Clinical Bacteriology (Lecture) Module 10: The Enterobacteriaceae PDF

MLS 3A CLINICAL BACTERIOLOGY (LECTURE) MODULE 10: 6. Cytochrome C oxidase negative (enteric always negative THE ENTEROBACTERIACEAE separates enterics from oxidase positive bacteria of genera...

MLS 3A CLINICAL BACTERIOLOGY (LECTURE) MODULE 10: 6. Cytochrome C oxidase negative (enteric always negative THE ENTEROBACTERIACEAE separates enterics from oxidase positive bacteria of genera INTRODUCTION Pseudomonas, Aeromonas, Vibrio, Alcaligenes, Achromobacter, Flavibacterium, Cardiobacterium which may have similar Enterobacteriaceae family contains a large number of genera that morphology.) are biochemically and genetically related to one another. Many of the 7. Usually reduces Nitrate to Nitrite (distinguishes enteric bacteria traditional or familiar bacteria are found in this family: from bacteria that reduce nitrate to Nitrogen gas, such as  Escherichia Pseudomonas and many other oxidase positive bacteria).  Shigella 8. Cell contain a characteristic antigen, called the enterobacterial  Salmonella common antigen.  Enterobacter  Proteus Enterobacterial common antigens are:  Yersinia O: Outer membrane H: Flagella COMMON CHARACTERISTICS OF FAMILY ENTEROBACTERIACEAE: K: Capsule 1. They are gram negative, short rods Vi: Capsule of Salmonella 2. They are non-sporulating, facultative anaerobes 3. These organisms have simple nutritional requirements and 9. Produces acid from glucose; ability to ferment lactose- MacConkey agar is used to isolate and differentiate organisms of distinguishes enteric from obligately aerobic bacteria. Enterobacteriaceae family (Pink colored colonies of lactose 10. Sodium neither required nor stimulatory for growth fermentercoliforms and pale colored colonies of Non lactose fermenter) BIOCHEMICAL TESTS FOR ENTEROBACTERIACEAE Lactose fermenters: (CEEK) Tests for identification of members of Enterobacteriaceae family: Citrobacter Escherichia Member of the Enterobacteriaceae family are identified based on their Enterobacter biochemical properties. Commonly used biochemical tests to identify them Klebsiella are: Non-lactose fermenters: (ShYPS) 1. Citrate utilization Test Shigella 2. Indole Test Yersinia 3. Motility Test Proteus 4. Methyl Red (MR) Test Salmonella 5. Voges–Proskauer (VP) Test 6. Triple Sugar Iron (TSI) Agar Test 4. Motility if present is by means of peritrichous (lateral) flagella. 7. Urease Test Motile by peritrichous flagella, except Shigella and Klebsiella which are non-motile 5. They are catalase positive #PadayonRMT | Page 1  MLS 3A 1. Citrate Utilization Test  Citrate utilization test is commonly employed as part of a group of Further metabolic breakdown is dependent upon the pH of the medium. tests, the IMViC (Indole, Methyl Red, VP and Citrate) tests, that A. Under alkaline conditions, pyruvate is metabolized to acetate and distinguish between members of the Enterobacteriaceae family formate. based on their metabolic by-products. Citrate utilization can be used o pyruvate = acetate + formate to distinguish between coliforms such as Klebsiella (formerly B. At pH 7.0 and below, lactate and acetoin are also produced. Enterobacter) aerogenes (+ve) which occur naturally in the soil and o pyruvate = acetate + lactate + CO2 in aquatic environments and fecal coliforms such as Escherichia coli o pyruvate = acetoin + CO2 (-ve) whose presence would be indicative of fecal contamination.  Citrate utilization test is used to determine the ability of bacteria to  The carbon dioxide that is released will subsequently react with utilize sodium citrate as its only carbon source and inorganic water and the sodium ion in the medium to produce sodium ammonium dihydrogen phosphate (NH4H2PO4) is the sole fixed carbonate, an alkaline compound that will raise the pH. In addition, nitrogen source. ammonium hydroxide is produced when the ammonium salts in the  In the citrate utilization test, the citrate medium most commonly medium are used as the sole nitrogen source. used is the formula of Simmons. The medium is poured into a tube  Growth usually results in the bromothymol blue indicator, turning on a slant. So therefore, it was called the “Simmon’s Citrate media”. from green to blue. The bromothymol blue pH indicator is a deep forest green at neutral pH. With an increase in medium pH to above Principle of Citrate Utilization Test: 7.6, bromothymol blue changes to blue.  When an organic acid such as citrate (remember Krebs cycle) is used as a carbon and energy source, alkaline carbonates and Procedure of Citrate Utilization Test: bicarbonates are produced ultimately. In addition, ammonium 1. Inoculate Simmons citrate agar lightly hydroxide is produced when the ammonium salts in the medium are on the slant by touching the tip of a used as the sole nitrogen source. needle to a colony that is 18 to 24 hours  Utilization of exogenous citrate requires the presence of citrate old. transport proteins (permeases). Upon uptake by the cell, citrate is 2. Incubate at 35°C to 37°C for 18 to 24 cleaved by citrate lyase to oxaloacetate and acetate. The hours. Some organisms may require up oxaloacetate is then metabolized to pyruvate and CO2. to 7 days of incubation due to their limited rate of growth on citrate Citrate = oxaloacetate + acetate medium. Oxalacetate = pyruvate + CO2 3. Observe the development of blue color; denoting alkalinization. Expected results in Citrate Utilization Test: Citrate positive: growth will be visible on the slant surface and the medium will be an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue. #PadayonRMT | Page 2  MLS 3A Citrate negative: trace or no growth will be visible. No color change  Indole test is a commonly used biochemical test (e.g. in IMVIC test, will occur; the medium will remain the deep forest green color of SIM test, etc). Indole test helps to differentiate Enterobacteriaceae the uninoculated agar. Only bacteria that can utilize citrate as the and other genera. sole carbon and energy source will be able to grow on the Simmons citrate medium, thus a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant. Citrate Test: POSITIVE  Klebsiella pneumoniae  Enterobacter species (minority of strains gives negative result)  Citrobacter freundii  Salmonella other than Typhi and Paratyphi A  Serratia marcescens  Proteus mirabilis (minority of strains gives negative result)  Providencia Citrate Test: VARIABLE (different strains give different results):  Proteus vulgaris Two methods are in use:  Vibrio cholerae A. A conventional tube method requiring overnight incubation,which  Vibrio parahaemolyticus identifies weak indole producing organisms and B. A spot indole test, which detects rapid indole producing organisms Citrate test: NEGATIVE o Escherichia coli A. Procedure of a Conventional Tube method for Indole Test o Shigella spp a) Inoculate the tryptophan broth with broth culture or emulsify o Salmonella Typhi isolated colony of the test organism in tryptophan broth. o Salmonella Paratyphi A b) Incubate at 37°C for 24-28 hours in ambient air. o Morganella morganii c) Add 0.5 ml of Kovac’s reagent to the broth culture. o Yersinia enterocolitica Expected results: (See picture above for reference) 2. Indole Test Positive: Pink colored rink after the addition of an appropriate  Indole test is used to determine the ability of an organism to split reagent amino acid tryptophan to form the compound indole. Tryptophan is Negative: No color change even after the addition of an appropriate hydrolyzed by tryptophanase to produce three possible end reagent. e.g. Klebsiella pneumoniae products – one of which is indole. Indole production is detected by Kovac’s or Ehrlich’s reagent which contains 4 (p)- dimethylamino- B. Spot Indole Test benzaldehyde, this reacts with indole to produce a red-colored  It is used to determine the presence of the enzyme tryptophanase. compound. Tryptophanase breaks down tryptophan to release indole, which when reacts with cinnamaldehyde produces a blue-green compound. The absence of enzyme results in no color production (i.e. indole negative). #PadayonRMT | Page 3  MLS 3A Procedure of Spot Indole Test: Point to remember: Indole test can also aid in species differentiation. 