AM17-4 Identificationpdf PDF

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Summary

Lectures of Analytical Microbiology...

Description

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Accurate and definitive microorganism identification is essential for: –

correct disease diagnosis,

– treatment of infection and

– trace-back of disease outbreaks associated with microbial infections.

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Bacterial identification is used in a wide variety of applications including: – microbial forensics, criminal investigations – Bio-terrorism threats – environmental studies – Food and pharmaceuticals industries • Desirable organisms • Undesirable organisms – Spoilage – Pathogens

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Phenotypic – Morphology, staining characteristics – Metabolism • Overall, e.g. Aerobic vs anaerobic • Metabolic pathways • enzyme activities

– Antigenic / surface markers • Immunological methods • Phage typing • Protein electrophoresis, amino acid sequencing

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Genotypic – Mostly not initial ID: usually strain ID – Nucleic acid sequences • Gene probes, • amplification and/or analyses • sequencing

• Initially based on morphological differences – Microscopy – Differential stains • Grams

• Growth characteristics – Simple media – Aerobic /anaerobic – Differential media • Bile • Blood • etcetera

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• Conventional Tests – Most look for enzyme based reactions • Single enzymes • Pathways • Some overall metabolism

• Commercial Identification systems – Kits – Automation – Computer aided Identification – Linked to databases and reporting software

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• Requirements for growth – Metabolic • Citrate utilisation – Environmental • (anaerobes) • Metabolic pathways – Carbohydrate utilisation – Amino acid utilisation • Single substrate utilisation – Carbohydrate ( e.g. Sugars) – Urea • Other characteristics – Motility – Nitrate reduction

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• Several tests are commonly used to determine the presence of a single enzyme. – Some rapid results can use existing cultures. – Some need a specific culture media

• Some tests are easy to perform and interpret and often play a key role in the identification scheme.

• Most single enzyme tests do not yield sufficient information to provide species identification, – used extensively to determine which subsequent identification steps should be followed. – For example, the catalase test can provide pivotal information and is commonly used in schemes for grampositive identifications

• Some single enzyme tests are confirmatory tests after other identification tests – Quick confirmation of the identity of important or target organisms

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The enzyme catalase

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2H2O2  22HO + 02 direct analysis of a bacterial culture

rapid bubbles when bacterial growth is mixed with a hydrogen peroxide is (+) No or weak effervescence is (-). • Catalase test key to the ID of many gram-positive organisms: – staphylococci are catalase-positive, whereas streptococci and enterococci are negative •

– Listeria monocytogenes and corynebacteria are (catalasepositive) most other gram-positive, non-spore-forming bacilli are negative Hint : care needed when testing on blood agar. Also putting 2H2O2 on Blood agar. Why?

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The oxidase test is a quick and simple 5 minute test done directly from a bacterial colony on a blood agar plate that helps differentiate Gram-negative rods. • Some bacteria possess a cytochrome oxidase system that facilitates the utilization of oxygen as a final hydrogen acceptor in reducing molecular oxygen to hydrogen peroxide. • The oxidase strips or swabs contain various reagents which support a series of reactions that lead to a final oxidase reaction and a purple color. • Oxidase negative organisms cause little or no color change. •

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• The enzyme tryptophanase degrades the amino acid tryptophan into pyruvic acid, ammonia and indole. • Indole reacts with the test reagent to give a blue green result. • The test is used in a number of identification schemes and is especially useful in identifying E. coli – Spot test used to confirm E. coli in urine analyses

• Tests for the enzyme urease which hydrolyses urea into ammonia and CO2 • The ammonia give an alkali reaction in the medium • + Proteus vulgaris + Helicobacter pylori • - E. coli

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• Differentiating between pathogenic (S. aureus) and nonpathogenic strains of Staphylococcus. • Defence mechanism by clotting the areas of plasma around them, to resist phagocytosis by the host's immune system. • sample is usually inoculated into 0.5 ml of rabbit plasma and incubated at 37C for one to four hours. • A positive test is denoted by a clot formation in the test tube after the allotted time.

