CLS463 Clinical Bacteriology II - Lecture 1 PDF

CLS463 Clinical Bacteriology II- Lecture 1 ENTEROBACTERIACEAE Done by: Dr.Ohoud Alhumaidan PhD. Molecular Microbiology  Enterobacteriaceae IT ES ❖Outline of our lecture : ❑What are th...

CLS463 Clinical Bacteriology II- Lecture 1 ENTEROBACTERIACEAE Done by: Dr.Ohoud Alhumaidan PhD. Molecular Microbiology  Enterobacteriaceae IT ES ❖Outline of our lecture : ❑What are they and where do we find them? ❑Are they medically important or not? ❑Characteristics shared by all Enterobacteriaceae ❑The classification schemes. ❑Different tests designed to identify Enterobacteriaceae Classification of bacteria on basis of Gram stain Gram positive Gram negative Rods Rods Cocci Cocci (bacilli) (bacilli) Enterobacteriaceae Pseudomonas spp. Vibrio spp.  Enterobacteriaceae ▪ Are a large family of Gram-negative bacteria(bacilli). ▪ They are classed into 31 genera and over 100 species. ▪ Epidemiology: notary Many members of this family are a normal part of the gut flora (Gut microbiota) ,found in the intestines ,of humans and other animals so sometimes referred to them as enterobacteria or "enteric bacteria ,while others are found in water or soil, or are parasites on a variety of different animals and plants.  Most common genera (genus) Escherichia Enterobacter Shigella Serratia Edwardsiella Proteus Salmonella Morganella Citrobacter Providencia Klebsiella Yersinia Coliform bacteria: Bacteria that are always present in the digestive tracts of animals, humans, and are found in their wastes. Coliform bacteria generally belong to four genera of the Enterobacteriaceae : Citrobacter , Enterobacter , E. coli, and Klebsiella pneumoniae. ▪ All coliforms are enteros but not all enteros are coliforms  Based on clinical infections produced, Enterobacteriaceae genera are divided into two categories: Webb II ▪ Opportunistic pathogens: (form part of the usual intestinal flora that may produce infection outside the intestine) e.g. Escherichia, Enterobacter and Citrobacter. 681 4 I ▪ Primary intestinal pathogens: (can cause disease in a host regardless of the host's resident microbiota or immune system) e.g. Salmonella, Shigella, and Yersinia  Enterobacteriaceae are associated with: Abscesses 269 Pneumonia 5114628 Meningitis 16WOWN Septicaemia MN infection of urinary tract (UTI), wound and intestine. awww 11  Characteristics shared by all Enterobacteriaceae Gram negative rods Facultative anaerobes I Non-spore forming All are Catalase positive All are Oxidase negative All can ferment glucose Noz No All reduce nitrate A to nitrite (nitrate reduction positive) SEE All grow on blood agar. All grow on Mac agar either as lactose fermenting or non lactose fermenting. Most motile except Klebsiella , Shigella and Y. pestis.  Antigenic structure 9 The Gram-negative outer membrane is coated in highly variable molecules or components that can cause immune activation, known as enterobacterial common antigen (ECA). The three major types of antigens: 1. O antigen: Outer membrane 2. H antigen : Flagella 3. K antigen : Capsule (Vi: Capsule of Salmonella) Classifications of Enterobacteriaceae by tribes The tribe concept proposed by Ewing. The Ewing classification :grouping the genera that share similar biochemical reactions in to tribes (8 tribes) a no  Classification of Enterobacteriaceae According to similar DNA composition Depend on the similarities between DNA structures and functional genes after genome sequencing. In Bergey's Manual of Systematic Bacteriology. Classification According to Lactose Fermentation Lactose fermenters: Non lactose fermenters: Escherichia Shigella Klebsiella Yersinia Enterobacter Proteus Citrobacter Salmonella Serratia Enterobacteriaceae Cultural Characteristics  Culture media 1-MacConkey Agar (Mac) Is a selective and differential culture medium designed to selectively isolate Gram negative and enteric bacilli and differentiate them based on lactose fermentation. Mac agar Contains : lactose, bile salts, neutral red (indicator) and crystal violet. The crystal violet and bile salts inhibit the growth of Gram positive organisms which allows for the selection and isolation of Gram negative bacteria. IF 1-MacConkey Agar (Mac) Enteric bacteria that have the ability to ferment lactose grow as pink and non-lactose fermenters grow as yellow. 