1. Saturate a piece of filter paper with the 1% para- dimethylaminocinnamaldehyde reagent. o Klebsiella species: 2. Use a wooden stick or bacteriologic loop to remove a small portion o Klebsiella oxytoca is indole positive of a bacterial colony from the agar surface and rub the sample on o Klebsiella pneumoniae is indole negative. the filter paper. o Citrobacter species: o Citrobacter Koseri is indole positive o Citrobacter freundii is indole negative o Proteus species: o Proteus Vulgaris is indole positive o Proteus mirabilis is indole negative Remember the phrase “OK VIP”  Where: O means: Oxytica (Klebsiella oxytoca), K means (koseri-i.e. Result: Citrobacter koseri), V means vulgaris (Proteus vulgaris) and IP means Positive: Development of a blue color within 30 seconds. Most Indole Positive. indole positive organisms turn blue within 30 seconds. Negative: No color development or slightly pink color. In Diagnostic laboratories, Indole test can is performed using multitest agar. Three most commonly used agar medium are: Uses:  Sulfide-indole-motility (SIM) medium: The SIM medium is a  Spot indole tests along with gram stain result and colony multitest agar used to test for indole production while characteristics can assist in the rapid identification of isolates. For simultaneously determining motility and hydrogen sulfide example: producing abilities of the isolate. o A flat, dry lactose fermenting (pink) colony on MacConkey  Motility-indole-urease (MIU) medium: MIU medium is used to test agar that is also spot indole positive and oxidase negative for indole and urease producing characteristics of the organism can be reported presumptively as E. coli. along with a test for motility.  Organisms that swarm on 5% sheep blood agar, exhibit a  Motility-indole-ornithine (MIO) medium: In addition to testing for characteristics odor, and are oxidase negative can be presumptively indole production, MIO agar is used to test for motility and identified as Proteus spp. With further testing by spot indole, the ornithine decarboxylase. positive isolates may be presumptively reported as Proteus vulgaris and the negative ones as Proteus mirabilis. 3. Motility Test  There are a variety of ways to determine the motility of a Indole: POSITIVE bacterium—biochemical tests as well as microscopic analysis. If a  Most strains of E. coli fresh culture of bacteria is available, microscopy is the most  P. vulgaris accurate way to determine bacterial motility, and ‘hanging drop  M. morganii method’ is a commonly used microscopic technique.  Providencia  Motile bacteria move with structures called flagella (a few exceptional bacteria move with the help of axial filaments, which cannot be seen in the microscope). In semi-solid agar media, motile #PadayonRMT | Page 4  MLS 3A bacteria ‘swarm’ and give a diffuse spreading growth that is easily Motility test is also used for the species differentiation of gram-positive recognized by the naked eye. cocci, enterococci. NON-MOTILE Procedure: o Enterococcus faecium 1. Prepare a semisolid agar medium in a test tube. o E. faecalis are non-motile, 2. Inoculate with a straight wire, making a single stab down the center GENERALLY MOTILE of the tube to about half the depth of the medium.  E. gallinarum 3. Incubate under the conditions favoring motility.  E. casseliflavus/E. flavescens 4. Incubate at 37°C 5. Examine at intervals, e.g. after 6 h, and 1 and 2 days (depends on Specific tests for bacterial motility determination: generation time of bacteria). Freshly prepared medium containing  Distilled water motility test: It is a simple and very useful test to 1% glucose can be used for motility tests on anaerobes. differentiate Vibrio species (gram-negative motile curved rod) and Aeromonas species (gram-negative motile rod). Aeromonas species Results: Hold the tube up to the light and look at the stab line to determine will grow on MacConkey agar and sometimes on TCBS, producing motility. yellow colonies. Both of them are oxidase positive. Non-motile bacteria generally give growths that are confined to the stab-line, have sharply defined margins, and leave the surrounding Procedure: medium clearly transparent. 1. Mix a loopful of growth from a nutrient agar subculture in a drop of Motile Bacteria typically give diffuse, hazy growths that spread sterile distilled water on one end of a slide. On the other end of the throughout the medium rendering it slightly opaque. slide, mix another loopful of growth in a drop of peptone water. 2. Cover each preparation with a cover glass. 3. Examine microscopically using the 40x objective. Results: All Vibrio species are immobilized in distilled water but remain motile in peptone water. Aeromonas species remain motile in distilled water and peptone water. 4. Methyl Red (MR) test Laboratory Use:  Methyl Red (MR) test determines whether the microbe performs In the laboratory, motility testing using a semi-solid medium is mixed acids fermentation when supplied glucose. Types and commonly used for the identification of gramnegative bacteria of proportion of fermentation products produced by anaerobic fermentation of glucose are one of the key taxonomic Enterobacteriaceae family. Motility testing is done in conjunction with other biochemical testing using special biochemical media. characteristics which help to differentiate various genera of enteric  Sulfide Indole Motility (SIM) Medium: It is a semisolid agar used to bacteria. determine hydrogen sulfide (H₂S) production, indole formation, and motility.  Motility Indole urease (MIU) test: It is used to determine motility, indole formation, and urease production (Urease Test.) #PadayonRMT | Page 5  MLS 3A 3. Add about 5 drops of the methyl red indicator solution to the first tube (for Voges-Proskauer test, Barrit’s reagent is added to another tube). 4. A positive reaction is indicated if the color of the medium changes to red within a few minutes.  