• Determines whether organism can use citrate as the sole carbon source for growth, and usually ammonia as the only nitrogen source.

+ -

Klebsiella sp. E. coli

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• Uses semi solid media and an indicator of growth such as tetrazolium • One of many methods: also hanging drop using microscopy • Examples

+ E. coli

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Klebsiella sp. + Listeria; characteristic umbrella shape

• Most pathways are common • Variable pathways are often related to organisms versatility and adaptations to their environment • The extent of metabolic diversity is often a good indicator of genetic diversity

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• Mixed acid fermentation: – ethanol +complex mixture of acids: acetic, lactic, succinic, and formic acids – This pattern is seen in Escherichia, Salmonella, Proteus, and other genera.

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• Butanediol fermentation pyruvate  acetoin, which is then reduced to 2,3-butanediol with NADH. – ethanol is also produced, plus smaller amounts of mixed acids. – characteristic of Enterobacter, Serratia, Erwinia, and some species of Bacillus

• 1. The Voges-Proskauer test detects acetoin a precursor to butanediol – positive with butanediol fermenters but not with mixed acid fermenters. – The Voges-Proskauer (VP) test is used by both the Enterotube II and API 20E microbial identification systems to identify enteric bacteria.

• 2. Mixed acid fermenters produce 4 times more acidic than neutral products , therefore pH drops more. This is the basis of the Methyl Red test. – positive when pH drops below 4.4 and the color of the indicator changes from yellow to red.

• 3. Alternate method is to detect gases produced – CO2 = H2 in mixed acid fermentation. – Butanediol fermenters [CO2/H2 ratio is 5: 1.

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• Tests whether the isolate has a mixed acid fermentation Three drops of methyl red indicator solution were pattern from added to each tube without shaking or mixing. A. glucose (positive Escherichia coli - E. coli is Methyl Red showing positive for mixed acid a low pH) or neutral production. B. Enterobacter cloacae is negative. (Methyl products such as red is red below pH 4.8 and acetoin yellow above pH 6.0) VP test 1. E. coli negative 2. Enterobacter positive

Scheme to identify Staphylococcus species

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1. Selection and inoculation of tests – Number and type of tests selected depend

on type of organism to be identified, clinical significance of isolates, and availability of reliable methods – Identification systems must be inoculated with pure cultures

2. Incubation for substrate utilization – Duration depends on whether bacterial multiplication is or is not required for substrate utilization (i.e., growth-based test vs. a non-growth-based test)

3. Detection of metabolic activity (substrate utilization) – Colorimetry, fluorescence, or turbidity are used to detect products – Detection is done visually or with the aid of various photometers

4. Analysis of metabolic profiles – Involves conversion of substrate utilization profile to a numeric code – Computer-assisted comparison of numeric code with extensive taxonomic data base provides most likely identification of the bacterial isolate – For certain organisms for which identification is based on a few tests, extensive testing and analysis are not routinely needed

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The number and types of tests that are selected depends on: • Type of bacteria to be identified – Organisms can have features that relatively few tests are required to establish identity • e.g. Staphylococcus aureus is the only: » gram-positive coccus that occurs microscopically in clusters, » Is catalase-positive,

» And produces produces coagulase » Therefore, identification usually requires only two tests coupled with colony and microscopic morphology.

– In contrast, identification of most clinically relevant gram-negative bacilli, such as those of the Enterobacteriaceae family, requires establishing metabolic profiles often involving 20 or more tests.

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Clinical significance of the bacterial isolate – The number of tests actually inoculated may depend on the clinical significance of an isolate. • e.g.if a Gram negative rod is mixed with several other bacterial species in a urine it is likely to be a contaminant. Multiple tests to establish species identity are not warranted should not routinely be performed. • However, if this organism is isolated in pure culture from cerebrospinal fluid full range of tests for identification would be justified.

