Identification of Bacterial Pathogens PDF

Identification of Bacterial Pathogens  Identification of pathogens is critical Use appropriate treatments Antibiotics don’t work on all bacteria Many bacteria are now drug-resistant Proper choice of antibiotics necessary Required for proper prognosis Streptococcal...

Identification of Bacterial Pathogens  Identification of pathogens is critical Use appropriate treatments Antibiotics don’t work on all bacteria Many bacteria are now drug-resistant Proper choice of antibiotics necessary Required for proper prognosis Streptococcal pharyngitis might appear like a mild infection Could cause serious heart, kidney complications Track spread of disease Allows faster treatment of others infected Allows identification of cause of infection  Identification of Microorganisms For many students and professionals the most pressing topic in microbiology is how to identify unknown microrganisms. Why is this important? Labs can grow, isolate and identify most routinely encountered bacteria within 48 hrs of sampling. The methods microbiologist use fall into three categories: ♣Phenotypic- morphology (micro and macroscopic)  Microbe TheIdentification successful identification of microbe depends on: ♥Using the proper aseptic techniques. ♥ Correctly obtaining the specimen. ♥ Correctly handling the specimen ♥ Quickly transporting the specimen to the lab. ♥ Once the specimen reaches the lab  Specimen Collection Successful identification depends on how the specimen is collected, handled and stored. It is important that general aseptic procedures be used including sterile sample containers and sampling methods to prevent contamination of the specimen. E.g. Throat and nasopharyngeal swabs should not touch the cheek, tongue or salvia. What other precautions must be taken when collecting specimens? Specim en Collect ion  Microbe Identification Identification measures include: ♣ Microscopy (staining) ♣ growth on enrichment, selective, differential or characteristic media ♣ specimen biochemical test (rapid test methods) ♣ immunological techniques ♣ molecular (genotypic) methods. After the microbe is identified for clinical samples it is used in susceptibility tests to find which method of control is most effective.  Phenotypic Methods ‘Old fashioned’ methods via biochemical, serological and morphological are still used to identify many microorganisms. Phenotypic Methods Microscopic Morphology include a combination of cell shape, size, Gram stain, acid fast rxn, special structures e.g. endospores, granule and capsule can be used to give an initial putative identification. PHENOTYPIC METHODS Macroscopic morphology are traits that can be accessed with the naked eye such as: Appearance of colony-size, shape, color. Pigment Speed of growth  COLONY MORPHOLO GY Form- shape of colony color, surface, texture and size Elevation-side view Edge- margin COLONY MORPHOLOGY Phenotypic Methods -microscopic appearance Microscopic Morphology include a combination of Cell shape Size Gram stain Acid fast reaction Special structures e.g. Endospores, Granules and Capsules can be used to give an initial putative identification. CELL SHAPE BACTERIAL MORPHOLOGY  Microscopy Magnification – enhancement of size using ocular and objective lenses. Ocular: eyepiece (10X) Objective: 4X – 100X – allows for visualization of bacteria, fungi, and parasites, not viruses Resolution – ability to distinguish two objects as distinct – resolving power is closest distance between two objects – immersion oil is added when using 100X objective to prevent light scatter Contrast – use stains to enhance visualization; allow organism to stand out from background Gram Sometimes highly suggestive stain of a particular microorganism e.g., Gram-negative rods in ♀ urine E. coli UTI e.g., Gram-positive encapsulated diplococci and numerous white blood cells in sputum Streptococcus pneumoniae Sometimes enough for complete diagnosis e.g., Gram-negative diplococci clustered in white blood cells of male urethral secretions Neisseria gonorrhoeae Gm+ve cocci & Gm- ve bacilli  Special  Somestains microbes have unique characteristics that can be detected with special staining procedures  e.g., Mycobacterium species possess cell walls with a high lipid content  Acid-fast stain on sputum is diagnostic for tuberculosis Culture and isolation of bacteria Principles of Cultivation – Nutritional requirements General concepts – non-fastidious: simple requirements for growth – fastidious: complex, unusual, or unique requirements for growth Phases of growth media – solid  agar; boil to dissolve, solidifies at 50ºC – liquid, broth  Media classifications and functions –Enrichment used to enhance growth of specific organisms –Selective contains agents that inhibit the growth of all organisms except that being sought (dyes, bile salts, alcohols, acids, antibiotics) –Differential