Mixed acid fermentation is one of the two broad patterns, 2-3- butanediol fermentation being another. In mixed acid fermentation, three acids (acetic, lactic and succinic) are formed in significant amounts. The mixed acid pathway gives 4 mol of acidic products Expected results: (mainly lactic and acetic acid), 1 mol of neutral fermentation The development of a stable red color on the surface of the medium product (ethanol), 1 mol of CO2, and 1 mol of H2 per mol of glucose indicates sufficient acid production to lower the pH to 4.4 and constitutes a fermented. positive test. Because other organisms may produce smaller quantities of  These large amounts of acid results significant decrease in the pH of acid from the test substrate, an intermediate orange color between yellow the medium below 4.4. This is visualized by using pH indicator, and red may develop. This does not indicate a positive test. methyl red (p-dimethylaminoaeobenzene-O-carboxylic acid), which is yellow above pH 5.1 and red at pH 4.4. MR TEST POSITIVE  The pH at which methyl red detects acid is considerably lower than  Escherichia coli: - the appearance of red color after the addition of the pH for other indicators used in bacteriologic culture media. methyl red reagent. Thus, to produce a color change, the test organism must produce large quantities of acid from the carbohydrate substrate being used. MR TEST NEGATIVE o Klebsiella (formerly Enterobacter) aerogenes: - the lack of color MR Positive: When the culture medium turns red after the addition change after the addition of methyl red. of methyl red, because of a pH at or below 4.4 from the fermentation of glucose. Methyl-Red (MR): POSITIVE MR Negative: When the culture medium remains yellow, which  Escherichia coli occurs when less acid is produced (pH is higher) from the  Shigella species fermentation of glucose.  Salmonella species  Citrobacter species Procedure for Methyl Red (MR) Test:  Proteus species MR-VP broth is used for both MR Test and VP test. Only the addition  Yersinia species of reagent differs, and both tests are carried out consecutively. Methyl-Red (MR): NEGATIVE 1. Inoculate two tubes containing MR-VP Broth with a pure culture of o Enterobacter species the microorganisms under investigation. o Hafnia species 2. Incubate at 35 °C for up to 4 days. o Serratia marcescens o Klebsiella pneumoniae #PadayonRMT | Page 6  MLS 3A 5. Voges Proskauer (VP) Test Procedure of Voges Proskauer Test  Voges-Proskauer is a double eponym, named after two 1. Inoculate a tube of MR/VP broth with a pure culture of the test microbiologists working at the beginning of the 20th century. They organism. first observed the red color reaction produced by appropriate 2. Incubate for 24 hours at 35°C culture media after treatment with potassium hydroxide. It was 3. At the end of this time, aliquot 1 mL of broth to clean test tube. later discovered that the active product in the medium formed by 4. Add 0.6mL of 5% α-naphthol, followed by 0.2 mL of 40% KOH. bacterial metabolism is acetyl methyl carbinol, a product of the (Note: It is essential that the reagents be added in this order.) butylene’s glycol pathway. 5. Shake the tube gently to expose the medium to atmospheric oxygen  Pyruvic acid, the pivotal compound in the fermentative and allow the tube to remain undisturbed for 10 to 15 minutes. degradation of glucose, is further metabolized through various metabolic pathways, depending on the enzyme systems possessed Results and Interpretation by different bacteria. One such pathway results in the production of Positive test - development of a red color 15 minutes or more after acetion (acetyl methyl carbinol), a neutral-reacting end product. the addition of the reagents indicating the presence of diacetyl, the oxidation product of acetoin. NOTE: The test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures may produce a copper like color, potentially resulting in a false positive interpretation. Voges-Proskauer (VP): POSITIVE - Enterobacteriaceae family  Klebsiella species  Enterobacter species  Hafnia species  Serratia species  Organisms such as members of the Klebsiella-Enterobacter-Hafnia- Serratia group produce acetoin as the chief end product of glucose 6. Triple Sugar Iron Agar (TSI) metabolism and form smaller quantities of mixed acids. In the  Whenever you see the name of this test i.e. “Triple Sugar Iron presence of atmospheric oxygen and 40% potassium hydroxide, Agar,” you have to remember that it’s a test which has three sugar acetoin is converted to diacetyl, and alpha-naphthol serves as a (lactose, sucrose, and glucose) and also iron; and it contains agar as catalyst to bring out a red complex. solidifying agent (TSI is a semi-solid media having slant and butt). Media:  Methyl red-Voges-Proskauer (MR/VP) broth (formulated by Clark Composition of Triple Sugar Iron Agar (TSI): and Lubs) is used in Voges–Proskauer test.  Lactose, sucrose and glucose in the concentration of 10:10:1 (i.e. 10 Reagents: part lactose (1%), 10 part sucrose (1%) and 1 part glucose (0.1%)). A. Alpha-naphthol, 5% color intensifier TSI is similar to Kligler’s iron agar (KIA), except that Kligler’s iron agar Alpha α-5g contains only two carbohydrates: glucose (0.1%) and lactose (1%). Absolute ethyl alcohol- 100 mL o 0.1% glucose: If only glucose is fermented, only enough acid B. Potassium Hydroxide, 40%, oxidizing agent is produced to turn the butt yellow. The slant will remain Potassium hydroxide 40g red Distilled water: 100 mL #PadayonRMT | Page 7  MLS 3A o % lactose/1.0% sucrose: If lactose or sucrose or both sugar are fermented, a large amount of acid will produce which turns both butt and slant yellow. So the appearance of yellow color in both slant and butt indicates that the isolate has the ability to ferment lactose or sucrose or both. o Iron (ferrous sulfate): Indicator of H2S formation o Phenol red: Indicator of acidification (It is yellow in acidic condition and red under alkaline conditions). o It also contains peptone which acts as a source of nitrogen. (Remember that whenever peptone is utilized under aerobic condition ammonia is produced) Expected results of TSI Agar test are: Procedure for Triple Sugar Iron Agar (TSI) Test: 1. Alkaline slant/no change in butt (K/NC) 1. With a sterilized straight inoculation needle touch the top of a well-  Red/Red = glucose, lactose and sucrose non-fermenter isolated colony 2. Alkaline slant/Alkaline butt (K/K) 2. Inoculate TSI agar by first stabbing through the center of the  Red/Red = glucose, lactose and sucrose non-fermenter medium to the bottom of the tube and then streaking on the 3. Alkaline slant/acidic butt (K/A) surface of the agar slant.  Red/Yellow = glucose fermentation only, gas (+ or -), H2S (+ 3. Leave the cap on loosely and incubate the tube at 35°C in ambient or -) air for 18 to 24 hours. 4. Acidic slant/acidic butt (A/A)  Yellow/Yellow = glucose, lactose and/or sucrose fermenter Interpretation of Triple Sugar Iron Agar Test: gas (+ or -), H2S (+ or -). 1. If lactose (or sucrose) is fermented, a large amount of acid is produced, which turns the phenol red indicator yellow both in the Some example of Triple Sugar Iron (TSI) Agar Reactions: butt and in the slant. Some organisms generate gases, which produces bubbles/cracks on the medium. 