Diagnostic Microbiology Lecture Notes PDF

Chapter 5: Diagnostic microbiology Dra Verónica Veses-Jiménez [email protected] Microbe Identification: arrival to the lab The successful identification of microbe depends on: – Using the proper aseptic techniques – Correctly obtaining and handling the specimen – Quickly transpo...

Chapter 5: Diagnostic microbiology Dra Verónica Veses-Jiménez [email protected] Microbe Identification: arrival to the lab The successful identification of microbe depends on: – Using the proper aseptic techniques – Correctly obtaining and handling the specimen – Quickly transporting the specimen to the lab 2 Identification of Microorganisms The methods used by microbiologists fall into three categories: – Classic microbiology – Molecular microbiology (genetic tests) – Immunological analysis 3 Where do we start? 4 CLASSIC MICROBIOLOGY 5 Classic microbiological route: the five Is Inoculation: producing a pure culture Incubation: growing microbes under proper conditions Isolation: Colony on media, one kind of microbe, pure culture Inspection: Observation of characteristics (data) Identification: use of data to identify organism to exact species 6  Inoculation Introduce bacteria into a growth medium using “aseptic technique” to prevent contamination. Tools: Bunsen burner, platinum loop 7 Incubation Allow organisms to grow under the optimal conditions Temperature, with or without oxygen 8 Isolation 9 Identification 10 Identification in selective media 11 Confirmation tests I: catalase Catalase is an enzyme, which is produced by microorganisms that live in oxygenated environments to neutralize toxic forms of oxygen metabolites; H2O2. The catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally lack the catalase enzyme. Catalase mediates the breakdown of hydrogen peroxide H2O2 into oxygen (bubbles) and water. 12  Confirmation tests II: oxidase The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain. When present, the cytochrome c oxidase oxidizes the reagent (tetramethyl-p- phenylenediamine) to (indoph enols) purple color end product. When the enzyme is not present, the reagent remains reduced and is colorless. 13 Confirmation tests III: API systems API test strips consists of wells containing dehydrated substrates to detect enzymatic activity, usually related to fermentation of carbohydrate or catabolism of proteins or amino acids by the inoculated organisms. A bacterial suspension is used to rehydrate each of the wells and the strips are incubated 14 Antimicrobial susceptibility testing Antimicrobial drugs are widely used for the treatment of infectious diseases Pathogens should be tested for susceptibility to individual antimicrobials to ensure appropriate chemotherapy. 15 The standard procedure: Kirby–Bauer method Step 1: A colony is picked from an agar plate and inoculated into a tube with sterile water, to density equivalent to 1 MacFarland 16 Step 2: A swab is Step 3: The swab is dipped in the liquid streaked evenly over a culture and incubated plate of sterile agar 17 Step 4: Discs containing known amounts of different antibiotics are placed on the plate. After incubation, inhibition zones are observed 18 19 MOLECULAR MICROBIOLOGY 20 Molecular (Genotyping) Methods These methods involve examining the genetic material of the microorganisms. Increasingly genotypic techniques are becoming the sole means of identifying many microorganisms because of its speed and accuracy. 21 Genotypic methods Genotypic methods of microbe identification include the use of: – PCR – Real time PCR – Reverse Transcriptase PCR – rRNA analysis (sequencing) – Plasmid fingerprinting 22 Polymerase Chain Reaction (PCR) PCR is widely used for the identification of microorganisms. Sequence specific primers are used with PCR in the amplification of DNA or RNA of specific pathogens. PCR allows the detection even if only a few cells are present and can also be used on viable non culturable specimens. The presence of the appropriate amplified PCR product confirms the presence of the organisms. Primers are available for food monitoring for the presence of Salmonella and Staphylococcus. 23 Polymerase Chain Reaction Kary Mullis developed the polymerase chain reaction (PCR) in 1983. PCR allows the rapid synthesis of designated fragments of DNA, solving the problem of specificity Using the technique, over one billion copies can be synthesized in a matter of hours, solving the problem of amplification 24 PCR Reaction Components Water Buffer: Stabilizes the DNA polymerase, DNA, and nucleotides DNA template: Contains region to be amplified Primers: Specific for ends of amplified region, forward and reverse Nucleotides: needed to synthesize the new DNA copies Mg++ ions: essential co-factor of DNA polymerase DNA Polymerase: the enzyme that does the extension 25 How PCR Works Step 1 - Denaturation (optimal temperature is 94ºC): by heating the DNA, the double strand melts and open to single stranded DNA 26 Step 2 Annealing (optimal temperature is 60ºC): the single- stranded primers bind to their complementary single- stranded bases on the denaturated DNA. 27 Step 3 Extension: 72°C is the ideal temperature for the DNA polymerase to attach and start copying the template. The result is two new helixes in place of the first. 28 Results By applying several times this cycle, the quantity of DNA obtained is quickly enough to perform any analysis. Starting with one DNA molecule after just 20 cycles there will be a million copies and after 30 cycles there will be a billion copies. 29 PCR: first cycle 30 PCR overview 31 Taq polymerase The bacterium Thermus aquaticus was first discovered in several springs in the Great Fountain area of the Lower Geyser Basin at Yellowstone National Park The Taq-polymerase does not get denatured at 94ºC 32 PCR: Visualizing results After thermal cycling, tubes are taken out of the PCR machine. Contents of tubes are loaded onto an agarose gel. DNA is separated by size using an electric field. DNA is then stained. PCR products are visible as different bands. 