0 0 LF NLF Myeloid input  2-Xylose Lysine Deoxycholate Agar (XLD) XLD selectively isolate Gram negative and used to so differentiate and isolate of Salmonella and Shigella species from other enteric bacteria After 24 hours at 37°C aerobically incubation Salmonella species: red or pink (since the background is red) colonies and some with black centers. Shigella species: red (since the background is red) colonies. Coliforms: yellow to orange colonies.  black pinkt Salmoneth other The figure shows three XLD agar plates. Bacteria were not cultivated on the plate in image (A). On the plate in image (B), Salmonella sp. has been cultivated. On plate ©, E. coli has been cultivated.  3-Hektoen Enteric Agar (HE) HE agar is a selective and differential media to isolate Salmonella and Shigella from clinical samples. 0 It is based on the use of bile salts for selective inhibition and two indicator systems (pH indicator system): Bromothymol blue and acid fuchsin as indicators of carbohydrate dissimilation (lactose, sucrose) Ferric iron as an indicator of the formation of hydrogen sulphide (H2S). is  After 24 hours at 37°C aerobically incubation Salmonella species: Blue-green to blue colonies, most strains being black in the centre or over their entire surface. Shigella species: Green colonies. Coliforms: yellow to salmon colour. a grant rather  Hektoen Enteric Agar Enteric organisms. e,g E.coli Salmonella Shigella  4-Eosin Methylene Blue Agar (EMB) EMB is the selective and differential medium for coliforms. The EMB agar is characterized by the presence of a combination of the two dyes eosin and methylene blue in the ratio of 6:1 to inhibits Gram positive organisms. After 24 hours at 37°C aerobically incubation: E.coli: metallic green sheen colonies. Other lactose fermenters : brown pink colonies (e.g., Klebsiella) Non lactose fermenters: appear as the color of the medium. (e.g., Salmonella, Shigella) 4-Eosin Methylene Blue Agar  5-Cystine Lactose Electrolyte Deficient Agar 00 CLED agar is differential (non selective) culture medium used for the isolation of bacteria from urine (urinary microbes). The medium supports the growth of all urinary potential pathogens and provides distinct colony morphology. It is electrolyte (salt) deficientwhy to prevents the swarming of Proteus species. Bromothymol blue is the indicator used in the agar, it changes to yellow in case of acid production during fermentation of lactose or changes to deep blue in case of alkalinization.  CLED agar Lactose fermenters produce colonies that appear as yellow, whereas colonies of non-lactose fermenters appear blue on CLED agar  Biochemical tests for Identification of Enterobacteriaceae Member of the Enterobacteriaceae family are identified based on their biochemical properties. Commonly used biochemical tests to identify them are: 1. Indole Test 2. Methyl Red (MR) Test 3. Voges–Proskauer (VP) Test 4. Citrate utilization Test 5. Triple Sugar Iron (TSI) Agar Test 6. Motility Test 7. Urease Test  Catalase Test Catalase is an enzyme, which is produced by microorganisms that live in oxygenated environments Anaerobes generally lack the catalase enzyme. Catalase mediates the breakdown of hydrogen peroxide H2O2 into oxygen and water. H2O2 + bacteria H2O + O2 Positive test: Air bubbles.  