Identification of Bacterial Pathogens basic skills in bacteriology

Identification of BacterialPathogensbasic skills in diagnostic bacteriologyDr.T.V.Rao MDDr.T.V.Rao MD 1

Identification of Microorganisms• For many students and professionals the mostpressing topic in microbiology is how to identifyunknown specimens.• Why is this important?• Labs can grow, isolate and identify most routinelyencountered bacteria within 48 hrs of sampling.• The methods microbiologist use fall into threecategories:♣Phenotypic- morphology (micro andmacroscopic)♣Immunological- serological analysis♣Genotypic- genetic techniquesDr.T.V.Rao MD2

MicrobeIdentificationDr.T.V.Rao MD5

Specimen Collection• Successful identification depends on how the specimenis collected, handled and stored.• It is important that general aseptic procedures be usedincluding sterile sample containers and samplingmethods to prevent contamination of the specimen.• E.g. Throat and nasopharyngeal swabs should not touchthe cheek, tongue or salvia.• What other precautions must be taken when collecting specimens?• After collection the specimen must be taken promptlyto the lab and stored appropriately (e.g. refrigeration).Dr.T.V.Rao MD6

SpecimenCollectionDr.T.V.Rao MD7

Phenotypic Methods of Identification• Microbiologists use 5 basic techniques to grow,examine and characterize microorganisms inthe lab.• They are called the 5 ‘I’s: inoculation,incubation, isolation, inspection andidentification.• Inoculation: to culture microorganisms a tinysample (inoculum) is introduced into medium(inoculation).• Isolation involves the separating one speciesDr.T.V.Rao MDfrom another.8

Phenotypic Methods• Macroscopic morphology are traits that can beaccessed with the naked eye e.g. appearance of colonyincluding texture, shape, pigment, speed of growth andgrowth pattern in broth.• Physiology/Biochemical characteristic are traditionalmainstay of bacterial identification.• These include enzymes (catalase, oxidase,decarboxylase), fermentation of sugars, capacity todigest or metabolize complex polymers and sensitivityto drugs can be used in identification.Dr.T.V.Rao MD12

Gm+ve cocci & Gm-ve bacilliDr.T.V.Rao MD17

StreptococcusDr.T.V.Rao MD 18

BacilliDr.T.V.Rao MD 19

Neisseria gonorrhea - Gram stainhttp://www.cdc.gov/STD/LabGuidelines/default.htmDr.T.V.Rao MD 20

Wound specimen - Gram stainhttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.htmlDr.T.V.Rao MD 21

Oral specimen - Gram stainDr.T.V.Rao MD 22http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html

Sputum specimen Gram stain—Streptococcus pneumoniaehttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.htmlDr.T.V.Rao MD 23

Sputum—cystic fibrosis patient, encapsulated gram negative rodshttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.htmlDr.T.V.Rao MD 24

Staining techniques forMycobacterial spp• Acid-fast stains– for staining of organisms with high degree of fatty(mycolic) acids—waxy– render the cells resistant to decolorization: “acid-fast”– Mycobacterium sp., Nocardia sp., Cryptosporidiumsp. are acid-fast– Procedure• Ziehl-Neelsen: heat drives in primary stain (carbolfuchsin)• Kinyoun: higher conc. of phenol does not require heat• Decolorize with acid-alcohol• Counterstain with methylene blue or malachite greenDr.T.V.Rao MD 25

1 2 34 5 67Ziehl-NeelsenstainDr.T.V.Rao MD26

How the Acid fast bacteria appearDr.T.V.Rao MD27

A Gram stain (left) of the abscess shows thin, gram positive rods in chains.An acid fast stain (right) was also positive.http://pathhsw5m54.ucsf.edu/overview/bacteria3.htmlDr.T.V.Rao MD 28

Sputum specimen—Acid fast stainhttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.htmlDr.T.V.Rao MD 29

Acid fast stain demonstrating chordingDr.T.V.Rao MD 30

Fluorochrome AFB Microscopy* More rapid andsensitive* Specificity : same withsufficient expérience* Equipment cost ,bulbs, technicaldemands* for busy labs* External qualityassessment should bedone if this method isperformed Dr.T.V.Rao MD31

Direct Fluorescent Antibodies32

Indirect Fluorescent Antibodies33

Mycobacterium – auramine stainhttp://www.lung.ca/tb/abouttb/what/causes_tb.htmlDr.T.V.Rao MD 34

Yeast—calcofluor whitehttp://www.med.sc.edu:85/mycology/mycology-3.htmDr.T.V.Rao MD 35

