Assistant Prof. Dr. Ayad almakki Department of Clinical …pharmacy.uobasrah.edu.iq/images/stage_two/Medical... · 2018-12-28 · Enterobacteriaceae Common Medically important Enterobacteriaceae

Assistant Prof. Dr. Ayad almakki

Department of Clinical Laboratory Science

College of Pharmacy

2 nd stage

Medical microbiology I

University of Basra

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Classification- more than 15 different generaEnterobacteriaceae

• Escherichia

• Klebsiella

• Entrobacter

• Salmonella

• Citrobacter

• Shigella

• Serratia

• Hafnia

• Proteus

• Yersinia

• Erwinia

• Morganella

• Providencia

• Edwardsiella

• Pectinobacterium

Dr. Ayad almakki

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Enterobacteriaceae Common Medically important Enterobacteriaceae

Escherichia coli

Shigella sonnei , Shigella flexneri

Klebsiella pneumonia, Klebsiella oxytoca

Salmonella enterica

Citrobacter freundii , Citrobacter koseri

Enterobacter aerogenes, Enterobacter cloacae

Proteus mirabilis

Serratia marcescens

Morganella morganii

Yersinia pestis, Yersinia enterocolitica & Yersinia pseudotuberculosis

Dr. Ayad almakki Ref : Abbott ( 2007)

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Characteristic features of Enterobacteriaceae

All Enterobacteriaceae• Gram-Negative rods• Ferment glucose with acid production• Reduce nitrates into nitrites• Oxidase negative (used to determine if a bacterium produces certain cytochrome C oxidases)

Facultative anaerobic

Motile (peritrichous flagella) except Shigella & Klebsiella

Non-sporing

Many are normal inhabitant of the intestinal tract of man and other animals

Dr. Ayad almakki Ref : Farmer et al.,2007

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Some are enteric pathogens and others are urinary or respiratory tract pathogens

Fig Sites of infections with commonmembers of the enterobacteriaceaelisted in order of prevalence

Dr. Ayad almakkiRef : Wanger et al.,2007

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Grow on bile containing media ( e.g. MacConkey agar)

Enterobacteriaceae

Non-lactose fermenter (NLF)

Salmonella,Shigella,Proteus,Yersinia

Lactose fermenters (LF)

E.coli,Klebsiella,Enterobacter,Citrobacter

There are several selective and differential media used to isolate and distinguish between LF &NLF

The most important media (MacConkey agar ,Eosin Methylene Blue(EMB) agar , Salmonella Shigella (SS)agar and Triple sugar Iron (TSI) agar


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Differentiation between LF & NLF by growth on MacConkey agar

MacConkey agar

Contain

Neutral red

pH indicator

Lactose

Cause of

differential

Crystal violetBile salts

Inhibit growth of Gm+ve bacteria

Lactose

ferementers

Pink colonies

Lactose non

ferementers

colorless

colonies

Dr. Ayad almakki

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The heat-stable Lipopolysaccharide(LPS) is the majorcell wall antigen and consists of three component:

1- O polysaccharide (outermost somatic) is important forthe epidemiologic classification of strains within a species.

2- Polysaccharide core (common antigen) is important forclassifying an organism as a member of theEnterobacteriaceae

3- Lipid A is responsible for endotoxin activity, an importantvirulence factor.

Differentiation is based on biochemical reactions and differences in antigenic structure

Continued

Dr. Ayad almakki

Ref : Nataro et al., 2007

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Many Enterobacteriaceae also possess fimbriae (alsoreferred to as Pili) , which have been subdivided intotwo general classes:

A- Common fimbriae (chromosomally mediated) areimportant for the ability of bacteria to adhere to specifichost cell receptors

B- Sex or conjugative pili ( encoded on conjugativeplasmids ) are important to facilitate genetic transferbetween bacteria

The epidemiologic (serologic) classification of theEnterobacteriaceae is based on three major groups ofantigen :

