading could reveal red slant, yellow butt throughout To inoculate: Stab, Stab, Streak very important that reactions be read within an 18- to 24-hour incubation period Test for decarboxylation and deamination Detects bacterial ability to decarboxylate or deaminate lysine and to reduce sulfur ○ Lysine deamination: Aerobic process, occurs at the slant. Positive reaction: Red color change. -Presence of color red in slant automatically deaminase ○ Lysine decarboxylation: Anaerobic process, occurs at the butt Positive reaction: Purple color. What is the result of E.coli on TSI? -Presence of color purple in butt automatically decarboxylate 5 of 13  Biochemical study of E.coli When inoculated with a lysine deaminase-positive organism Oxidative deamination will produce a compound that react with ferric ammonium citrate and a coenzyme, flavin mononucleotide, forming a red color on the slant Positive results When inoculated with a lysine decarboxylase-positive organism Initial yellowing of butt (glucose fermentation) Cadaverine neutralizes acids produced by glucose fermentation, Medium reverts to alkaline = purple color on the butt Sample LIA tubes Let’s practice reading: How to read deamination, How to read decarboxylation In the 4th tube (labeled as “4,” when you see the black part it is opposite with the TSI. If in TSI the H2S only occurs in 6 of 13  Biochemical study of E.coli acidic environment in LIA the H2S is formed in alkaline environment Therefore, in the 4th tube (+)H2s (so it is alkaline, and is color purple) so (+) decarboxylase. What is the result of E. Coli on LIA Doc did not explain this. What happens Citrate permease brings citrate into the bacterial cell: Other organism: Ex. Shigella on LIA Bromthymol blue in alkaline pH changes to blue. What about other organisms? Results Note: LIA is really confusing so better practice! The important thing here is to memorize the characteristics of E. Coli. CITRATE UTILIZATION TEST Composition: Simmon’s Citrate Agar ○ Carbon source: Sodium Citrate ○ Determines if bacteria can use citrate as a sole carbon source. Indicators: Bromthymol blue (pH indicator) ○ Neutral pH: Green ○ Alkaline pH: Blue To Inoculate: Streak 7 of 13  Biochemical study of E.coli What is the result of E. Coli on citrate? Results of various enteric bacteria What is the result of E. Coli on urease? Results of various enteric bacteria UREASE TEST SULFIDE INDOLE MOTILITY TEST Composition: Christensen’s Urea Agar (solid) / Stuart’s Tests three different parameters, which are represented Urea Broth (liquid) by the three letters in the name: Urea, peptone, glucose ○ Indicators: Phenol red: pH indicator ○ Alkaline (>pH 8.4): Pink/magenta ○ Acidic(