Mould—calcofluor whiteDr.T.V.Rao MD 36

Influenza virus infected cells, fluorescent antibody stainDr.T.V.Rao MD 37

Better Use Microscopy• Phase Contrast Microscopy– shift in light allows visualization of organism; canvisualize viable organisms• Fluorescent Microscopy– certain dyes (fluorochromes) give off light whenexcited (fluorescence)– color of light depends on the dye and the filters used– Staining techniques• Fluorochroming: direct chemical interaction with organism– Acridine orange: stains nucleic acid; useful for cell-wall deficientorganisms– Auramine-rhodamine: bind to mycolic acids in nearly allMycobacteria– Calcoflour white: binds to chitin in cell walls of fungi• Immunofluorescence: fluorochrome is bound to an antibody;can detect/identify specific organismsDr.T.V.Rao MD 38

Culture and isolation of bacteria• Principles of Cultivation– Nutritional requirements• General concepts– non-fastidious: simple requirements for growth– fastidious: complex, unusual, or uniquerequirements for growth• Phases of growth media– solid agar; boil to dissolve, solidifies at50ºC– liquid, brothDr.T.V.Rao MD 39

• Media classifications and functions–Enrichment• used to enhance growth of specificorganisms–Supportive• support growth of most non-fastidiousorganisms–Selective• contains agents that inhibit the growth of allagents except that being sought (dyes, bilesalts, alcohols, acids, antibiotics)–Differential• contains factor(s) that allow certainorganisms to exhibit different metaboliccharacteristicsDr.T.V.Rao MD 40

According to Use• Enriched Medium –broth or solid, containsrich supply of specialnutrients that promotesgrowth of a particularorganism while notpromoting growth ofother microbes thatmay be present (e.g BAP& chocolate agar)Dr.T.V.Rao MD 41

• Types of artificial 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 forvisualization 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 andHaemophilus– MacConkey agar• selective for Gram-negative rods (GNRs)because of crystal violet and bile salts; differentialdue to lactose, fermenters lower pH changingneutral red indicator pink/redDr.T.V.Rao MD 42

According to Use• Selective Medium -contains inhibitorsthat discourage thegrowth of certainorganisms &enhances thegrowth of themicrobe sought(e.g. SSA, MannitolSalt Agar)Dr.T.V.Rao MD 43

According to Use• Differential Medium -contains dyes, indicatorsor other constituentsthat give colonies ofparticular organismsdistinctive and easilyrecognizablecharacteristics (e.g.McConkey Agar)Dr.T.V.Rao MD 44

Hemolysis a guiding factorDr.T.V.Rao MD 45

Hemolysis a guiding factorDr.T.V.Rao MD 46

Enriched MediaChocolate agar, a mediumthat gets brown fromheated blood. Used forisolation of N. gonorrhea.Blood agar plate withbacteria from human throat.This media differentiatesamong different colonies byappearanceDr.T.V.Rao MD47

Testing for Bacitracin and OptochinSensitivityDr.T.V.Rao MD 48

Dr.T.V.Rao MD 49

Mac Conkey Agar a Minimaldifferentiating MediumDr.T.V.Rao MD 50

MacConkey AgarDr.T.V.Rao MD 51

General vs Selective MediaDr.T.V.Rao MD52

Differential Media• Differential media growseveral types oforganisms and displayvisible differencesamong organisms.• Differences may showup as colony size, mediacolour, gas bubbleformation andprecipitate formation.Dr.T.V.Rao MD53

Salmonella-Shigella AgarDr.T.V.Rao MD 54

Dr.T.V.Rao MD 55

• Types of artificial media– Hektoen enteric agar• contains bile salts and dyes(bromothymol blue and acid fuchsin) toinhibit non-pathogenic GNRs; nonpathogens ferment lactose changingBTB to orange; pathogens Salmonellaand Shigella are clear; ferric ammoniumcitrate detects H 2 S production ofSalmonella (black colonies)– Columbia colistin-nalidixic acid (CNA)agar• Columbia agar base, sheep blood,colistin and nalidixic acid; selectiveisolation of gram-positive cocciDr.T.V.Rao MD 56

• Types of artificial media– Hektoen enteric agar• contains bile salts and dyes (bromothymolblue and acid fuchsin) to inhibit nonpathogenicGNRs; non pathogens fermentlactose changing BTB to orange; pathogensSalmonella and Shigella are clear; ferricammonium citrate detects H 2 S production ofSalmonella (black colonies)– Columbia colistin-nalidixic acid (CNA)agar• Columbia agar base, sheep blood, colistinand nalidixic acid; selective isolation of grampositivecocciDr.T.V.Rao MD 57