1- Somatic O polysaccharides (O antigens) are present ineach genus and species, although cross-reactions betweenclosely related genera are common ( e.g. Salmonella withCitrobacter, Escherichia with Shigella)

2- K antigens in the capsule (type-specific polysaccharides)

3- H antigens are heat-labile flagellin proteins

O antigen

side chain

Dr. Ayad almakki


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Common Virulence factors Associated with Enterobacteriaceae1- Endotoxin

Is a virulence factor shared among aerobic and some anaerobic gram-negative bacteria . Theactivity of this toxin depends on the Lipid A component of LPS, which is released at cell lysis

2- Capsule

Encapsulated Enterobacteriaceae are protected from phagocytosis by the hydrophilic capsularantigens, which interfere with the binding of antibiodies to the bacteria and are poorimmunogens or activators of complement.

3- Antigenic phase Variation

The expression of the somatic O antigens, Capsular K antigens and flagellar H antigens is underthe genetic control of the orgamism. Each of these antigens can be alternately expressed or notexpressed(phase variation), a feature that protects the bacteria from antibody-mediated celldeath.

Continued

Dr. Ayad almakki Ref : Nataro et al., 2007

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4- Type III Secretion system

Is a molecular syringe consisting of approximately 20 proteins that facilitate transfer of bacterial virulence factorsinto the targeted host cells. In the absence of the type III secretion system, the bacteria have diminished virulence

5- Sequestration of growth factors

Iron is an important growth factor required by bacteria, but it is bound in heme proteins (e.g. hemoglobin ormyoglobin) or in iron-chelating proteins. The bacteria counteract the binding by producing their own competitivesiderophores or iron-chelating compound (e.g. enterobactin, aerobactin).Iron can also be released from host cellsby hemolysins produce by the bacteria.

6- Resistance to serum Killing

The bacterial capsule can protect the organism from serum killing as well as other factors that prevent the binding ofcomplement components to the bacteria and subsequent complement-mediated clearance.

7- Antimicrobial Resistance

As rapidly as new antibiotics are introduced, organisms can develop resistance to them. This resistance can beencoded on transferable plasmids and exchanged among species, genera, and even families of bacteria.


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Modes of infection

Water & food contamination ( Salmonella spp., Shigella spp.,Yersinia enterocolitica and Escherichia coli O157:H7)

Endogenous ( Urinary tract infection “UTI” , primary bacterial peritonitis, abdominal abscess)

Abnormal host colonization (nosocomial pneumonia)

Transfer between debilitated patients

Insect (flea) vector (unique for Yersinia pestis)

Dr. Ayad almakki

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A-Escherichia coli

There are six distinct groups of E. coli with different pathogenetic mechanisms

1- Enteropathogenic E.coli (EPEC)

2- Enterotoxigenic E.coli (ETEC)

3- Enterohemorrhagic (verotoxin-producing) E.coli (EHEC)

4- Enteroinvasive E.coli (EIEC)

5- Enteroaggregative E.coli (EAEC)

6- Diffuse-aggregative E.coli (DAEC)

Other infection with E.coli , Urinary tract infection, neonatal meningitis & Septicemia

Dr. Ayad almakki Ref : Qadri et al., 2005

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1-1 Site of Action: Small intestine

1-2 Disease:

Infant diarrhea in developing countries; watery diarrhea and vomiting , non-bloodystools.

1-3 Pathogensis:

Plasmid-mediated, with disruption of normal microvillus structure resulting inmalabsorption and diarrhea


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1-4 Pathogenic mechanism :

- EPEC do not appear to make any toxins

- They do produce bundle-forming pili (Bfp), intimin (an adhesin)and an associatedprotein (translocated initimin receptor, Tir). These virulence factors allow bacterialattachment to epithelial cells of the small intestine, leading to disruption of themicrovillus leading to diarrhea

Fig Electron micrograph ofEPEC adhering to the brushborder of intestinal mucosalcells with localizeddestruction of microvilli


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1-5 Epidemiology :

- EPEC strains belong to particular O serotypes cause sporadic cases and outbreaks ofinfection in babies and young children, importance in adults is less clear