2. If lactose is not fermented but the small amount of glucose is, the oxygen-deficient butt will be yellow (remember that butt has comparatively more glucose than slant i.e. more media more glucose), but on the slant the acid produced (less acid produces in slant as media in slant is less) will be oxidized to carbon dioxide and water by the organism and the slant will be red (alkaline or neutral 7. Urease test pH). Medium used for urease test: 3. If neither lactose/sucrose nor glucose is fermented, both the butt  Any urea medium, agar (Christensen’s Urea Agar) or broth (Stuart’s and the slant will be red. The slant can become a deeper red-purple Urea Broth). Urease test medium can be a sole medium or part of (more alkaline) as a result of the production of ammonia from the panel like motility indole urease (MIU) test. oxidative deamination of amino acids (remember peptone is a Indicator used in urease test: major constituent of TSI agar).  Phenol red 4. 4. if H2S is produced, the black color of ferrous sulfide is seen. #PadayonRMT | Page 8  MLS 3A Urease test principle: o Urea is a diamide of carbonic acid. It is hydrolyzed with the release In routine diagnostic laboratories the urease test result is read within 24 of ammonia and carbon dioxide. Many organisms especially those hours. that infect the urinary tract, have a urease enzyme which is able to  If organism produces urease enzyme, the color of the slant changes split urea in the presence of water to release ammonia and carbon from light orange to magenta. dioxide.  If organism do not produce urease the agar slant and butt remain o The ammonia combines with carbon dioxide and water to form light orange (medium retains original color). ammonium carbonate which turns the medium alkaline, turning the indicator phenol red from its original orange yellow color to bright pink. Procedure for urease test:  For Christensen’s Urea Agar 1. Streak the entire slant surface with a heavy inoculum from an If Stuart’s Urea Broth is used; rapidly urease positive organisms (Proteus 18-24 hour pure culture (Do not stab the butt as it will serve as spp., Morganella morganii) will produce a strong positive reaction within 8- a color control). 24 hours of incubation but delayed positive organisms (e.g., Enterobacter) 2. Incubate tubes with loosened caps at 35°C. will not produce a positive reaction due to high buffering capacity of this 3. Observe the slant for a color change at 6 hours and 24 hours medium. unless specified for longer incubation.  For Stuart’s Urea Broth Diagnostic utility of Urease test: 1. Inoculate the broth with a heavy inoculum from an 18-24 hour 1. Urease test helps for the identification of Proteus species (urease pure culture positive) and to differenitate it from other non-lactose fermenting 2. Shake the tube gently to suspend the bacteria members of the Enterobacteriaceae family. 3. Incubate the tubes with loosened caps at 35°C. 2. Urease test is used for the presumptive evidence of the presence of 4. Observe the broth for a color change at 8, 12, 24 hours. Helicobacter pylori in tissue biopsy material. This is done by placing a portion of crushed tissue biopsy material directly into urease Result and Interpretation: broth. A positive urease test is considered presence of Helicobacter HYDROLYZE UREA RAPIDLY (within 1 or 6 hours of incubation) pylori. Commercially available urease agar kits are also available.  Proteus spp., 3. Rapid Urease test is can be used to differentiate between the  Morganella morganii yeasts, Candida albicans and Cryptococcus neoformans. A  some Providencia stuartii strains presumptive identification of C. neoformans may be based on rapid DELAYED POSITIVE (6 hours of incubation which will be intense during urease production, whereas Candida albicans do not. further incubation) 4. Urea breath test: A common noninvasive test to detect Helicobacter  Klebsiella spp. pylori also based on urease activity. This is highly sensitive and  Enterobacter species specific test. NEGATIVE (remain a yellowish color) o Escherichia coli. #PadayonRMT | Page 9  MLS 3A Name of urease positive organisms (Bacteria):  Most commonly found in the colon of the large intestine.  Proteeae  Most of them are harmless and opportunistic. o Proteus spp.  E. coli form a mutual relationship with its host. o Morganella morganii  E. coli helps with the absorption of vitamin K and other vitamins in o some Providencia stuartii strains the colon.  Cryptococcus spp.  It is the largest group of bacteria living in the intestine.  Corynebacterium spp.  E. coli constitute about 0.1% to 1% of GI tract bacteria.  Helicobacter pylori  It is a facultative aerobe.  Brucella spp.  E. coli is also found in human feces.  When E. coli is excreted from the intestinal tract, the bacteria are Note: Mneomonics to remember urease positive organisms: PUNCH (Similar able to survive only for a few hours. mneomonic for oxidase positive organism is PVNCH)  E. coli is found outside the body in the faecally contaminated o P: Proteus environments such as water or mud or sediments. o U: Ureaplasma  If E. coli comes in contact with raw vegetables, it has the potential o N: Nocardia to attach itself to the leaves of the vegetable. o C: Cryptococcus neoformans/Corynebacterium spp.  E. coli can also be found in environments at a higher temperature, o H: Helicobacter pylori such as on the edge of hot springs.  E. coli is also found on ground meats due to slaughterhouse processing.  Humans are most likely to be infected with E. coli O157: H7 found in cattle. Morphology of E. coli:  E. coli is gram-negative rod-shaped bacteria.  It is 1-3 x 0.4-0.7 µm in size  It is arranged singly or in pairs.  It is motile due to peritrichous flagella.  Some strains are non-motile.  Some strains may be fimbriated. The fimbriae are of type 1 Escherichia coli (hemagglutinating & mannose-sensitive) and are present in both motile and non-motile strains.  E. coli was discovered by Theodor Escherich in 1885 after isolating it  Some strains of E. coli isolated from extra-intestinal infections have from the feces of newborns. a polysaccharide capsule.  E. coli is the normal flora of the human body.  They are non-spore forming.  The niche of E. coli depends upon the availability of the nutrients  They have a thin cell wall with only 1 or 2 layers of peptidoglycan. within the intestine of host organisms.  They are facultative anaerobes.  The primary habitat of E. coli is in the gastrointestinal (GI) tract of  Growth occurs over a wide range of temperatures from 15-45°C. humans and many other warmblooded animals.  It is found in the mucus or the epithelium on the wall of the intestine. #PadayonRMT | Page 10  MLS 3A Cultural Characteristics of E. coli:  E. coli is a facultative anaerobes.  