Types of artificial media– Thayer-Martin agar• CAP with antibiotics (colistin inhibits gramneg, vancomycin inhibits gram pos,nystatin inhibits yeast); for N.gonorrhoeae and N. meningitidis; Martin-Lewis has similar function but differentantibiotics• Preparation of artificial media– Sterilization• autoclave: pressurized steam at 121ºC for15-30 min.Dr.T.V.Rao MD 58

• Environmental requirements– Oxygen and Carbon dioxide availability• aerobic: room air• facultative: aerobic or anaerobic• microaerophilic: reduced oxygen tension• anaerobic– strict or aerotolerant• capnophilic: increased C0 2 (5-10%)– Temperature• 35-37ºC• 30ºC• cold• 42ºCDr.T.V.Rao MD 59

• Bacterial Cultivation– Isolation of bacteria from specimens• streaking for isolation• streaking for quantitation– Evaluation of colony morphologies• Type of media supporting growth• Relative quantities of each colony type• Colony characteristics– colony form: pinpoint, circular, filamentous,irregular– colony elevation: flat, raised, convex– colony margin: smooth, irregular• Gram stain and subcultures– sterile loop, isolated coloniesDr.T.V.Rao MD 60

http://science.nhmccd.edu/biol/wellmeyer/media/media.htmDr.T.V.Rao MD 61

Chocolate agarUninoculatedHaemophilushttp://www.hardydiagnostics.com/catalog2/hugo/ChocolateAgar.htmDr.T.V.Rao MD 62

MacConkey agarhttp://science.nhmccd.edu/biol/wellmeyer/media/media.htmDr.T.V.Rao MD 63

Hektoen agarhttp://medic.med.uth.tmc.edu/path/hekto.htmDr.T.V.Rao MD 64

• Nutritional requirements and metaboliccapabilities– Single enzyme tests• Catalase: H 2 O 2 + catalase = O 2 and H 2 0;differentiates Staphylococcus v. Streptococcus,Listeria and Corynebacterium v. other non sporeforming gram-positive bacilli• Oxidase: detection of cytochrome oxidase thatparticipates in nitrate metabolism; Pseudomonas,Aeromonas, Neisseria• Indole: tryptophanase degrades tryptophan intopyruvic acid, ammonia, and indole; indole isdetected by aldehyde indicator; presumptive id forE. coli• Urease: hydrolyzes urea into ammonia, water andCO 2 ; increase pH changes causes bright pink colorof indicator• PYR: hydrolysis of PYR, indicator turns pink; GroupA Strep and enterococci are +Dr.T.V.Rao MD 65

Biochemical Tests• The microbe is cultured in a media with a specialsubstrate and tested for an end product.• Prominent biochemical tests include carbohydratefermentation, acid or gas production and thehydrolysis of gelatin or starch.• Many of these test in rapid miniaturized system thatcan detect for 23 characteristics in small cups calledRapid test.• The info from the rapid test are input into a computerto help in identification of the organisms.Dr.T.V.Rao MD66

CarbohydrateFermentation . This medium showfermentation (acidproduction) and gasformation. The small Durham tube forcollecting gas bubbles. Left- right:Uninoculated negativecontrolCentre, positive for acid(yellow) and gas (openspace).Growth but no gas oracid.Dr.T.V.Rao MD67

Methyl Red Test• This is a qualitativetest for acidproduction.• The bacteria is grownin MR-VP broth.• After addition ofseveral drops ofmethyl red solution abright red colour ispositive and yelloworangenegative.Dr.T.V.Rao MD68

Nitrate Reduction ♫After 24-48 hrs ofincubation, nitratereagents are added.♫Left to right:♫Gas formation (positivefor nitrate reduction).♫positive for nitratereduction to nitrite (red colour).♫Negative controlDr.T.V.Rao MD69

Biochemical Tests• Other biochemical tests of interest include:‣H 2 S production‣Indole test‣Oxidase test‣Oxidation fermentation‣Phenylalanine deaminase test‣Antibiotic susceptibility tests• Principle, procedure, most common use.Dr.T.V.Rao MD70

Dr.T.V.Rao MD 71

Dr.T.V.Rao MD 72

Coagulase TestDr.T.V.Rao MD 73

–Tests for presence ofmetabolic pathways• Oxidation and fermentation: oxidationof glucose requires oxygen,fermentation does not; pH decreasescausing yellow color• Amino acid degradation: detection ofamino acid decarboxylase enzymesDr.T.V.Rao MD 74