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Availability of reliable testing methods – Unusual organisms: often require a wider variety of tests, • immunocompromised patients – Organ transplants; HIV; radio/chemotherapy

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• The time required to obtain ID depends on incubation time needed before the test result is available. • Incubation time depends on whether the test is measuring: – metabolic activity that requires bacterial growth or – measuring the presence of a particular enzyme or cellular product that can be detected without the need for bacterial growth.

• Conventional Identification – The generation time for most clinically relevant bacteria is 20 to 30 minutes, growth-based tests usually require hours of incubation before the presence of an end product can be measured. – Many conventional identification schemes require 18 to 24 hours of incubation, or longer, before the tests can be accurately interpreted.

• There is a need for simpler cheaper and faster ID hence the development of newer, quicker ID’s

• Rapid Identification: in diagnostic bacteriology the term rapid is relative. – In some instances a rapid method is one that provides a result the same day that the test was inoculated. – Alternatively, the definition may be more precise whereby "rapid" is only used to describe tests that provide results within 4 hours of inoculation.

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• Two general approaches have been developed to obtain more rapid identification results. – One has been to vary the conventional testing approach by decreasing the test substrate medium volume and increasing the concentration of bacteria in the inoculum. • Several conventional methods, such as carbohydrate fermentation use this strategy for more rapid results.

– Secondly use substrates that can measure enzyme activity without extra growth e.g. catalase and oxidase tests

• Accuracy of ID schemes depends on reliably detecting whether an isolate has utilized the substrates within the tests. • The sensitivity and strength of the detection signal can also contribute to how rapidly results are available. • No matter how quickly an organism may metabolise a particular substrate, if the end products are slowly or weakly detected, the ultimate production of results will still be "slow." – Detection strategies for determining the end products of different metabolic pathways use one of the following: • colorimetry • turbidity • fluorescence

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Colorimetry •

Many identification systems measure color change to detect the presence of metabolic end products. – Most frequently the color change is produced by pH indicators in the media. – An alternative to the use of pH indicator is the oxidation-reduction potential indicator tetrazolium violet. • Organisms are inoculated into wells containing a single, utilizable carbon source. Metabolism of the substrate generates electrons that reduce the tetrazolium violet, resulting in production of a purple color (positive reaction) that can be spectrophotometically detected.

– In a third approach, the substrate themselves may be chromogenic so that when they are "broken down" by the organism a colour is produced.

• Fluorescence: There are two basic strategies for using fluorescence to measure metabolic activity. – In one approach, substrate-fluorophore complexes are used. If a bacterial isolate processes the substrate, the fluorophore is released and becomes fluorescent – Alternatively, pH changes resulting from metabolic activity can be measured by changes in fluorescence of certain fluorophore markers.

• Turbidity – Less commonly used for bacterial identifications – Widespread application for determining inhibitors, including antimicrobial agents – Detecting bacteria/yeast present in certain clinical specimens.

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• The metabolic profile obtained of an organisms is the

phenotypic fingerprint, or signature, of that organism. – Typically, the profile is recorded as a series of pluses (+) and minuses (-) for each test.

• Databases for identification – Established by the commercial companies selling identification systems – The pluses and minuses converted to a numerical value – The identification only applies to the test system used (with some exceptions)

• Databases tend to be of “like groups”, such as gram positive rods, or yeasts. – Most rely on supplementary tests, such as gram stains.

% positive reactions for 100 strains Organism

Lactose

Sucrose

Indole

Ornithine

Escherichia

91

49

99

63

Shigella

1

1

38

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Probability that unknown strain of known genus will give a result

Organism Unknown

Escherichia Shigella

Lactose +

Sucrose +

Indole +

Ornithine +

0.91 0.01

0.49 0.01

0.99 0.38

0.63 0.20

Identification schemes are based on probabilities derived from the cumulative probabilities of each test.

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Commercially available identification systems have largely replaced compilations of conventional test media and substrates prepared in-house for bacterial identification. – Increased laboratory workloads make it economic to use the commercial systems

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Simple tests economize by doing multiple tests on one well e.g. indole and nitrate, or motility.