Its optimum growth temperature is 37°C and ranges from 10°C to 40°C. E. coli on Nutrient Agar (NA): o They appear large, circular, low convex, grayish, white, moist, smooth and opaque. o They are of 2 forms: Smooth (S) form and Antigenic Structures: Rough (R) form. 1. Heat Stable Lipopolysaccharide (LPS) is the major cell wall antigen of o Smooth forms are emulsifiable in saline. E. coli. o Due to repeated subculture, there is smooth 2. E. coli possesses 4 antigens; H, O, K and F. to rough variation (S-R variation). a. H or Flagellar Antigen i. Heat and alcohol labile protein E. coli on Blood Agar (BA): ii. Present on the flagella o Colonies are big, circular, gray and moist. iii. Genus specific o Beta (β) hemolytic colonies are formed. iv. Present as monophasic v. 75 ‘H’ antigens have been recognized b. O or Somatic Antigen i. Heat stable, resistant to boiling up to 2 hrs. 30 minutes ii. Occur on the surface of the outer membrane iii. An integral part of the cell wall iv. 173 ‘O’ antigens have been recognized c. K or Capsular Antigen E. coli on MacConkey Agar (MAC): i. Heat labile o Colonies are circular, moist, smooth and of entire margin. ii. Acidic polysaccharide antigen present in the o Colonies appear flat and pink. envelope o They are lactose fermenting colonies. iii. Boiling removes the K antigen iv. Inhibit phagocytosis v. 103 ‘K’ antigens have been recognized d. F or Fimbrial antigen i. Heat labile proteins ii. Present in the Fimbriae #PadayonRMT | Page 11  MLS 3A E. coli on Eosin Methylene Blue (EMB) Agar: Typical biochemical properties of E.coli are: o Green Metallic sheen colonies are formed. 1a. E. coli Pathotypes- ETEC, EPEC, EHEC, EAEC, EIEC, DAEC:  Human Escherichia coli strains are classified base on their genetic E. coli on m-ENDO Agar: features and clinical outcomes. o Colonies are green metallic sheen.  Commensal microbiota E. coli, o Metabolize lactose with the production of aldehyde and acid.  Enterovirulent E. coli, and  Extraintestinal pathogenic E. coli (ExPEC)  Most infections (with the exception of neonatal meningitis and gastroenteritis) are endogenous.  E. coli that are part of the patient’s normal microbial flora are able to establish infection when the patient’s defenses are compromised (e.g., through trauma or immune suppression). The effectiveness of E. coli as a pathogen is illustrated by the fact that: o the bacteria are the most common gram-negative rods isolated E. coli on Liquid Media: from patients with sepsis o They show homogenous turbid growth within 12-18 hours. o responsible for causing more than 80% of all community-acquired o R form agglutinate spontaneously, forming sediment on the bottom UTIs as well as many hospital-acquired infections, of the test tubes. o a prominent cause of gastroenteritis. o After prolonged incubation (>72 hrs), pellicle are formed on the surface of liquid media.  The strains of E. coli that cause gastroenteritis are subdivided. For o Heavy deposits are formed which disperses on shaking. the pathogenic enteric E. coli strains, six pathotypes:  Enterotoxigenic E. coli (ETEC)  Enteropathogenic E. coli (EPEC)  Enterohemorrhagic E. coli (EHEC)  Enteroaggregative E. coli (EAEC)  Enteroinvasive E. coli (EIEC)  Diffusely adhering E. coli (DAEC)  They are commonly referred to as the diarrheagenic E. coli. Diarrheagenic E. coli are antigenically distinct from the commensal E. coli which colonize the intestine. Only few serotypes of E. coli which express the enterotoxin or other virulence mechanisms can cause diarrhea. #PadayonRMT | Page 12  MLS 3A  The different types are recognized on the basis of distinct chromosomal pathogenicity island called the locus of enterocyte epidemiological and clinical features, specific virulence effacement (LEE). determinants, and an association with certain serotypes.  EPEC frequently causes infantile diarrhea (outbreaks) and A. Enterotoxigenic E. coli (ETEC) occasionally cause sporadic diarrhea in adults.  Enterotoxigenic E. coli (ETEC) is one of the most common causes of  Disease is transmitted by fecal-oral exposure to contaminated bacterial diarrheal disease in developing countries (estimated 840 surfaces or food products. Person-toperson spread is seen. million cases annually) and in an estimated 30% of travelers to  However, it is non- toxigenic and non- invasive. these countries with diarrheal disease.  Mechanism of diarrhea involves the adhesion to intestinal mucosa,  It causes acute watery diarrhea in infants and adults. mediated by plasmid coded bundleforming pill, which form cup-like  Because the inoculum for disease is high, infections are primarily projections called pedestals. acquired through consumption of fecally contaminated food or  A/E lesions (attaching and effacing lesions) are produced on the water. Person-to-person spread does not occur. intestinal epithelium (coded by chromosomal LEE gene, i.e. locus for  Common serotypes associated are-06, 08, 0 15, 025, 0 27, 0 153, 0 enterocyte effacement); which leads to disruption of brush border 159, etc. epithelium causing increased secretion and watery diarrhea.  It is toxigenic, but not invasive.  The onset of disease may be as rapid as a few hours after ingestion  Pathogenesis of ETEC involves attachment to the intestinal mucosa of EPEC, and although most infections resolve after a few days, mediated by fimbrial protein called CFA (colonization factor persistent diarrhea requiring hospitalization can occur. antigen) and elaboration of heat-stable and heat-labile enterotoxins. C. Enteroaggregative E. coli (EAEC)  ETEC produce two classes of enterotoxins: heat-stable toxins (STa  Enteroaggregative E. coli (EAEC) are a heterogeneous collection of and STb) and heat-labile toxins (LT-I, LT-II). strains characterized by their autoagglutination in a “stacked-brick”  Heat-stable toxin STa but not STb is associated with human disease, arrangement over the epithelium of the small intestine and, in is found in 75% to 80% of ETEC either alone or associated with LT, some cases, the colon. That is, it is so named because it adheres to and is responsible more commonly for severe disease than LT-only HEP-2 cells in a distinct pattern, layering of the bacteria aggregated ETEC strains. in a stacked-brick fashion.  Secretory diarrhea caused by ETEC develops after a 1- to 2-day  Most strains are “O” untypeable but “H “ typeable. incubation period and persists for an average of 3 to 5 days.  The prevalence of disease caused by EAEC is unclear because a  The symptoms (watery, non-bloody diarrhea and abdominal single molecular marker for these bacteria has not been cramps; less commonly nausea and vomiting) are similar to those of discovered. Genes encoding adhesins, toxins including Shiga toxin, cholera but are usually milder, although mortality is high in and other virulence proteins are highly variable among EAEC. malnourished individuals and in those with underlying diseases,  The intestinal colonization is mediated by aggregative adhesion particularly children and the elderly. fimbriae I (regulated by aggR gene). It also produces EAST l toxin  Diagnosis is done by detection of toxins by in vitro and in vivo (entero-aggregative heat stable enterotoxin l). methods.  Manifestation of the disease include persistent and acute diarrhea are; especially in developing countries. B. Enteropathogenic E. coli (EPEC)  These are one of the few bacteria associated with chronic diarrhea  Two groups of E. coli are responsible for enteric disease and growth retardation in children. (Enteropathogenic E. coli [EPEC] and some Shiga toxin–producing E. coli [STEC]) possess a cluster of virulence genes located on a #PadayonRMT | Page 13  MLS 3A  E. coli 0104: H4 is an enteroaggregative strain that has caused E. Enteroinvasive E. coli (EIEC) major outbreaks in Germany in 2011. One peculiar feature of this  Enteroinvasive E. coli (EIEC) strains are rare in both developed and strain is, it produces Shiga-like toxin and can cause HUS. developing countries.  