BIOL 2010 Lecture 9: Identification Methods for Enterobacteriaceae

BIOL 2010 Lecture 9 – Overview of identification methods for Enterobacteriaceae BAILEY AND SCOT T’S – CHAPTER 12, 19 DIAGNOSTIC MICROBIOLOGY – CHAPTER 9, 19 In this lecture Introduction to Enterobacteriaceae Common ID methods for Enterobacteriaceae Special media Tests Test systems Enter...

BIOL 2010 Lecture 9 – Overview of identification methods for Enterobacteriaceae BAILEY AND SCOT T’S – CHAPTER 12, 19 DIAGNOSTIC MICROBIOLOGY – CHAPTER 9, 19 In this lecture Introduction to Enterobacteriaceae Common ID methods for Enterobacteriaceae Special media Tests Test systems Enterobacteriaceae The family Enterobacteriaceae (also called enterics) includes many genera and species. Clinical isolates in acute-care settings consist PRIMARILY of three organisms: Escherichia coli Klebsiella pneumonia Proteus mirabilis Bacterial Species and the Infections They Commonly Produce Clinical Significance Two major types Opportunistic pathogens (This lecture) Normal flora Infections in other “nonnormal” sites Ex. E. coli is normal in the gut, but can cause, septicemia, wounds, urinary tract infections (UTIs), meningitis Primary pathogens (i.e. ALWAYS pathogen) Salmonella spp. Shigella spp. Yersinia spp.  Clinical Significance – another way to look at it Opportunistic Pathogens Pathogens Escherichia Salmonella Klebsiella Shigella Enterobacter Yersinia Serratia Pathogenic E. coli Citrobacter Proteus Morganella Providencia General Characteristics of ALL Enterobacteriaceae All ferment glucose All reduce nitrate to nitrites All are oxidase negative Motility All motile at body temperature except Klebsiella Shigella Yersinia Key Characteristics of the Family Enterobacteriaceae COPYRIGHT © 2015 BY Copyright SAUNDERS, © 2019 ANInc. by Elsevier IMPRINT OFreserved. All rights ELSEVIER INC. Enterobacteriaceae – ID Cellular morphology: Gram-negative coccobacilli or straight rods Not very useful in identification other than ruling out other organisms Growth conditions: Facultatively anaerobic Molecular methods: modern methods Culture** Macroscopic morphology on BAP Not very useful in identification other than ruling out other organisms Large moist, gray colonies - some can be mucoid Selective plates are utilized to ID pathogenic species in stool specimens. MacConkey Agar Enterobacteriaceae grow on MAC and serve as first point for ID Review of MacConkey (MAC) agar Selective and differential Bile salts and crystal violet inhibit gram-positive Lactose fermentation is differential. Colonies can be LF or Lactose fermenters (Pink) - A NLF or Non-Lactose Fermenters (Yellow) – B LLF or Late Lactose Fermenters – these are NLF at 24h, but become LF at 48h  Lactose Non-Lactose Fermenters Fermenters 1.Escherichia 1.Shigella ** 2.Klebsiella 2.Citrobacter 3.Enterobacter (LLF) 4.Serratia 3.Salmonella (LLF) ** 4.Proteus 5.Providencia 6.Morganella 7.Yersinia ** EMB Agar Eosin-methylene blue (EMB) agar Selective and differential Methylene blue inhibits gram-positive. Lactose and sucrose fermentation is differential. Hektoen Enteric Agar Media designed for Salmonella/Shigella Hektoen enteric (HE) agar A Selective and differential B Bile salts inhibit gram-positive, some gram-negative. Lactose and sucrose fermentation is differential. Colonial morphology Orange color (lactose fermentation, low pH). = nonpathogens (A) Blue green color = target pathogen Salmonella will produce H2S and have a black center C (B) Shigella will NOT produce H2S and appear blue green (C) Xylose Lysine Deoxycholate Agar Another media designed for Salmonella/Shigella Xylose lysine deoxycholate (XLD) agar Selective (less so than MAC and HE) and differential Sodium deoxycholate Inhibits gram-positive, some gram-negative Three carbohydrates, sucrose and lactose in excess, and xylose with a phenol red indicator Lysine present to detect lysine decarboxylation Thiosulfate present to detect hydrogen sulfide (H2S) Xylose Lysine Deoxycholate Agar Colonial morphology Yellow colonies = nonpathogens (A) Fermenters or those not producing lysine decarboxylase Escherichia coli, Citrobacter A B Red colonies with black centers = target pathogen (B) Initially yellow then revert to red When lysine is decarboxylated, causing alkaline pH Salmonella C Colorless or red colonies = target pathogen (C) Shigella Salmonella Shigella Agar SS agar designed to isolate and differentiate Salmonella and Shigella Selective ingredients Bile salts, sodium citrate, and brilliant green Inhibit gram-positive organisms and some lactose-fermenting, gram-negative rods normally found in the