1-6 Laboratory diagnosis:

- DNA-based assays for detection of attachment (virulence) factors



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-Isolate organisms from feces

-Determine serotype of several colonies with polyvalent antisera for known EPEC types

-Adhesion to tissue culture cells can be demonstrated by a fluorescence actin staining test


2-2 Disease:

Traveler’s diarrhea; infant diarrhea in developing countries; watery diarrhea,vomiting, cramps, nausea and low-grade fever

2-3 Pathogenesis:

Plasmid-mediated, heat-stable and/or heat labile enterotoxins that stimulatehypersecretion of fluids and electrolytes



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- ETEC possess colonization factors (fimbrial adhesins)

- These bind the bacteria to specific receptors on the cell membrane of the smallintestine. These organisms produce powerful plasmid-associated enterotoxins which arecharacterized as being either heat labile (LT) or heat stable (ST)

• Heat-labile enterotoxin LT-I is very similar in structure and mode of action to choleratoxin produced by V. cholera, and infections with strains producing LT-I can mimiccholera, particularly in young and malnourished children

• Heat-stable enterotoxin (STs) activates guanylate cyclase activity, causing an increasein cyclic guanosine monophosphate (cGMP), which results in increased fluid secretion.

Electron micrograph of ETEC ,showing pili necessary foradherence to mucosal epithelial cells.

Dr. Ayad almakki

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2-5 Epidemiology :

- Most important bacterial cause of diarrhea in children in developing countries

- Most common cause of traveler’s diarrhea

- Water contaminated by human or animal sewage may be an important sourcebacterial spread


- Immunoassays are commercially available for the identification of ETEC


Dr. Ayad almakki

Ref : Qadri et al., 2005

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-Isolate organisms from feces , food and water.

3-1 Site of Action: Large intestine

3-2 Clinical Features

Disease caused by EHEC ranges from mild , uncomplicated diarrhea tohemorrhagic colitis.Initially: watery diarrhea with abdominal pain, vomiting (in about 50% ofpatients), low grade fever.Bloody diarrhea & severe abdominal pain developed in 30% – 65% of patients

within 2 days of onset.Complete resolution of symptoms occurs in most untreated patients.However, hemolytic uremic syndrome (HUS) develop in about 5%-10% of infectedchildren younger than 10 years.



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The most common strain of EHEC is serotype O157:H7.After bacterial attachment to the mucosa of large intestine (by attaching –effacing mechanism) , EHEC would produce shiga toxins ( Stx-1, Stx-2, or both)with destruction of intestinal microvilli resulting in decreased absorption anddiarrhea.

3-3 Pathogenesis:

HUS characterized by:

Resolution of symptoms occurs in most patientsDeath can occur in 3%-5% Severe sequelae can occur in as many as 30% of HUS patients (renal impairment, hypertention, CNS manifestations)

-Acute renal failure-Thrombocytopenia- Microangiopathic hemolytic anemia



3-4 Pathogenic mechanism :- Stx-1 is essentially identical to the shiga toxin produced by Shigella dysentriae; Stx-2

has 60% homology.- EHEC strains with both Shiga toxins and attaching-effacing activity are more

pathogenic than strains producing only one Shiga toxin.

- HUS has been preferentially associated with the production of Stx-2, which hasbeen shown to destroy glomerular endothelial cells platelets activation andthrombin deposition decreased glomerular filtration acute renal failure.


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3-5 Epidemiology :

- Serotype O 157:H7 most important EHEC in human infections

- Outbreaks and sporadic cases occur worldwide



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- Person –to- Person spread can occur.

-The ingestion of fewer than 100 bacteria can produce disease

- Most infections are attributed to the consumption of undercooked ground beefor other meat products, water, unpasteurized milk or fruit juices, uncookedvegetables.