contains factor(s) that allow certain organisms to exhibit different metabolic characteristics  According to Enriched Use Medium – broth or solid, contains rich supply of special nutrients that promotes growth of a particular organism while not promoting growth of other microbes that may be  Types of media – Brain-heart infusion nutritionally rich supportive media used in broths, blood culture systems and susceptibility testing – Sheep blood agar supportive media containing 5% sheep blood for visualization of hemolysis – Chocolate agar same as sheep blood agar except blood has been “chocolatized” RBCs lysed by heating; releases X (hemin) and V (NAD) factors for Neisseria and Haemophilus – MacConkey agar selective for Gram-negative rods (GNRs) because of crystal violet and bile salts; differential  According to Use Selective Medium - contains inhibitors that discourage the growth of certain organisms & enhances the growth of the microbe sought (e.g. Salmonella  According to Use Differential Medium - contains dyes, indicators or other constituents that give colonies of particular organisms distinctive and easily recognizable characteristics Hemolysis a guiding factor Hemolysis a guiding factor  Enriched Media Chocolate agar, a medium that gets brown from heated blood. Used for isolation of N. gonorrhea. Blood agar plate with bacteria from human throat. This media differentiates among different colonies by appearance 47 Testing for Bacitracin and Optochin Sensitivity Mac Conkey Agar a Minimal differentiating Medium 5 0 MacConkey Agar General vs Selective Media Salmonella-Shigella Agar Differential Media Differential media grow several types of organisms and display visible differences among organisms. Differences may show up as colony size, media colour, gas bubble formation and  Types of artificial media – Hektoen enteric agar contains bile salts and dyes (bromothymol blue and acid fuchsin) to inhibit non-pathogenic GNRs; non pathogens ferment lactose changing BTB to orange; pathogens Salmonella and Shigella are clear; ferric ammonium citrate detects H2S production of Salmonella (black colonies) – Columbia colistin-nalidixic acid (CNA) agar Columbia agar base, sheep blood, colistin and nalidixic acid; selective isolation of gram-positive cocci  Types of artificial media – Thayer-Martin agar Chocolate agar with antibiotics (colistin inhibits gram neg, vancomycin inhibits gram pos, nystatin inhibits yeast); for N. gonorrhoeae and N. meningitidis; Martin- Lewis has similar function but different antibiotics  Devices for cultivation of microorganisms from clinical specimens Jars for anaerobic and microaerophil atmosphere Thermostat for aerobic cultivation СО2 thermostat for cultivation of microaerophils and tissue cultures  Phenotypic Methods- Biochemical Tests The microbe is cultured in a media with a special substrate and tested for an end product. Prominent biochemical tests include: Enzymes Carbohydrate fermentation Acid production Gas production Sensitivity to drugs  Phenotypic Methods- Biochemicals…. Enzymes  Catalase test  Oxidase test  Urease test  Coagulase test  Phenotypic Methods- Biochemicals….  CATALASE TEST  Catalase is present in most aerobic and facultative anaerobic bacteria (except streptococcus spp).  Hydrogen peroxide forms as one of the oxidative end product of aerobic carbohydrate metabolism.  If this is allowed to accumulate in the bacterial cells it becomes lethal to the bacteria  Catalase thus helps in converting H2o2 to H2o and o2  The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide and the rapid effervescence of O2 bubbles occurs. picture s  Catalase test…. Positive Micrococcus Staphylococcus Negative Bacillus Streptococcus Listeria sp Clostridium monocytogenes Enterobacteriacae Gonococcus & Meningococcus Vibriocholerae Pseudo/Aero/  Phenotypic Methods OXIDASE TEST This test depends on the presence of cytochrome oxidase in bacteria Procedure- Place a piece of filter paper in petri dish and add 3 drops of freshly prepared oxidase reagent (1% solution of tetramethyl-p- phenylene diamine) Using a sterile glass rod, remove a colony of test organisms from a culture plate and smear it on the filter paper Oxidase positive organisms give blue/ dark purple color within 5-10 seconds, and in oxidase negative organisms, color does not change. Oxidase test ….. Positive Negative Pseudomonas Enterobacteriace spp. ae Aeromonas spp. Acenitobactoer Vibrio spp. spp. Alcaligenes spp. Neisseria spp. Haemophilus sps Oxidase picture Phenotypic Methods Coagulase test  This test is used to differentiate Staphylococcus aureus (positive) from coagulase negative Staphylococci  When a bacterial suspension is mixed with plasma, this enzyme causes alteration in fibrinogen.  leading to precipitation on the staphylococcal cells, causing the cells to clump.  