Oxidase Test The oxidase test is used to identify bacteria that produce cytochrome 0 D c oxidase enzyme. It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae. Oxidase reagent (tetramethyl-p phenylenediamine) If blue or purple color appears, then the microorganisms are oxidase positive. When the enzyme is not present, the reagent remains reduced and is colorless( oxidase negative)  Nitrate Reduction Test It is used to determine if an organism has the ability to produce a nitrate reductase enzyme capable of reducing nitrate (NO3–) to nitrite (NO2–) After 24h incubation : If the nitrate broth turns red after the addition of the nitrate reagents, it is considered a positive result for nitrate reduction. If the medium does not turn red after the addition of the reagents, it can mean that the organism was unable to reduce the nitrate  Urease Test This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease (which is able to split urea in the presence of water to release ammonia and carbon dioxide) The ammonia combines with carbon dioxide and water to form ammonium carbonate which turns the medium alkaline, turning the indicator phenol red from its original orange yellow color to bright pink.  Indole Test This test demonstrate the ability of certain bacteria to decompose the amino acid tryptophane to indole, which accumulates in the medium. If an organism has the ability to produce a tryptophanase enzyme capable of hydrolyzing tryptophan, three possible end products will be produced, one of which is indole. reagent i g Indole production is detected by Kovac’s or Ehrlich’s reagent, this reacts with indole to produce a red colored compound. Indole Test After 24h Kovac’s reagent Red colour near Top principle  Voges–Proskauer (VP) Test VP is a testprinciple used to detect the ability of bacteria to convert the glucose to acetoin in a bacterial broth culture. The test is performed by adding alpha-naphthol and potassium hydroxide to the VP broth which has been inoculated with the bacteria. A cherry red color indicates a positive result, while a yellow-brown color indicates a negative result.  Citrate Utilization Test This is a defined medium used to determine if an organism can use citrate as its only carbon source. indicator :bromthymol blue Citrate positive: the medium will be an intense blue color. Citrate negative: No color change will occur (the medium will remain the deep green color)  IMViC Tests Each of the letters in “IMViC” stands for one of these tests: “I” is for indole; “M” is for methyl red; “V” is for Voges-Proskauer, and “C” is for citrate, lowercase “i” is added for the ease of pronunciation. IMViC is an acronym that stands for four different tests.  Triple Sugar Iron (TSI) Agar Test It tests for organisms’ abilities to: 1- ferment glucose and lactose. 2- produce gas during fermentation. 3. Produce of H2S from the sulfur source  Triple Sugar Iron (TSI) Agar Test TSI medium contain : red 0.1% glucose: If only glucose is fermented, yellow only enough acid is produced to turn the bottom yellow. The slant will remain red 1% lactose & 1% sucrose: If lactose are fermented, a large amount of acid will produce which turns both bottom and slant in yellow. Iron (ferrous sulfate): Indicator of H2S formation give black color. Phenol red indicator (It is yellow in acidic condition and red under alkaline conditions). A TSI allows for determination of: 1. Fermentation of glucose, lactose 2. Production of gas during fermentation 3. Production of H2S from the sulfur source n it t I  Motility Test In semi-solid agar media, motile bacteria ‘swarm’ and give a diffuse spreading growth that is easily recognized by the naked eye. Motile: organisms will spread out (diffuse, hazy ) growths into the politin medium from the site of Negatin inoculation, diffuse zone Non-motile: organisms remain at the site of inoculation  SIM/MIU Tests Sulfide Indole Motility (SIM) Medium: It is a semisolid agar used to determine hydrogen sulfide (H2S) production, indole formation, and motility. Motility Indole urease (MIU) test: It is used to determine motility, indole formation and urease test. sif gymotile  API (Analytical Profile Index) 20E The API 20 E is a strip consists of 20 microtubes containing dehydrated substrates used for identification and differentiation of members of the Enterobacteriaceae. M negative if positive t.ws  API 20E Watch the videos: 1. Performing an API 20E strip test https://youtu.be/BDVpQLyktkk 2. Reading an API20E https://youtu.be/ew3amo02_b0  Other techniques ▪ Automation VITEK-2 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF MS) ▪ Molecular Methods Used to Detect Bacteria PCR, real-time PCR. VITEK-2 MALDI-TOF MS