- Isolate organisms from feces , proportion of EHEC in fecal sample may be very low(often < 1% of E. coli colonies)


Dr. Ayad almakki

Ref : Qadri et al., 2005

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- Shiga toxin production and associated genes detected by biological, immunological andnucleic-acid-based assays

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- Usually sorbitol non-fermenters (Colonies colorless)



Sorbitol-fermenting (Pink)Non-fermenting (colorless)

4-1 Site of Action: Large intestine

4-2 Disease:Rare in developing and developed countries.Initial watery diarrhea; may progress to dysenteric form of disease consisting offever, abdominal cramps, and blood and leukocytes in stool.


Plasmid-mediated invasion and destruction of epithelial cells lining colon, thenbacteria destroy the phagocytic vacuole and moved within the cytoplasm toinvade adjacent epithelial cell.The process of epithelial cell destruction with inflammatory infiltration canprogress to colonic ulceration.



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4-5 Epidemiology :- Important cause of diarrhea in areas of poor hygiene

- Infections usually foodborne; no evidence of animal or environmental reservoir


-Cells or nucleic –acid-based assays for invasion associated genes


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- Isolate organisms from feces

- Test for enteroinvasive potential in tissue culture


5-2 Disease:

Infant diarrhea in developing and probably developing countries, traveler’sdiarrhea ; persistent watery diarrhea with vomiting, dehydration , and low-gradefever

5-3 Pathogenesis:

Plasmid-mediated aggregative adherence of rods with shortening of microvilli,mononuclear infiltration, and hemorrhage; decreased fluid absorption


Fig EAEC formstacked brick


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- EAEC derive their name from their characteristic attachment pattern to tissue culture cells

- The pattern is an aggregative or “stacked brick” formation. These organisms act in thesmall intestine to cause persistent diarrhea especially in children in developing countries.Their aggregative adherence ability is due to plasmid-associated fimbriae. EAEC alsoproduce heat-labile toxins but their role in diarrheal disease is uncertain.



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Dr. Ayad almakki

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5-5 Epidemiology :

- Characteristic attachment to tissue culture cells


- Tissue culture assays for aggregative adherence


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6- Diffuse-aggregative E.coli (DAEC)


- DAEC produce an alpha hemolysin and cytotoxic necrotizing factor 1

- They are also known as diffuse-adherent or cell-detaching E. coli . Their rolein diarrheal disease , especially in young children , is incompletely understoodand somewhat controversial , with some studies reporting no association.

6-2 Epidemiology :


- Cause diarrhea in children in developing countries

- Role of toxins uncertain

- Tissue culture assays for diffuse adherence


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B-Shigella

- Gram-negative ,Facultatively anaerobic rods and oxidase negative- Four species consisting of almost 50 O-antigen-based serogroupshave been described :

S. sonnei responsible for most infections in developed countries ; S.flexneri for infections indeveloping countries; S. dysenteriae for the most sever infections; and S. boydii not commonlyisolated

Biology

Virulence- Exotoxin (Shiga toxin) produced by S. dysenteriae disrupts protein synthesis andproduces endothelial damage

Note: Shiga toxin has one A subunit and five B subunits. The B subunits bind to a

host cell glycolipid (Gb3) and facilitate transfer of the A subunit into the cell


33 Fig S. sonnei colonymorphology on blood agar

Disease

B-Shigella

- Most common form of disease is gastroenteritis (shigellosis), an initial waterydiarrhea progressing within 1-2 days to abdominal cramps and tenesmus (with orwithout bloody stools); severe form of disease is caused by S. dysenteriae (bacterialdysentery); asymptomatic carriage develops in a small number of patients( reservoirfor future infections)

Pathogenesis

- Shigellae cause disease by invading and replicating in cells lining the colon. Shigellaspecies appear unable to attach to differentiated mucosal cells; rather, they firstattach to and invade the M cells located in peyer patches.


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Pathogenesis

- Shigellae survive phagocytosis by inducing programmed cell death (apoptosis). Thisprocess also leads to the release of IL-1β, resulting in the attraction ofpolymorphonuclear leukocytes into the infected tissues. This , in turn, destabilizes theintegrity of the intestinal wall and allows the bacteria to reach the deeper epithelialcells

B-Shigella

- Shiga toxin can mediate damage to the glomerular endothelial cells, resulting in renalfailure

- The type III secretion system mediates secretion of four proteins(IpaA,IpaB, IpaC &IpaD) into epithelial cells and macrophages.