O-F glucose mediahttp://academic.mwsc.edu/jcbaker/bio390sec01/bio390_laboratory_study_images.htmDr.T.V.Rao MD 75

Chromagar orientationuses colour-formation todistinguish at least 7common urinarypathogens.DifferentialMediaChromagarThis allow for rapididentification andtreatment. The bacteria werestreaked as to spell theirnames.Dr.T.V.Rao MD76

• Principles of Phenotype-based IDschemes– Selection and inoculation of ID test battery• Type of bacteria to be identified• Clinical significance of isolate• Availability of reliable testing methods– Incubation for substrate utilization• Conventional ID• Rapid ID– Detection of metabolic activity• Colorimetry: pH change of indicators• Fluorescence: release of fluorophore from substrate orchanges in fluorescence due to pH changes• Turbiditiy: growth or no-growth– Analysis of metabolic profiles• ID databases• Use of databases to ID unknowns– Confidence in IDDr.T.V.Rao MD 77

Rapid Tests• Rapid test: a biochemical system for the identificationof Enterobacteriaceae and other Gram –ve bacteria.• It consist of plastic strips with 20 μl of dehydratedbiochemical substrates used to detect biochemicalcharacteristics.• The biochemical substrates are inoculated with purecultures and suspended in physiological saline.• After 5 hrs-overnight the 20 tests are converted to 7-9digital profile.Dr.T.V.Rao MD78

Rapid TestspositivenegativeONPG (β galactosidase); ADH (arginine dihydrolase); LDC (lysine decarboxylase); ODC(ornithine decarboxylase); CIT (citrate utilization); H 2 S (hydrogen disulphide production); URE(urease); TDA ( tryptophan deaminase); IND (indole production); VP (Voges Proskauer test foracetoin); 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).Dr.T.V.Rao MD79

Bacteriophage Typing• What are bacteriophages?• Bacteriophage typing is based on the specificity ofphage surface receptor for the cell surface receptor.• Only those phages that can attach to the surfacereceptors can cause lysis.• The procedure involves:• A plate is heavily inoculated so that there is nouninoculated areas.• The plate is marked off in squares (15-20 mm) andeach square inoculated with a drop of suspension fordifferent phages.Dr.T.V.Rao MD80

Heavily Inoculated PlateDr.T.V.Rao MD81

Bacteriophage Typing• The plate is incubated for 24 hrs then observedfor plaques.• The phage type is reported as a specific genusand species followed by the types that can infectthe bacterium.• E.g. 10/16/24 means that the bacteria issensitive to phages 10, 16 and 24.• Phage tying remain a tool for research andreference labs.Dr.T.V.Rao MD82

Uncultivable Organisms• Environmentalresearchers estimatethat < 1% ofmicroorganisms areculturable andtherefore it is notpossible to usephenotypic methods ofidentification.• These microorganismsare called viablenonculturable (VNC).Dr.T.V.Rao MD83

Flow Cytometry• Classical techniques are not successful in identificationof those microorganisms that cannot be cultured.• What do is the name of microorganisms that cannot be cultured?• Flow cytometry allows single or multiplemicroorganisms detection an easy, reliable and fast way.• Inflow cytometry microorganisms are identified on thebasis of the cytometry parameters or by means ofcertain dyes called fluorochromes that can be usedindependently or bound to specific antibodies.Dr.T.V.Rao MD84

Flow Cytometry• The cytometer forces a suspension ofcells through a laser beam and measuresthe light they scatter or the fluorescencethe cell emits as they pass through thebeam.• The cytometer also can measure thecell’s shape, size and the content of theDNA or RNA.Dr.T.V.Rao MD85

Recent Advances in Flowcytometry• Flow cytometry (FCM) allows single- or multiplemicrobedetection in clinical samples in an easy,reliable, and fast way. Microbes can be identifiedon the basis of their peculiar cytometryparameters or by means of certain fluorochromesthat can be used either independently or boundto specific antibodies or oligonucleotides. FCMhas permitted the development of quantitativeprocedures to assess antimicrobial susceptibilityand drug cytotoxicity in a rapid, accurate, andhighly reproducible wayDr.T.V.Rao MD 86

Immunological Methods• Immunological methods involve the interactionof a microbial antigen with an antibody(produced by the host immune system).• Testing for microbial antigen or the productionof antibodies is often easier than test for themicrobe itself.• Lab kits based on this technique is available forthe identification of many microorganisms.Dr.T.V.Rao MD87