Pathogenic strains are primarily associated with a few restricted O D. Shiga Toxin–Producing E. coli (STEC/VTEC/EHEC) serotypes: O124, O143, and O164.  Nomenclature for this group of E. coli is confusing, referring to them  The strains are closely related by phenotyping and pathogenic as Shiga toxin–producing E. coli (STEC), verocytotoxin-producing E. properties to Shigella. coli (VTEC), and enterohemorrhagic E. coli (EHEC).  The bacteria are able to invade and destroy the colonic epithelium,  All members of this group are defined by the presence of Shiga toxin producing a disease characterized initially by watery diarrhea. 1 (Stx1) or 2 (Stx2). Some but not all EHEC strains are LEE positive  A minority of patients progress to the dysenteric form of disease, and form A/E cytopathology, resembling EPEC strains. consisting of fever, abdominal cramps, and blood and leukocytes in  Serotypes associated with EHEC are: 0157:H7 (most common stool specimens. serotype). Other serotypes are rarely associated such as 026:H11,  A series of genes on a plasmid mediate bacterial invasion (pInv 06, 055, 091, 0103, 0 111 and 0113. genes) into the colonic epithelium.  Diagnosis of STEC disease is now based on detection of the Shiga  The bacteria then lyse the phagocytic vacuole and replicate in the toxins rather than serotyping suspected isolates. cell cytoplasm.  STEC disease is most common in the warm months, and the highest  Movement within the cytoplasm and into adjacent epithelial cells is incidence is in children younger than 5 years. regulated by formation of actin tails (similar to that observed with  Most infections are attributed to the consumption of undercooked Listeria). ground beef or other meat products, water, unpasteurized milk or  This process of epithelial cell destruction with inflammatory fruit juices (e.g., cider made from apples contaminated with feces infiltration can progress to colonic ulceration. from cattle), uncooked vegetables such as spinach, and fruits.  Ingestion of fewer than 100 bacteria can produce disease, and F. Diffusely-adherent E. coli (DAEC) person-to person spread occurs.  Recently, diffusely adherent E. coli (DAEC) strains have been  Disease caused by STEC ranges from mild uncomplicated diarrhea to recognized as the sixth class of diarrheagenic E. coli and appear as a hemorrhagic colitis with severe abdominal pain and bloody heterogeneous group. diarrhea. Severe disease is more commonly associated with STEC  It is characterized by the ability to adhere to HEp-2 cells in a diffuse O157:H7. pattern in which the bacteria uniformly cover the entire cell surface.  Hemolytic uremic syndrome (HUS), a disorder characterized by  It expresses diffuse adherence fimbriae which contribute to the acute renal failure, thrombocytopenia, and microangiopathic pathogenesis. hemolytic anemia, is a complication in 5% to 10% of infected  These strains are age dependently involved in diarrhea in children, children younger than 10 years. are apparently not involved in diarrhea in adults, and can also be  Resolution of symptoms occurs in uncomplicated disease after 4 to asymptomatic intestinal microbiota strains in children and adult. 10 days in most untreated patients; however, death can occur in 3%  Hence, the pathogenesis and pathogenicity of the strains are still to 5% of patients with HUS, and severe sequelae (e.g., renal under question. impairment, hypertension, central nervous system [CNS]  DAEC is however thought to be capable of causing diarrheal disease, manifestations) can occur in as many as 30% of HUS patients. primarily in children aged 2-6 years. #PadayonRMT | Page 14  MLS 3A Prevention and Control Klebsiella pneumoniae accounts for a small percentage of  It is widely recommended that caution be observed in regard to pneumonia cases, the case fatality rates are high (up to 90% in food and drink in areas where environmental sanitation is poor and untreated cases). that early and brief treatment (eg, with ciprofloxacin or o Klebsiella pneumoniae is a major threat to public health with the trimethoprimsulfamethoxazole) be substituted for prophylaxis. emergence of multidrug-resistant strains, infection by these strains  Their control depends on handwashing, rigorous asepsis, very challenging to treat. Strains of K. pneumoniae producing sterilization of equipment, disinfection, restraint in intravenous extended-spectrum beta-lactamases (ESBLs) or carbapenemases therapy, and strict precautions in keeping the urinary tract sterile are considered global priority pathogens. (ie, closed drainage). General Properties of Klebsiella pneumoniae:  Gram–negative The Genus Klebsiella  Non-spore-forming rods  The genus Klebsiella, family Enterobacteriaceae, was named by V.  Facultative anaerobes Trevisan in 1885 in honor of German bacteriologist Theodor  Catalase Test: Positive Albrecht Edwin Klebs (1834–1913). Dr Klebs is known for his  Oxidase Test: Negative pioneering work demonstrating that microorganisms are  Lactose fermenter (forms pink colored colonies on MacConkey responsible for infectious diseases. Agar).  The genus Klebsiella consists of non-motile, aerobic and  Presence of polysaccharide capsule (in the culture plate mucoid facultatively anaerobic, Gram negative rods. At the time of writing, colonies are seen). the genus Klebsiella comprises K. pneumoniae subsp. pneumoniae,  Non-motile (Klebsiella species are nonmotile and non-flagellated K. pneumoniae subsp.ozaenae, K. pneumoniae subsp. and thus have no H antigens). rhinoscleromatis, K. oxytoca, K. ornithinolytica, K. planticola, and K. terrigena. Virulence Factors of K. pneumoniae: o Capsule 1. Klebsiella pneumoniae o Cell wall receptors o Klebsiella pneumoniae is a Gram-negative rod shaped bacteria of o Lipopolysaccharide (endotoxin) genus Klebsiella and family Enterobacteriaceae. They are members o Fimbriae of normal intestinal flora of humans and animals and may be o Siderophores isolated from a variety of environmental sources. o K. pneumoniae was first isolated in the late 19th century and was initially known as Friedlander’s bacterium. Classic cases of pneumonia, characterized by production of brick-red or “currant jelly” sputum, were known to be caused by Friedlander’s bacillus (Klebsiella pneumoniae). o Klebsiella pneumoniae causes infections in people of all age groups especially in infants, elderly, immunocompromised and alcoholics. It is one of the leading cause of hospital-acquired (nosocomial) infections. The range of infections includes pneumonia (it is a frequent cause of ventilator-associated pneumonia), urinary tract infection, bloodstream infection (BSI), and liver abscesses. Though #PadayonRMT | Page 15  MLS 3A Laboratory diagnosis of K. pneumoniae infection: The Genus Enterobacter Sample: SPECIES: o Sputum (Red currant-jelly sputum may be seen in a patient  Enterobacter aerogenes infected with K.pneumoniae)  Enterobacter agglomerans o Mid-stream urine  Enterobacter amnigenus o Blood (depending on the suspected illness/clinical  Enterobacter asburiae presentation).  