stool. Differential ingredients Lactose and neutral red Sodium thiosulfate, ferric ammonium citrate Salmonella Shigella Agar Colonial morphology Lactose fermenters (pink) = non- A B pathogens (A) Non-Lactose Fermenters (Colorless) = pathogens Colorless with black center = Salmonella (B) Colorless = Shigella (C) (Note: Not all Shigella grow on SS) C CIN media Cefsulodin, irgasan, novobiocin media Designed to isolate Yersinia spp. Inhibitory agents: incorporates cefsulodin, irgasan, novobiocin, bile salts, and crystal violet MAC selective agents plus antimicrobials Inhibits normal intestinal microbiota Differential ingredient: mannitol with phenol red Many formulations of agar available CIN media Presentations: Mannitol fermenters Decrease in pH around colony due to mannitol fermentation = causes phenol red to turn red Bile precipitates in the middle of the colony Bullseye colonies (image) Mannitol non-fermenters are translucent, colorless Selenite Broth Selective inhibition of gram-positive and many gram-negative organisms. Selective enrichment for the growth of Salmonella spp. Some Shigella also grow Useful if Salmonella is in small numbers in stool Procedure generally involves incubating sample in Sel broth for 16-20 hours, then subculturing on another selective/differential media Other media for Salmonella Brilliant Green Agar Selective ingredients: Brilliant Green, which is inhibitory to most gram-positive and gram-negative bacteria Salmonella grow as white to pink or red colonies surrounded by a bright halo (Top image) Bismuth Sulfite Agar Selective ingredients: Bismuth and Brilliant Green Salmonella grow as black colonies (Bottom image) May inhibit some Salmonella strains though Enterobacteriaceae - Biochemical tests Oxidase (Note all should be neg) Urea TSI (Screening test) Deaminase ONPG Indole Decarboxylase (Adenine, Lysine, VP Ornithine) Gelatin Citrate Sugar fermentation H2S *We will learn about these using API Lysine Iron Agar (LIA) and TSI Screening Identification Determine if Enterobacteriaceae Gram-negative Oxidase negative Except for Plesiomonas shigelloides Always use young colonies from sheep blood agar (SBA) plates Ferment glucose Reduce nitrate to nitrite Serologic Grouping Serologic grouping Salmonella 60 types of O antigens 95% are serogroups A through E1 Direct or latex agglutination tests for serogroup Shigella A through D serogroups Historical Methodologies HISTORICAL METHODOLOGIES CURRENT METHODOLOGIES Biochemical testing Newer technology available: matrix- Based on phenotypic characteristics assisted laser desorption–ionization- time-of-flight mass spectrometry o Miniaturized multitest systems (MALDI-TOF MS) Serotyping (a.k.a., serogrouping) Based on characterization of microbial proteins Use of antibodies to detect specific antigens located on the bacterial Molecular biology surface Based on genotype, nucleic acid sequences o Highly sensitive, specific, and rapid, providing accurate results in a few hours or less 26 Traditional biochemical tests Carbohydrate Amino Acid Miscellaneous utilization Utilization tests ONPG Decarboxylase Citrate Lactose utilization Dihydrolase DNAse OF Lysine Iron Agar Indole Glucose Deaminase MIO fermentation Nitrate TSI Oxidase MRVP Urease Oxidase Test - review Purpose Determines the presence of cytochrome oxidase activity for oxidase-negative enteric bacteria from other gram-negative rods Gram negative identification should start here Principle Determines the presence of cytochrome oxidase using tetramethyl-p-phenylenediamine dihydrochloride to indophenol (bluish-purple) Limitation Nickel-base alloy wires that contain chromium and iron may cause a false-positive result Oxidase Test - review Results Positive: Purple within 60s (A) Negative: NO purple – colony may stay colorless or pink (B) Quality control Positive—P. aeruginosa Negative—E. coli Lactose Utilization The most important carbohydrate determination Can be used to differentiate lactose fermenting (LF) bacterial species from nonlactose fermenters (NLFs) Lactose is a disaccharide bond consisting of glucose and galactose connected by a galactoside bond. An enzyme degrades lactose into glucose and galactose – hence glucose becomes available for metabolism. What is a common way to test this? (Hint: Media) 30 Lactose Degradation Requires two enzymes β-galactoside permease Transports lactose through the cell wall β-galactosidase Breaks lactose to yield subunits Some bacterial species lack permease but have β-galactosidase. Known as late or delayed LFs 31 ONPG Test Purpose of test To determine if an organism found to be a dLF (one that lacks the