Slide test: Positive when there is Macroscopic clumping in 10 seconds or less in a plasma drop and no clumping in a saline or water drop.  Tube test: Positive when there is a Clot of any size  picture s Tube test: Coagulase Positive : Staphylococcus aureus Coagulase -Positive: Clot of any size (a) negative: Staphylococcus epidermidis -Negative: No clot (b) Phenotypic Methods- Biochemicals…. Enzymes Urease test Some bacteria produce urease an enzyme that hydrolyzes urea into ammonia and carbon dioxide. The test for urease production relies on the fact that the ammonia produced upon hydroysis is alkaline. The test organism is inoculated into a urea broth that contains phenol red, a pH indicator, and has a pH of 6.8. At this pH phenol red is salmon color. However, when the pH rises above 8.1 phenol red turns a cerise (hot pink) color. The urease test is useful for differentiating Salmonella which is urease negative, from Proteus which is urease positive. Urease Test Phenotypic Methods Fermentation of sugars and Gas production Triple Sugar Iron Agar (TSI) test  TSI agar is used to determine whether a gram negative rod utilizes glucose and lactose or sucrose fermentatively and forms hydrogen sulphide (H2S).  TSI contains 10 parts lactose: 10 parts sucrose: 1 part glucose and peptone. Phenol red and ferrous sulphate serves as indicators of acidification and H2S formation, respectively.  The formation of CO2 and H2 is indicated by the presence of bubbles or cracks in the agar or by separation of the agar from the sides or bottom of the tube.  The production of H2S requires an acidic environment and is Results interpretation:  Alkaline slant/no change in the butt (K/NC) = Glucose, lactose and sucrose non-utilizer (alkaline slant/alkaline butt)  Alkaline slant/acid butt (K/A) = Glucose fermentation only.  Acid slant/acid butt (A/A), with gas production = Glucose, sucrose, and/or lactose fermenter.  Alkaline slant/acid butt (K/A), H2S production = Glucose fermentation only.  E.g:  A/A, with gas: E. coli  K/A, H2S: Salmonella typhi  K/NC: Pseudomonas aeruginosa TSI results A/A, with gas: E. coli K/A, H2S: Salmonella typhi K/NC: Pseudomonas aeruginosa  SIM- SULFIDE,INDOLE,  ThisMOTILITY is a differential medium. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar (be motile). SIM is commonly used to differentiate members of Enterobacteriaceae.  If sulfide is produced, a black color forms in the medium. Proteus mirabilis is positive for H2S production.  Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole.  Indole reacts with added Kovac’s reagent to form rosindole dye which is red in color (indole +).  Escherichia coli is indole positive.  SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile then the growth will radiate from the stab mark and make the entire tube appear turbid.  Pseudomonas aeruginosa and Proteus mirabilis are motile. SIM Test  MOTILITY  If bacteria is motile, there will be growth going out away from the stab line, and test is positive.  If bacteria is not motile, there will only be growth along the stab line.  A colored indicator can be used to make the results easier to see. CITRATE  The citrate test is commonly employed as part of a group of tests distinguish between members of the Enterobacteriaceae family based on their metabolic by- products.  The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source.  Simmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6.  Bromthymol blue is yellow at acidic pH's (around 6), and gradually changes to blue at more alkaline pH's (around 7.6).  Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color.  Growth of bacteria in the media leads to development of a blue color (positive citrate).  Enterobacter and Klebsiella are citrate positive while E.coli is negative. Citrate Test  Rapid Rapid test:Tests a biochemical system for the identification of Enterobacteriaceae and other Gram –ve bacteria. It consist of plastic strips with 20 μl of dehydrated biochemical substrates used to detect biochemical characteristics. The biochemical substrates are inoculated with pure cultures and suspended in physiological saline. After 5 hrs-overnight the 20 tests are converted to 7-9  Methods for identification of the bacterial species  Biochemical identification - Determination of the metabolic characteristics of different bacteria Pannels for biochemical identification  Rapid Tests positiv e negati ve ONPG (β galactosidase); ADH (arginine dihydrolase); LDC (lysine decarboxylase); ODC (ornithine decarboxylase); CIT (citrate utilization); H2S (hydrogen disulphide production); URE (urease); TDA ( tryptophan deaminase); IND (indole production); VP (Voges Proskauer test for acetoin); GEL ( gelatin liquefaction); the fermentation of glucose (GLU), mannitol (MAN), inositol (INO), sorbitol (SOR), rhamnose (RHA), sucrose (SAC); Melibiose (MEL), amygdalin (AMY), and arabinose (ARA); and OXI (oxidase). Instrumentation, Automation and Computer Analysis In recent years, new diagnostic systems have been developed. Instrumentation, automation, and computer analysis has been applied for identification of bacteria. Bacterial families such as Enterobacteriaceae or others have a great diversity of biochemical activity and their identification is often difficult and time-consuming procedure.  