Enterobacter cancerogenus  Enterobacter cloacae Colony characteristics of Klebsiella pneumoniae :  Enterobacter dissolvens  Blood Agar: Mucoid, non-hemolytic colonies  Enterobacter gergoviae  MacConkey Agar: Mucoid, lactose-fermenting (pink colored)  Enterobacter hormaechei colonies  Enterobacter intermedium Various biochemical tests using conventional methods or miniature  Enterobacter nimipressuralis commercial system (API-20E or Enterotube test) is done to identify the  Enterobacter sakazakii suspected colony as Klebsiella pneumoniae. Some of the commonly used  Enterobacter taylorae tests for the identification of Klebsiella pneumoniae is given below. HABITAT  All species are found in the natural environment including water, sewage, soil, plants, human and animal feces. E. cloacae is the most frequently isolated Enterobacter species form man and animals. It is found in human and animal feces. Several species have been isolated from urine, sputum and the respiratory tract, pus, and occasionally from blood or spinal fluid. PATHOGENICITY  Several species, most notable E. cloacae , E. sakazakii , E. aerogenes , E. agglomerans and E. gergoviae , are opportunistic pathogens of animals and humans; they are not considered to enteric pathogens. Enterobacter species are often implicated in burn, wound, and urinary tract infections. They occasionally cause septicemia and meningitis. E. sakazakii is often commensal without clinical significance and is occasionally a pathogen causing neonatal meningitis and bacteremia. E. agglomerans can behave as an opportunistic pathogen in immunologically compromised patients such as neonates, premature infants, burned or multiply traumatized patients, and patients with leukemia or who are undergoing immunosuppressive therapy. This species is also isolated by blood culture because they are generally introduced by such invasive procedures as catheterization and intubation. Such contaminations result in transitory bacteremia occasionally #PadayonRMT | Page 16  MLS 3A septicemia. E. gergoviae have been implicated in a long-term NEGATIVE REACTIONS: nosocomial outbreak of urinary tract infections. o Oxidase  Other species, such as E. amnigenius and E. intermedium have not o DNAse been isolated from human infection. o Tween ® 80-esterase o Indole MICROSCOPIC APPEARANCE o Lysine (except E. gergoviae ) Gram Stains: Negative. o Methyl-Red. Morphology: Straight rods. Differential Tests for commonly isolated Enterobacter species: Size: 0.6-1.0 micrometers by 1.2-3.0 micrometers. Motility: Some are motile by four to six peritrichous flagella. Spores: None MACROSCOPIC APPEARANCE  Enterobacter spp. grow rapidly on the usual enteric media. In The Genus Serratia general, the strains from environmental sources grow better at 20-  Once considered a harmless saprophyte, Serratia marcescens is 30 degrees C., whereas strains from clinical sources grow better at now recognized as an important opportunistic pathogen combining 37 degrees C. Colonial morphology differs greatly among a propensity for healthcare-associated infection and antimicrobial Enterobacter spp. ranging from smooth, irregularly round to rough resistance. "cauliflower" type colonies. Anaerogenic strains often exhibit  Serratia marcescens is a member of the genus Serratia, which is a yellow pigmented colonies. part of the family Enterobacteriaceae.  Currently 14 species of Serratia are recognized within the genus, METABOLIC PROPERTIES eight of which are associated with human infection. Of the eight  Aerobic; Facultatively anaerobic. species implicated in clinical infection S. marcescens, S. liquefaciens  Chemoorganotrophic. and S. odorifera are best known. Of all Serratia species, S.  Both respirative and fermentative. marcescens is the most common clinical isolate and the most  Glucose is fermented with the production of acid and gas. important human pathogen.  As members of the Enterobacteriaceae family, Serratia spp. are RECOMMENDED MEDIA: motile, non-endospore forming Gram-negative rods. In the For culture: Blood Agar 5%, TSA Agar, Nutrient Agar. laboratory Serratia are routinely isolated from bloodstream and For selective isolation: MacConkey Agar, Hektoen Enteric (HE) Agar, wound sites using blood agar culture or from respiratory and EMB Agar. urinary sites using selective culture methods. Common selective For maintenance: Blood Agar 5%, TSA Agar, Nutrient Agar. agar cultures include MacConkey agar which categorizes Serratia isolates with the other non-lactose fermenting Enterobacteriaceae KEY BIOCHEMICAL REACTIONS or chromogenic agars, which classifies them into a broad Klebsiella, POSITIVE REACTIONS: Enterobacter, Serratia and Citrobacter (KESC) grouping. o ONPG Biochemical Tests: Positive for = DNAse, Gelatinase, Lipase and o Voges-Proskauer ONPG o Simmons-Citrate IMVIC reaction: - - + + o Ornithine (except E. agglomerans ) TSIA Reaction: K/A, (+)gas, (-)H2S #PadayonRMT | Page 17  MLS 3A Pathogenecity Motility: Motility occurs at 30 degrees C. by peritrichous flagella.  S. marcescens is rarely associated with primary invasive infection. It Capsules: None operates as a true opportunist producing infection whenever it Spores: None gains access to a suitably compromised host. Patients most at risk include those with debilitating or immunocompromising disorders, MACROSCOPIC APPEARANCE those treated with broad-spectrum antibiotics and patients in ICU  Colonies are non-lactose-fermenters and may resemble who are subjected to invasive instrumentation. The indwelling Salmonellae. Most strains are translucent or colorless; rare strains urinary catheter is a major risk factor for infection. The risk of a may produce red or pink colonies on media containing sucrose. catheterized patient becoming infected with S. marcescens has been directly related to the proximity of other catheterized patients METABOLIC PROPERTIES colonized or infected with the organism. The respiratory tract is also  Facultatively Anaerobic recognized as a major portal of entry with S. marcescens being  Chemoorganotrophic isolated from the respiratory tract of up to 80% of post-operative  fermentative and respiratory type metabolisms. patients developing S. marcescens bacteremia. Not surprisingly,  Utilizes citrate, acetate and malonate as sole source of carbon common infections include urinary tract infection in patients with  Glucose is fermented with the production of acid and gas. indwelling catheters, respiratory tract infection in intubated  Acid is not produced from sorbitol, raffinose, melibiose, adonitol, patients and bloodstream infection in post-surgical patients, and myo -inositol. especially in those with intravenous catheters. RECOMMENDED MEDIA The Genus Hafnia For culture:Tryptic Soy Agar, or Blood Agar 5%.  Hafnia alvei For selective isolation: EMB, MacConkey Agar, Hektoen Enteric HABITAT Agar, SS or XLD Agar.  