enzyme β- galactoside permease but possesses β-galactosidase) is a true NLF Principle Substrate is Ortho-Nitrophenyl-β-D-Galactopyranoside ONPG is structurally similar to lactose, but ONPG is more readily transported through the bacterial plasma membrane. Therefore NO permease enzyme required. Simply tests for B-galactosidase enzyme β-galactosidase hydrolyzes ONPG into Galactose and O-nitrophenol (Yellow color) 32  ONPG Test Steps to perform ONPG Test Make a heavy suspension of bacteria in sterile saline. Add ONPG disk/tablet. Incubate at 35° C. Positive results usually seen within 6 hours Results Positive: Yellow Negative: Clear Limitations: Yellow pigment producing bacteria should not be tested because of potential false-positive results. 33 Two Pathways of Glucose Metabolism 34 Glucose fermentation tests Oxygen is not required Glycolysis pathway Glucose to pyruvate Oxidized to other acid products and gases Acid is detected by pH indicators. Remember CTA sugars? 35 Oxidation–Fermentation (O/F) Tests Bacterial sugar utilization patterns Oxidation Utilize carbohydrates aerobically Produce weak acids Ex. OF test with high sugar content Fermentation Utilize carbohydrates anaerobically Strong acid producer Ex. CTA sugar with lower sugar content Asaccharolytic Do not utilize carbohydrates 36 O/F Basal Media (OFBM) Lower concentration of peptones Help classify as oxidizer or fermenter Detects small amounts of acids Contents pH indicator is bromothymol blue. Uninoculated is green. Acid pH is yellow. Alkaline pH is blue. 37 O/F Basal Media (OFBM) Uses two tubes Tube #1 is Aerobic Aerobic: only open tube produces acid (oxidizer) Tube #2 is Anaerobic (overlaid with sterile mineral oil) Anaerobic: only closed tube produces acid (fermenter) Facultative: both tubes produce acid (both) 38 O/F Basal Media (OFBM) Organism 1 Both are yellow = sugar is being used with and without oxygen Organism is a fermenter Organism 2 Only the tube exposed to air is yellow = sugar is only being used with oxygen Organism is an oxidizer Organism 3 NO tube is yellow – sugar is not utilized, and organism is asaccharolytic 39 Triple Sugar Iron Agar (TSI) Media and Principle TRIPLE sugars: sucrose, glucose, and lactose Lactose and sucrose present in 10:1 ratio to glucose. Kligler iron agar (KIA) is similar but no sucrose. Ferrous sulfate and sodium thiosulfate Detect production of hydrogen sulfide (H2S) as a black color Phenol red Indicator Below 6.8 yellow (acidic) Above 6.8 red (alkaline) Reaction chambers Slant is aerobic. Butt is anaerobic. 40 Triple Sugar Iron Agar (TSI) Purpose Determines whether a gram-negative rod ferments glucose and lactose or sucrose and forms hydrogen sulfide Principle Triple sugar iron (TSI) agar contains 10 parts lactose; 10 parts sucrose;1 part glucose and peptone; and ferrous sulfate Butt of tube turns yellow if glucose is fermented (acid) Slant of tube turns yellow if lactose or sucrose are fermented (acid) Tubes turns black if hydrogen sulfide is produced Gas formation results in bubbles or breaking of the agar 41 TSI - results Lactose or sucrose (or both) fermentation A/A after using glucose then use lactose or sucrose Both stay yellow Hydrogen sulfide Production Black precipitate – can mask acid reaction Acid from bacteria + sodium thiosulfate H2S gas + ferric ions ferrous sulfide (black ppt) Gas production Bubbles or splitting of media is due to gas generated. 42 TSI - reactions Examples A: A/A, Gas B: K/A, Gas, H2S C: K/K D: K/K 43 TSI - reactions 44 Examples of TSI Reactions 45 Glucose Metabolism Occurs via the Embden–Meyerhof pathway Produces several intermediate by-products, including pyruvic acid Further degradation of pyruvic acid can produce mixed acids as end products. Enterics take two separate pathways Mixed acid fermentation Butylene glycol pathway The methyl red (MR) and Voges–Proskauer (VP) test detect end products of glucose fermentation. One broth including glucose inoculated, then separated into two tubes One tube = MR test Second tube = VP test 46 MR test Principle Detects mixed acids pathway Test read directly after incubation Results Negative: MR-negative cultures remain yellow after addition of the pH indicator (pH 6.0). Positive: Glucose pyruvic acid mixed acid ferm pH 4.4 red color with MR (positive test) VP test Principle Tests butylene glycol pathway products (acetoin and glycol) MUST add reagents first, then gently shake tube to increase oxygenation. α-naphthol which serves as a catalyst/color intensifier. Add 40% potassium hydroxide (KOH) or sodium hydroxide (NaOH). Results Positive: Produces butylene glycol and