Instrumentation, Automation and Computer Analysis The new diagnostic identification kits and systems are based on biochemical characteristics of cultures, but usually use micromethods for the biochemical identification of bacteria. They are based on commercial identification kits of miniaturized reaction cuvettes with biochemical tests. The color reactions of the isolate with the substrates in the tests of the identification kit are red visually or by automated systems. Even using the most reach database, the final diagnosis may need additional testing of biochemical and cultural characteristics of the isolate, which is due to the phenotypic variations of bacterial species. Automated kits and systems for diagnostic identification of microorganisms Mini API Phoenix BD  Immunological Methods Immunological methods involve the interaction of a microbial antigen with an antibody (produced by the host immune system). Testing for microbial antigen or the production of antibodies is often easier than test for the microbe itself. Lab kits based on this technique are available for the identification of many microorganisms Immunological Methods for Identification and Detection of Microorganisms’ Antigens in the Pure Culture or in the Specimen Bacteria possess many highly specific structures that may be antigenic (capsular polysaccharides, flagellar proteins, exotoxins, and several cell wall components. These antigens, which present on whole bacteria or are at free state as bacterial exotoxins may be demonstrated with specific antigen-antibody reactions in vitro. Using the antibodies of known specificity bacterial identification can be made by formation of antigen-antibody complex.   Immunological Methods for Identification and Detection of Microorganisms’ Antigens (determination of species, serogroup, serotype) are:  Precipitation  Agglutination  Immunofluorescention  ЕLISA Slide agglutination for determination of salmonella serogroup  Precipitation Tests Precipitation is an antigen-antibody reaction, in which thousands of molecules of soluble antigen and IgG, IgM or IgA antibodies cross-link to form visible aggregates known as precipitates. e.g., - streptococcal group antigens from Lancefield classification, diphtheria toxin Agglutination Tests Agglutination is an antigen-antibody reaction when particulate antigen is combined with specific and IgG, IgM or IgA antibody cross-link to form visible aggregates known as agglutinates. E.g. Salmonella strain, Influenza virus. Genotypic/ Molecular  Genotypic methods involve Methods examining the genetic material of the organisms and has revolutionised bacterial identification and classification.  Genotypic methods include PCR (RT- PCR, RAPD-PCR), use of nucleic acid probes, RFLP and plasmid fingerprinting.  Genotypic techniques are becoming Challenges in Bacterial Identification Traditional methods of bacterial identification rely on phenotypic identification of the causative organism These methods of bacterial identification suffer from two major drawbacks. They can be used only for organisms that can be cultivated in vitro. Second, some strains exhibit unique biochemical characteristics that do not fit into patterns that have been used as a characteristic of any known genus and species.  Bacterial infections: direct identification & characterization methods Microscopy Culture Antimicrobial sensitivity  Antimicrobial sensitivity testing for the sensitivity of microbes to various antibiotics Kirby-Bauer antibiotic testing (KB testing or disk diffusion antibiotic sensitivity testing) is a test which uses antibiotic-impregnated paper disks to test whether particular bacteria are susceptible to specific antibiotics. A known quantity of bacteria are grown on agar plates in the presence of thin filter paper discs containing relevant antibiotics. If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of inhibition). Kirby-Bauer disk diffusion Testing Results Measure the diameters of the zone of inhibition Interpret the results as “resistant” or “susceptible” according to the guideline provided by the CLSI Interpretation of the zone of inhibition is different for each bacteria-antibiotic combination 79  Patient results may be incorrect if: The organism was misidentified A clerical error was made Inappropriate choice of antimicrobials were tested and reported The wrong patient’s sample was examined The wrong test was ordered The sample was not preserved properly 80