Dr. Ayad almakkiRef : Nataro et al., 2007

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B-Shigella

The mechanism of Shigellosis in the colon Dr. Ayad almakki


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Epidemiology

B-Shigella

- Human are only reservoir for these bacteria

- Disease spread person to person by fecal-oral route

- Patients at highest risk for disease are young children in daycare centers, nurseries, andcustodial institutions; siblings and parents of these children ; male homosexuals

- Relatively few organisms can produce disease (highly infectious)

- Disease occurs worldwide with no seasonal incidence ( consistent with person-to-personspread involving a low inoculum)


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Treatment

B-Shigella

Antibiotic therapy shortens the course of symptomatic disease and fecal shedding

Treatment should be guided by in vitro susceptibility tests

Empiric therapy can be initiated with a fluoroquinolone or trimethoprim-sulfamethoxazole

Control

Hand washing

Proper disposal of soiled linens


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C-Klebsiella

- Gram-negative ,Facultatively anaerobic rods and oxidase negative

Biology

- Four species recognized:

K. pneumonia and K. oxytoca , which can cause community or hospital-acquired primary lobarpneumonia.

Virulence

- Capsule is responsible for mucoid appearance of isolated colonies and the enhancedvirulence of the organisms in vivo

K. rhinoscleromatis , cause of a granulomatous disease of the nose and K. ozaenae , cause ofchronic atrophic rhinitis.


39Fig Klebsiella pneumoniacolonies on MacConkeyagar

Disease- Most common form of disease is Pneumonia caused by Klebsiella spp. frequentlyinvolves the necrotic destruction of alveolar spaces, formation of cavities , and theproduction of blood-tinged sputum. These bacteria also cause wound , soft-tissue, andUTIs.

C-Klebsiella

Pathogenesis

- The organism formerly called Donovania granulomatis or K. granulomatis( granulomainguinal) , a granulomatous disease affecting the genitalia and inguinal area.

- Granuloma inguinal can be transmitted after repeated exposure through sexualintercourse or nonsexual trauma to the genitalia.

- After a prolonged incubation of weeks to months, subcutaneous nodules appear onthe genitalia or in the inguinal area.


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C-Klebsiella Pathogenesis

- The nodules subsequently break down, revealing one or more painlessgranulomatous lesions that can extend and coalesce into ulcers resembling syphiliticlesions.

Epidemiology

- Granuloma inguinale is a rare disease in the United States but is endemic in parts ofPapua New Guinea, the Caribbean, South America, Southern Africa, Vietnam andAustralia.


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Diagnosis

- Granuloma inguinale is diagnosis by microscopy ( a smearfrom the lesion stained with Wright’s or Giemsa stain ,Donovan bodies appear as clusters of blue- or black-stained)

C-Klebsiella

Treatment

- Tetracyclines or Co-trimoxazole


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C-Proteus

- Gram-negative ,Facultatively anaerobic rods , oxidase negative and highly motileand swarms on some media .

Biology

- Genus contains several species, of which two are of medical importance:

P. mirabilis and P. vulgaris

Virulence- O (somatic) and H (flagellar) antigens

Disease

- Urinary tract infection (e.g. bladder infection or cystitis; kidney infection orpyelonephritis); hospital-acquired wound infection and septicemia

Fig:Swarmingproteus onBlood agar

Dr. Ayad almakkiRef : Farmer et al., 2007

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C-Proteus

Pathogenesis

- P. mirabilis produces large quantities of urease, which splits urea into carbon dioxideand ammonia. This process raises the urine pH, precipitating magnesium and calciumin the form of struvite and apatite crystals, respectively , and results in the formationof renal (kidney) stones.

- The increased alkalinity of the urine is also toxic to the uroepithelium.