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Inoculation can be simplified eg. One inoculum for system

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Some systems enable the user to specify what substrates and tests are incorporated

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Can be safety benefits, due to less handling

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Can reduce human error

Product

Scope

Format

Time

Detection

Analyses

API bioMerieux

wide

Strip wells

2-72

Manual Color, Fluo, Turb

Computer codebook

Oxoid Microbac

wide

Microtitre

18-48

Manual

Computer codebook

BBL Enterotube

Narrow

Strip chambers

18-48

Manual Colorimeter

Codebook

GN-GP Biolog

broad

Microtitre plate

4-24

Auto Colorimeter

Computer directed

Rap-ID (REMEL)

narrow

Strip chambers

4

Manual Colorimeter

Computer codebook

Microscan

wide

Microtitre plate

2-24

Auto,Manual Color, Fluo,

Computer automated

Vitek bioMerieux

wide

Cards, mini wells

2-18

Auto Colorimeter

Computer automated

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• Uses 96 Well Format/plate reader • A variety of different products for bacteria fungi environmental profiling • Most tests are based on:

– Utilisation of different classes of carbon sources such as sugars, sugar alcohols sugar acids, carboxylic acids etcetera

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– In addition GEN III – Determines In addition GEN III determines other important physiological properties such as pH, salt, and lactic acid tolerance ,reducing power, chemical sensitivity

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• Total of 2000+ species covering, veterinary organisms, environmental isolates, plant pathogens, human pathogens and food isolates can be identified. • Bacteria 1350+ – Aerobic • Gram (-)

42%

• Gram (+)

28%

– Anaerobic • All

30%

• Yeast 267 • Filamentous Fungi 618

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• 17 different products covering Gm+, Gmbacteria, anaerobic bacteria, and yeasts

Enterobacteriaceae

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• Gram Negative nonfermenting bacteria • Substrate utilisation tested by turbidity

• Based on strips of wells • 12 or 24 biochemical tests per sample • Gram Negative rods – Enterbacteriaceae; Oxidase negative, nitrate-positive glucose fermenters comprising 15 genera ) • Acinetobacter spp. Shigella , Enterbacter, Klebsiella, Esherichia ,Yersinia , Proteus Salmonella

– Oxidase positives nitrate-negative, and glucosenonfermenters • Pseudomonas, Moraxella, Burkholderia , Alcaligenes, Vibrio

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• Microbact™ TM 12L Listeria Identification System – The Microbact™ Listeria 12L System has 12 wells, 11 wells are carbohydrate utilisation tests. The final well is a rapid micro-haemolysis test, as recommended by recognised international standard methods.

• Microbact™ TM Staphylococcal 12S Identification System – The Microbact™ Staphylococcal 12S system uses a combination of sugar utilisation and colorimetric enzyme detection substrates in a test strip format for the identification of 22 clinically significant staphylococcal species, including both coagulase-negative and coagulase-positive species.

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Similar to API but uses chromogenic reagents to test for enzyme activity rather than for growth

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Results in 4hrs

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Kits for Medically important species ( No. Species) – Anaerobes (90)

Gm(+) rods (50)

– Enterobacteriaceae (70)

Yeast ( 40)

– Gm(-) non Enterobacteriaceae (70)

Streptococci (30)

– Staphylococci (40)

Fastidious Gm (-) (30)

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UTI kit IDs 12 most common organisms in 2hrs

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Easy to inoculate; one tray inoculates all wells.

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No need for oil overlays or supplementary reagents

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Integrated automated system of inoculation, detection and results, in an average 5-8hrs.

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Results monitored every 15mins

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Identification and antibiotic susceptibility test (AST) cards – Gram negative bacteria – Gram positive bacteria – Yeast

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Identification – Neisseria, Haemophilus and other fastidious Gram negative bacteria identification – Anaerobic bacteria and coryneform bac...