acetoin, which is further converted to diacetyl Diacetyl + KOH + -naphthol red complex Negative: NO Diacetyl to react with reagents, colorless Decarboxylase and Dihydrolase Tests Purpose This test is used to differentiate decarboxylase-producing Enterobacterales from other gram-negative rods Decarboxylase principle Test the presence of enzymes capable of removing carboxyl group (COOH) Specific for amino acids  Lysine, ornithine, arginine Lysine lysine decarboxylase cadaverine + CO2 Ornithine Ornithine decarboxylase putrescine + urea Dihydrolase principle Arginine arginine dihydrolase citrulline ornithine putrescine 49 Decarboxylase and Dihydrolase Tests (Moeller’s) Broth medium used to detect decarboxylation Contains glucose, peptones pH indicators Bromocresol purple Cresol red Specific amino acid at a concentration of 1% Overlay with oil Initial pH of 6.0 Un-incoulated media is yellow 50 Decarboxylase and Dihydrolase Tests Results Positive: If amino acid is broken down, amines cause an alkaline pH shift, causing a purple color Negative: If color remains yellow. Note: Two conditions must be met for decarboxylation to occur. An acid pH An anaerobic environment (hence the oil overlay) A control tube is usually inoculated 51 Phenylalanine Deaminase Test Purpose: This test is used to determine the ability of an organism to oxidatively deaminate phenylalanine to phenylpyruvic acid. The genera Morganella, Proteus, and Providencia are positive. Principle Microorganisms that produce phenylalanine deaminase remove the amine (NH2) from phenylalanine. The reaction results in the production of ammonia (NH3) and phenylpyruvic acid. The phenylpyruvic acid is detected by adding a few drops of 10% ferric chloride; a green-colored complex is formed between these two compounds. Results: MUST add 10% ferric chloride Positive: Green Negative: colorless 52 Tryptophan Deaminase Test Purpose: This test is used to determine the ability of an organism to oxidatively deaminate Tryptophan Most strains of E. coli, P. vulgaris, P. rettgeri, M. morgani and Providencia are positive Principle Microorganisms that produce tryptophanase remove the amine (NH2) from tryptophan to produce indole, pyruvic acid, ammonium. Results: Positive: Reddish brown Negative: LIGHT brown (can sometimes be confusing) 53  Citrate Utilization Purpose Citrate test determines whether an organism can use sodium citrate as a sole carbon source. Principle Use of citrate results in an alkaline pH. Media is green when uninoculated. It is inoculated with a light inoculum Too heavy = organisms will die and become source of carbon Incubate in aerobic conditions Limitations: Some organisms are capable of growth on citrate and do not produce a color change - Growth is considered a positive Results Positive: blue Negative: Green 54  Indole Purpose This test is used to identify organisms that produce the enzyme tryptophanase Principle Organisms that possess tryptophanase can deaminate tryptophan resulting in indole. Bacteria are inoculated into tryptophan or peptone broth and incubated for 48 hours before indole testing can be done. Result is based on reagent used Ehrlich’s reagent (more sensitive) = red is positive Kovac’s reagent = red is positive Spot indole (quick, done on filter paper with DMACA reagent) = blue is positive (Bottom image) 55 Motility-Indole-Ornithine (MIO) Agar MIO agar Semi-solid Used to detect Motility and Indole and Ornithine decarboxylase production Useful in differentiation Klebsiella spp. from Enterobacter and Serratia spp. Results Positive Motility is shown by a clouding or medium or spreading growth from the inoculation line. Positive Ornithine decarboxylation is indicated by a purple color throughout medium. Positive Indole is detected by adding Kovac’s reagent. Pink to red color in reagent area is positive test. 56 Nitrate and Nitrite Reduction Purpose Determines whether an organism can reduce nitrites Principle Microorganisms capable of reducing nitrite to nitrogen will not turn a color and will produce gas in the nitrate reduction (nitrate to nitrite) test Limitation Zinc dust is added if broth does not become red or no gas is observed Quality control Positive—Proteus mirabilis (ATCC 12453); colorless gas Negative—Acinetobacter baumannii (ATCC 19606); red, no gas 57  Urease (Christensen’s) Purpose This test is used to determine an organism’s ability to produce the enzyme urease, which hydrolyzes urea. Proteus spp. may be presumptively identified by the ability to rapidly hydrolyze urea. Principle Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, causing a pH shift. Limitations