Principles of Serologic Test Methods– Methods for antibody detection• Direct whole pathogen agglutination assays– pos patient sera causes organism to clump• Particle agglutination tests– latex beads or RBCs coated with Ag• Flocculation tests– RPR – precipitation of soluble Ag with Ab» charcoal particles coated with cardiolipin-lecithinbinds reagin• ELISAs• IFAs– organism/antigen on slides; patient Ab detected withfluorescent secondary Ab• Western blotsDr.T.V.Rao MD 88

API strips – bioMerieuxDr.T.V.Rao MD 89

Commercial ID systems– Advantages andexamples ofcommercialsystems• ARIS (Trek)• MicroScan (Dade-Behring/Seimens)• Phoenix (Becton-Dickinson)• Vitek(bioMérieux)Dr.T.V.Rao MD 90

Immunochemical methods for ID• Principles of immunochemical methods– Particle agglutination• Latex agglutination– Immunofloresecent assays• Direct immunofluorescence assay (DFA)• Indirect immunofluorescence assay (IFA)• Enzyme immunoassays– Solid-phase immunoassays• Membrane bound immunoassays• Immunochromatographic assays– Optical immunoassaysDr.T.V.Rao MD 91

Serologic methods for diagnosis• Features of the Immune Response– Characteristics of antibodies• Features of immune response useful in diagnostictesting– Acute v. anamnestic response– IgM v. IgG– IgM can’t cross placenta– immunocompetent v. immunocompromised• Interpretation of serologic tests– single v. paired sera; rare pathogen– 4-fold rise in titer v. qualitative testing– cross reactivity (herpes viruses, heterophile Abs,pregnancy)Dr.T.V.Rao MD 92

Principles of Serologic Test Methods– Methods for antibody detection• Direct whole pathogen agglutination assays– pos patient sera causes organism to clump• Particle agglutination tests– latex beads or RBCs coated with Ag• Flocculation tests– RPR – precipitation of soluble Ag with Ab» charcoal particles coated with cardiolipin-lecithinbinds reagin• ELISAs• IFAs– organism/antigen on slides; patient Ab detected withfluorescent secondary Ab• Western blotsDr.T.V.Rao MD 93

Genotypic methods• Genotypic methods of microbe identificationinclude the use of :Nucleic acid probesPCR (RT-PCR, RAPD-PCR)Nucleic acid sequence analysisrRNA analysisRFLPPlasmid fingerprinting.94

Genotypic Methods• Genotypic methods involve examining thegenetic material of the organisms and hasrevolutionized bacterial identification andclassification.• Genotypic methods include PCR (RT-PCR, RAPD-PCR),use of nucleic acid probes, RFLP andplasmid fingerprinting.• Increasingly genotypic techniques are becomingthe sole means of identifying manymicroorganisms because of its speed andaccuracy.Dr.T.V.Rao MD95

Real Time PCR and RT-PCR• Currently many PCR tests employ real time PCR.• This involves the use of fluorescent primers.• The PCR machine monitors the incorporation of the primers anddisplay an amplification plot which can be viewed continuouslythru the PCR cycle.• Real time PCR yields immediate results.• Another application of PCR is RT-PCR (reverse trancriptase PCR).• During RT-PCR an RNA template is used to generate cDNA andfrom this dsDNA is generated.• The enzyme used is reverse transciptase.• RT-PCR is used to detect for HIV and to monitor the progress ofthe disease.96

RAPD-PCR• Random amplified polymorphic DNA PCR uses arandom primer (10-mer) to generate a DNA profile.• What are random primers?• The primer anneals to several places on the DNAtemplate and generate a DNA profile which is used formicrobe identification.• RAPD has many advantages:Pure DNA is not neededLess labour intensive than RFLP.There is not need for prior DNA sequence data.• RAPD has been used to fingerprint the outbreak ofListeria monocytogenes from milk.97

Plasmid fingerprinting• The procedure involves:• The bacterial strains aregrown, the cells lysed andharvested.• The plasmids are separatedby agarose gelelectrophoresis• The gels are stained withEtBr and the plasmidslocated and compared.98

Computer and Bacteria Identification• Computers improvethe efficiency of thelab operations andincrease the speedand clarity with whichresults can bereported.• Computers are alsoimportant for theresult entry, analysisand preparation.99

• Programme created byDr.T.V.Rao MD for MedicalMicrobiologists in Developingworld• Email• doctortvrao@gmail.comDr.T.V.Rao MD 100