Treatment

- P. mirabilis is generally susceptible to most antibiotics apart from tetracycline andnitrofurantoin, but 10–20% of P. mirabilis strains are also resistant to first-generationcephalosporins and ampicillin.

- P. vulgaris commonly more resistant to antibacterials than P. mirabilisDr. Ayad almakki

Ref : Farmer et al., 2007

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Many of the members of the Enterobacteriaceae are part of the normal population of bacteria that colonize the human body.

Give three example of organisms that are normal flora in healthy individuals and an example of disease caused by each

organism. What condition leads to disease with each?

Dr. Ayad almakki

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46

Dr. Ayad almakki

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1- Kligler test (Triple Sugar Iron –TSI)

- TSI are useful in presumptive identification of gram-negative enteric bacteria,particularly in screening for intestinal pathogens.

- Lactose is present in a concentration 10 times that of glucose(1% Lactose and 0.1%Glucose) and Sucrose is also present in a 1% concentration.

- Ferrous sulfate and Sodium thiosulfate are added to detect the production ofhydrogen sulfide gas(H2S). Phenol red is used as the pH indicator, which is yellowbelow the pH of 6.8.

Dr. Ayad almakki Ref : Donald ( 2015)

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- Uninoculated medium is red because the pH is buffered at 7.4.

- If Glucose fermented , the Alkaline slant “red”/ Acid butt “yellow” K/A

- If Lactose and/or Sucrose fermentation , the Acid slant “yellow”/ Acid butt “yellow” A/A

- No fermentation, the Alkaline slant “red”/ Alkaline butt “red” K/K

- H2S production :Alkaline slant “red”/ Acid butt “yellow” , H2S in butt “Black”K/A,H2S Or Acid slant “yellow”/ Acid butt “yellow” H2S in butt “Black” A/A H2S


- Gas production : The production of gas results in the formation of bubbles orsplitting of the medium in the butt or complete displacement of the medium fromthe bottom of the tube.


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1 2 3 4 5


1- A/A gas 2- A/A H2S 3- K/A 4- K/A H2S 5- K/K


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Glucose metabolism and its metabolic products

2-Methyl Red Test (MR)

- Glucose metabolized via the Embden-Meyerhof pathway produces severalintermediate by-produce , including pyruvic.

- Bacteria are incubated in a broth medium containing glucose. The broth should beincubated 3 to 5 days at 35 °C. After incubation, approximately half the broth istransferred to a clean dry tube for the Voges-Proskauer (VP).

- If glucose is metabolized by the mixed acid fermentation pathway, stable acid endproducts are produced , which results in a low pH.

- A red color develops after addition of the pH indicator MR (positive).

- MR-negative cultures remain yellow after addition of the pH indicator (pH 6.0)


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2-Methyl Red Test (MR)

Uninoculated + -W

Glucose Pyruvic acid Mixed acid fermentation (pH 4.4)

Red color with methyl indicator


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3- Voges-Proskauer test (VP)

- In some bacteria, acids formed during fermentation can be metabolized further to 2,3-butanediol through the intermediate acetoin.

- After incubation,α-naphthol is added first as a catalyst or color intensifier. Next , 40%potassium hydroxide (KOH) or Sodium hydroxide (NaOH) is added, and the tube is gentlyshaken to increase oxygenation.

- Under these condition , acetoin is oxidized to diacetyl.

- Diacetyl in the presence of KOH and α-naphthol forms a red complex.

- The pH remain relatively neutral.

Note: Bacteria tend to be positive for either MR or VP but not both. Some bacteria are negative for

both tests


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Glucose Pyruvic acid Acetoin Diacetyl+ KOH+α-Naphthol Red complex

2,3- Butanediol

3- Voges-Proskauer test (VP)

+ -Uninoculated

Dr. Ayad almakki

Ref : Donald ( 2015)

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Miscellaneous Tests

4- Citrate Utilization

- The citrate test determines whether an organism can use sodium citrate as a solecarbon source. Simmons’ citrate medium is frequently used to determine citrateutilization.