False positive can occur upon prolonged incubation Result Positive: BRIGHT pink (right) Negative: light yellow (left) Quick note about IMVIC Test Stands for Indole Methyl Red Voges Proskauer Citrate Quick panel of tests to screen for organisms, Examples: I MR VP C E. coli + + - - K. pneumoniae - - + +  Multi-test systems Microbial ID methods fall into one of five categories (or combination): Methods can be manual or automated System Pure Examples culture required 1. pH-based Yes Analytical Profile Index (API) – manual method 2. Enzyme profile No Microscan rapid panels 3. Carbon Yes Color change based on transfer of electrons to colorless utilization indicator Ex. Biolog 4. Visual Yes Measurement of turbdity ex. MicroScan detection 5. Molecular Yes MALDI-TOF – protein patterns assays – include No PCR – DNA materials characterization of molecules like fatty Yes Immunoassay Analytical Profile Index (API) Released in 1970 System for the identification of gram-negative fermentative bacteria (the family Enterobacteriaceae) is called API 20E. API 20E system highlights Consists of 20 cupules attached to plastic strip each with a specific lyophilized, pH- based substrate (miniaturized versions of biochemical tests we just discussed) A liquid bacterial suspension is used to rehydrate the cupules. 61 API 20E - example API 20E System Highlights Inoculation Prepare a bacterial suspension and inoculate all cupules Some cupules require mineral oil overlay. Principles of the tests are the same for similar tests performed in test tubes. Strip is incubated for 18 to 24 hours. Interpretation (AFTER incubation) Reagents are added to some cupules, as appropriate Results are recorded and a 7-digit code profile number is determined. The code profile number is checked against a database provided by manufacturer for identification. Results = organism ID, with a confidence score Additional testing may be needed. 63 Rapid identification systems Traditional methods to ID require growth and isolation of bacteria – this could take days, maybe weeks Why would this be an issue? Rapid identification systems (since the 1970s) aim to get ID quicker in comparison to traditional method The phrase “rapid method” is quite loose, and encompasses a wide variety of procedures and techniques For example, a 3-hour enzyme immunoassay method to detect Clostridioides difficile toxin is rapid compared with a 48-hour cell culture cytotoxicity assay. Rapid identificati on system - Examples Automated Identification Systems Examples MicroScan System TREK Diagnostic System Vitek 2 System BD Phoenix Automated Microbiology System Biolog OmniLog ID System Sherlock Microbial Identification System 66 Evaluation of Identification Systems Compare side by side to current methods of reference procedures. Test Accuracy Cost effectiveness Work flow Systems can provide decreased sensitivity or specificity for some bacteria. Usually supplemental and differential media and biochemicals will still need to be kept on hand. 67 Knowledge Check Which of the following tests detects the production of mixed acids from glucose because of subsequent metabolism of pyruvate? a. Methyl red test b. Voges-Proskauer test c. Citrate test d. Indole test Knowledge Check A gram-negative bacillus that produces an acid slant and acid butt on triple sugar iron agar can ferment which of the following carbohydrates? a. Glucose only b. Glucose and lactose or sucrose or both c. Lactose only d. Lactose and sucrose, but not glucose Knowledge Check Tryptophan broth is inoculated and incubated 24 hours. After incubation, Kovac’s reagent is added. A red color develops at the surface of the broth. Which product of metabolism was formed? a. Mixed acids b. H2S c. Phenylpyruvate d. Indole Knowledge Check Indole-positive bacteria: a. Produce tryptophanase b. Deaminate lysine c. Decarboxylate tryptophane d. Decarboxylate ornithine Knowledge Check What type of ID method is employed by the API system? a. Immunoassay b. Enzyme characterization c. Carbohydrate utilization and pH d. Molecular methods Knowledge Check What do black colonies on XLD agar indicate? a. Shigella b. E. coli c. Salmonella d. Non-pathogenic Enterobacteriaceae Knowledge Check What do orange colonies on HEK agar indicate? a. Shigella b. E. coli c. Salmonella d. Non-pathogenic Enterobacteriaceae Knowledge Check Most pathogenic Enterobacteriaceae are a. Lactose fermenters on MAC b. Non-Lactose Fermenters on MAC c. Alpha hemolytic on BAP d. Pink on XLD Knowledge Check Pink (lactose fermenting) colonies on SS agar could be: a. Salmonella b. Shigella. c. E. coli d. All of the above