H. alvei occurs not only in man and animals and birds, but also in For maintenance: CTA at room temperature for up to 1 year. natural environments such as soil, sewage and water. H. alvei is Lyophilization is required for long- term storage. found in clinical specimens, especially from feces in healthy humans, occasionally blood, sputum, urine, and wounds, abscesses, the KEY BIOCHEMICAL REACTIONS throat, abdominal cavity and autopsies. POSITIVE REACTIONS: o Lysine- and ornithine-decarboxylase PATHOGENICITY o ONPG  H. alvei seem to be opportunistic pathogens which produce o Catalase infections in patients with underlying illness or predisposing factors. NEGATIVE REACTIONS: H. alvei has been reported as a possible causative agent of intestinal o Oxidase disorders. However, no conclusive evidence has been obtained o H2S (Triple Sugar Iron - TSI) regarding its enteropathogenicity. o Urease o Indole MICROSCOPIC APPEARANCE o DNase Gram Stain: Negative. Reduces Nitrate to Nitrite Morphology: Straight rods. Size:1.0 micrometer by 2.0-5.0 micrometers. #PadayonRMT | Page 18  MLS 3A The Genus Proteus Biochemical Properties of Proteus mirabilis and Proteus vulgaris  Proteus are Gram-negative rod-shaped bacteria of the family Enterobacteriaceae. They are widely distributed in nature and also occur as normal intestinal flora of humans. An opportunistic pathogen, they are one of the common cause of urinary tract infections (UTIs) and are associated with infection-induced renal stones. Other infections caused by  Proteus species are pyogenic lesions, infection of ear, respiratory tract infections and nosocomial infections.  Proteus species have pili (fimbriae). Pili are associated with adhesive properties and, in some cases, are correlated with virulence. Laboratory Diagnosis & Identification: Sample: Depends on the nature of the disease/site of infections. Antigenic characteristics: o UTI - midstream urine  The bacilli possess thermostable, ‘O’ (somatic) and thermostable ‘H’ o Pyogenic lesions - pus aspirate or swab (flagellar) antigens, based upon which several serotypes have been  Sample should be collected in the sterile container recognized. maintaining aseptic conditions and should reach  Certain strains of Proteus vulgaris (OX-19, OX-2, and OX-K) produce laboratory within an hour of collections. O antigens that are shared by some rickettsiae. These Proteus Culture: The choice of the culture media used for the isolation of strains are used in an agglutination test (the Weil-Felix test) for the etiological agents depend on the nature of the specimen and serum antibodies produced against rickettsiae of the typhus and suspected pathogens. spotted fever groups. o Pus & urine sample - Blood Agar and MacConkey agar  Blood Agar plate - successive waves to form a thin filmy layer of General Properties: concentric circles (swarming).  Gram negative  Proteus do not swarm in the MacConkey agar medium - form  Non-spore-forming rods smooth, pale or colorless (NLF) colonies.  Facultative anaerobes  Urease positive (strong)  Oxidase test: Negative  Nitrates are reduced to nitrites  Ferments glucose but does not ferment lactose  Deaminates phenylalanine to phenylpyruvic acid Urease enzyme produced by Proteus species is thought to play a major role in the production of infectioninduced urinary stones. The ammonia produced after the breakdown of the urea results in struvite (magnesium  Swarming properties of Proteus presents problems in the diagnostic ammonium phosphate) stone formation. Recurrent urinary tract infections laboratory when mixed growth is present in which Proteus is one of with a urease-producing organism (mostly Proteus species) results in the isolates Several methods have been used to inhibit swarming. formation of staghorn calculi in the kidney. These are: #PadayonRMT | Page 19  MLS 3A o increasing the concentration of agar in the medium, raising it to 6% instead of 1-2%. PATHOGENICITY o incorporation of choral hydrate (1:500), sodium azide (1:500),  There is considerable evidence that Morganella species play a boric acid (1:1000) in the medium pathogenic role in urinary tract infections, particularly for those of o Using Cysteine Lactose Electrolyte Deficient (CLED) as a sole nosocomial origin. It is an opportunistic secondary invader rather medium instead of MacConkey Agar and Blood Agar for the than a primary pathogen at other sites and has been isolated from processing of urine samples. bacteremia, respiratory tract, and wound infections. Dienes Phenomenon MICROSCOPIC APPEARANCE o Proteus mirabilis is well known for its ability to differentiate into Gram Stain: Gram-negative. hyperflagellated, motile, and elongated swarmer cells that rapidly Morphology: Straight rods. spread over a surface. Size: 0.6-0.7 micrometers by 1.0-1.7 micrometers. o When two different strains of P. mirabilis swarm on the same agar Motility: Motile by peritrichous flagella, swarming does not occur. plate, a visible demarcation line with lower cell density forms at the Capsules: None. intersection, and this line is known as a Dienes line (after Louis Spores: None. Dienes, who described the phenomenon in 1946) BUT when two Other: Originally classified in the genus Proteus as Proteus morganii. identical isolates meet, the swarming edges merge without formation of a Dienes line. MACROSCOPIC APPEARANCE  Some Morganella strains appear hemolytic when cultured on Blood Agar, while others produce a reddish-brown pigmentation. METABOLIC PROPERTIES  Facultatively anaerobic  Chemoorganotrophic Differential Tests for the Commonly Isolated Proteus Species  Fermentative and respiratory type metabolism  Acid production from mannose, but gas is not produced RECOMMENDED MEDIA For culture:Tryptic Soy Agar (TSA), Blood Agar 5%. For selective isolation: MacConkey Agar, EMB Agar, Selenite Broth, Tetrathionate Broth The Genus Morganella For maintenance: Tryptic Soy Agar for short-term maintenance and  Morganella morganii subsp. morganii lyophilization for long-term preservation.  Morganella morganii subsp. Sibonii KEY BIOCHEMICAL REACTIONS: HABITAT POSITIVE REACTION:  Intestinal and feces pathogen of man, other mammals, and reptiles. o Methyl-Red #PadayonRMT | Page 20  MLS 3A o Ornithine-decarboxylase o Catalase MACROSCOPIC APPEARANCE o Urease  Relatively large, dull gray colonies; non-swarming. Providencia spp. o Indole usually appear colorless on enteric agars such as Eosin Methylene o Voges-Proskauer Blue (EMB) Agar, Hektoen Enteric (HE) Agar, and Salmonella Shigella NEGATIVE REACTION: (SS) Agar. o Simmons-Citrate METABOLIC PROPERTIES o Oxidase  Facultatively anaerobic. o H 2 S Produces acid from mannose.  Chemoorganotrophic IMVIC reaction: ++--  Fermentative and respiratory type metabolism The Genus Providencia RECOMMENDED MEDIA  Providencia alcalifaciens For culture: Tryptic Soy Agar or Blood Agar 5%.  Providencia heimbachae For selective isolation: Simmons Citrate Agar, MacConkey Agar,  Providencia rettgeri Tergitol Agar, or CHROM™ UTI.  Providencia rustigianii For maintenance: Tryptic Soy Agar or Blood Agar 5%. Brucella with  Providencia stuartii 20% Glycerol or Skim Milk for long-term storage at -70 degrees C. HABITAT Lyophilization may be used for preservation  The most common site of P. stuartii and P. rettgeri infections is the urinary tract of the catheterized or compromised patient. KEY BIOCHEMICAL REACTIONS Extraintestinal tracts (stool samples) of human beings and other POSITIVE REACTION: animals, in particular poultry, are also common sites of Providencia o Catalase spp. o Indole (except P. heimbachae ). o Citrate PATHOGENICITY o Methyl-Red  Strains of P. stuartii and P. rettgeri may produce wound and burn o (P. stuartii may be urease-positive, approximately 15%) infections. The rise in medical i