- In addition to citrate, the test medium contains ammonium salts as the sole nitrogensource.

- Bacteria able to use citrate will use the ammonium salts, releasing ammonia

- The alkaline pH that results from use of the ammonium salts changes the pH indicator(bromthymol blue) in the medium from green to blue.


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4- Citrate Utilization

Uninoculated + -

Fig Citrate utilization test


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Miscellaneous Tests

5- Indole Production

- Indole is one of the degradation products of the amino acid tryptophan . Organismsthat possess the enzyme tryptophanase are capable of deaminating tryptophan withthe formation of the intermediate degradation products of indole, pyruvic acid, andammonia.

- Bacteria are inoculated into tryptophan or peptone broth.

- After inoculation, indole is then tested for by a colourmetric reaction with Kovac’sreagent ,containing para-dimethylaminobenzaldehyde (PDAB)

- Approximately 5 drops of Kovac’s reagent is added directly to the broth culture. Thetube is shaken , and if indole is present, a red color develops ( A red color indicates apositive reaction).


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5- Indole Production

+ -UninoculatedFig Indole broth

CH2-CH-COOH

NH2

CH3-C-COOH + Indol + NH3

tryptophan

tryptophanase Pyuvic acid Ammonia


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Miscellaneous Tests

6- Oxidase test

- The oxidase test determines the presence of the cytochrome oxidase system thatoxidizes reduced cytochrome with molecular oxygen

- The oxidase test is helpful in differentiating between the Enterobacteriaceae, whichare oxidace- negative, and the pseudomonads, which are oxidase-positive.

- The oxidase test is also useful in identifying Neisseria spp. which are oxidase-positive.

- Kovac’s oxidase test uses a 0.5% or 1% aqueous solution of tetramethyl-ρ-phenylenediamine dihydrochloride.

- A drop of the reagent is added to filter paper, and a wooden applicator stick is used torub a colony onto the moistened filter paper

- The development of a lavender color within 10-15 seconds is a positive reaction.


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Miscellaneous Tests

7- Urease test

- The urease test determines whether a microorganism can hydrolyze urea, releasing asufficient amount of ammonia to produce a color change by a pH indicator.

- Urease hydrolyzes urea to form ammonia, water , and CO2

- Different formulations of urea agar are available, but Christensen’s urea agar isgenerally preferred.

- The surface of the agar slant is inoculated but not stabbed.

- The medium contains phenol red as the pH indicator.

- The resulting alkaline pH from hydrolysis of urea is indicated by a bright pink color


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7- Urease test

- +


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O= C

NH2

NH2

2H2O+ 2NH3 + CO2 + H2O

Urea

pH= 6.8

Ammonia

pH=8.1

Urease

Summary of Biochemical Reactions of Enterobacteriaceae

ShigellaSalmnellaProteusKlebsiellaE.coliEnterobacter

Alkaline (Red)Alkaline (Red)Alkaline (Red)Acid (Yellow)Acid (Yellow)Acid (Yellow)Slant

TSIAcid (Yellow)Acid (Yellow)Acid (Yellow)Acid (Yellow)Acid (Yellow)Acid (Yellow)Butt

-±±±++Gas

-±+---H2S

--±±+-Indole

--+±--Urease

-±±+-+Simmon’s

citrate

+++-+-MR

---+-+VP


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Triple sugar iron (TSI) agar to determine glucose and lactose, or sucrose, utilization

utilization (sucrose in TSI only) and hydrogen sulfide production

Methyl red and vogas-Proskauer tests to determine end products of glucose fermentation

Indole test to determine if indole is formed from tryptophan by tryptophanase

Urease test to determine hydrolysis of urea

Simmon’s citrate to determine if citrate can be used as the sole carbon source

Dr. Ayad almakki

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Documents

Assistant Prof. Dr. Ayad almakki Department of Clinical …pharmacy.uobasrah.edu.iq/images/stage_two/Medical... · 2018-12-28 · Enterobacteriaceae Common Medically important Enterobacteriaceae