33 Real Time PCR Real-time involves the use of fluorescent primers The PCR machine monitors the incorporation of the primers and display an amplification plot which can be viewed continuously through the PCR cycle Real time PCR yields immediate results 34 Reverse Trancriptase-PCR an RNA template is used to generate cDNA and from this dsDNA is generated. The enzyme used is reverse transcriptase Example: detection of HIV and monitorization of the progress of the disease 35 DNA sequencing Small amounts of DNA can be used for microbial identification. The most common sequence used for microbe identification is DNA sequence of the 16s rRNA gene. PCR is used to amplify the 16S rRNA gene and the sequence determined (Sanger, NGS). 36 Plasmid fingerprinting Plasmid fingerprinting identifies microbial species or similar strains, as related strains often contain the same number of plasmids with the same molecular weight. Fingerprinting of plasmids of many strains and species such as E. coli, Salmonella, Campylobacter and Pseudomonas has demonstrated that this methods is more accurate than other classic methods such as antibiotic resistance patterns or immunological tests. 37 Plasmid fingerprinting The procedure involves: The bacterial strains are grown, the cells lysed and harvested. The plasmids are separated by agarose gel electrophoresis The gels are stained with EtBr and the plasmids located and compared 38 IMMUNOLOGICAL ROUTE 39 An immune response is a natural outcome of infection 40 Immunological methods Immunological methods involve the interaction of a microbial antigen with an antibody (produced by the host immune system) You can test for microbial antigen or for the production of antibodies Lab kits based on this technique are available for the identification of many microorganisms 41 Immunological Methods (II) The study of antibody (Ab)- antigen (Ag) reactions in vitro is called serology (serological reactions) The usefulness of serological test is dependent on its sensitivity and specificity – Sensitivity is the ability to detect small amounts of Ab or Ag – Specificity is the ability to detect one particular Ag or Ab amongst others 42 False Negatives/Positives High sensitivity prevents false negatives – False negatives occurs when there is not reaction but the Ag or Ab are present High specificity prevents false positives – False positives occurs when there is cross reaction with another molecule 43 Immunological tests Precipitation Agglutination Fluorescent ELISA Immunoxpert TM 44 Precipitation Reactions Precipitation is the interaction of a soluble Ag with an soluble Ab to form an insoluble complex (an aggregate of Ag and Ab). Example: precipitation reactions test use antibodies to detect for streptococcal group antigens. Precipitation reactions occurs maximally only when the optimal proportions of Ag and Ag are present. 45 Precipitation Reactions 46 Agglutination Reactions Agglutination is the visible clumping of an Ag when mixed with a specific Ab. Agglutination tests are widely used because they are simple to perform, highly specific, inexpensive and rapid. Standardized tests are available for the determination of blood groups and identification of pathogens and their products. 47 Agglutination Reaction 48 Direct Agglutination Direct agglutination occurs when a soluble Ab results in clumping by interaction with an Ag which is part of a surface of a cell – Blood typing and detection of Mycoplasma pneumonia 49 Indirect or passive Agglutination Soluble Ag is adsorbed or chemically coupled to latex beads or charcoal particles which serve as an inert carrier. Commercial suspensions of latex beads are available for the detection of Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenza and Campylobacter spp Passive agglutination tests are simple, specific and inexpensive which make them suitable for large scale screening purposes. 50 51 Fluorescent Antibodies Antibodies can be chemically modified with fluorescent dyes such as rhodamine B, fluorescent red, fluorescein isothiocynate and fluoresce yellow, green or red. Cells with bound fluorescent antibodies emit a bright red, orange, yellow or green light depending on the dye used. There are two distinct fluorescent antibody procedures: direct and indirect. 52 Fluorescent Antibodies (II) In the direct method the fluorescent antibody is directed to a surface antigen of the organism In the indirect method a non-fluorescent antibody reacts with the organism's antigen and a fluorescent antibody reacts with the non-fluorescent antibody. Fluorescent antibodies can be used to detect microorganisms directly in tissue, long before a classic isolation technique yield the suspected pathogen, Fluorescent antibodies have been used for the detection of Bacillus anthracis and HIV virus 53 Direct Fluorescent Antibodies 54 Indirect Fluorescent Antibodies 55 ELISA (Enzyme-Linked Immunosorbent Assay) The antigen (microbe) is attached to the plate A solution with a specific antibody is added After washing away any unbound material, a second Ab with a conjugated enzyme is added. The second Ab is specific for the first Ab and it is link to an enzymatic activity A substrate is added which reacts with the enzyme to give a colored reaction. ELISA tests are available for the detection of many microorganisms including S. aureus, E. coli and Salmonella. 56 57 ImmunoXpert tm is a pioneering in-vitro diagnostic test that accurately distinguishes between bacterial and viral infections based on the patient’s immune response It uses a computer algorithm to combine immunoassay measurements of three host immune response proteins present in human serum, in order to help distinguish between bacterial and viral etiologies. 58