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Culture and identification of infectious agents, Lecture 25

Dr. Alvin Fox

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Key TermsKey TermsIsolation (culture)• Agar plate plate/colonies• Liquid media

Identification & taxonomy• Family• Genus• Species• Type• Strain

After culture• Biochemical (physiological) tests• Genetic tests

- Polymerase chain reaction (PCR)- Sequencing- DNA-DNA homology/arrays- Restriction enzymes (digests)

• Chemical - fatty acid/protein profiling

• Immunological

Direct detection (i.e. without culture)• PCR• Antigen detection • Staining (e.g. Gram stain)• Serology (antibody detection)

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Taxonomy

• Defines common traits among strains for a bacterial species

• Usually genetic

• Allows development of diagnostic kits

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Species versus strains- selecting discriminating featuresselecting discriminating features

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ClassificationClassification

• Strain: one single isolate or lineStrain: one single isolate or line

• Type: sub-set of speciesType: sub-set of species

• Species: related strainsSpecies: related strains

• Genus: related speciesGenus: related species

• Family: related generaFamily: related genera

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Streptococcus pyogenes

Both terms define the species name

Genus part Species part

So Streptococcus pyogenes and Streptococus agalactiae would be two species in the same genus

In a report On first usage: Streptococcus pyogenes On second usage: S. pyogenes

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Identification of infectious agentsIdentification of infectious agentsin the diagnostic laboratoryin the diagnostic laboratory

• Aids treatmentAids treatment• Helps antibiotic selectionHelps antibiotic selection• General hospital laboratoryGeneral hospital laboratory

– physiological testsphysiological tests• More fully equipped laboratoriesMore fully equipped laboratories

– Genetic testsGenetic tests

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Steps in isolation and identificationSteps in isolation and identification

• Step 1: Step 1: Streaking culture plates Streaking culture plates – colonies on incubation (e.g 24 hr)colonies on incubation (e.g 24 hr)– size, texture, color, hemolysis size, texture, color, hemolysis – oxygen requirement oxygen requirement

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CDC/Dr. James Feeley

Sheep blood agar plate culture

Bacillus anthracisBacillus cereus.

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Mixed colonies

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Isolation and identificationIsolation and identification

• Step 2: Colonies Gram stained Step 2: Colonies Gram stained – cells observed microscopicallycells observed microscopically

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Gram negativeGram negative Gram positiveGram positive

Heat/DryHeat/Dry

Crystal violet stainCrystal violet stain

IodineIodine FixFix

Safranin stainSafranin stain

AlcoholAlcohol dede-stainstain

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Gram stain morphologyGram stain morphology

• ShapeShape

– cocci (round)cocci (round)

– bacilli (rods)bacilli (rods)

– spiral or curved (e.g. spirochetes)spiral or curved (e.g. spirochetes)

• Single or multiple cellsSingle or multiple cells

– clusters (e.g. staphylococci)clusters (e.g. staphylococci)

– chains (e.g. streptococci) chains (e.g. streptococci)

• Gram positive or negativeGram positive or negative

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Step 3:Step 3:

Isolated bacteria are speciatedIsolated bacteria are speciated

• Generally using physiological testsGenerally using physiological tests

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Clinical Microbiology Clinical Microbiology Laboratory BenchLaboratory Bench

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Step 4:Step 4: Antibiotic susceptibility testing Antibiotic susceptibility testing

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No No growthgrowth

Antibiotic susceptibility testingAntibiotic susceptibility testing

SusceptibleSusceptible Not susceptibleNot susceptible

BacterialBacterial lawnlawn

GrowthGrowth

Antibiotic diskAntibiotic disk

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Molecular Molecular differentiationdifferentiation• GenomicsGenomics

• Gene characterization Gene characterization – PCRPCR– SequencingSequencing– Restriction digestionRestriction digestion

• Hybridization (probes, arrays)Hybridization (probes, arrays)

• % guanine-cytosine% guanine-cytosine

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16S rRNA Sequencing16S rRNA Sequencing

• Differentiates bacterial species Differentiates bacterial species

• Development of clinical tests based Development of clinical tests based on sequence (e.g. PCR)on sequence (e.g. PCR)

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Real-time PCR

Cycle one

Cycle two

Cycle 30

2 2 3030

ds DNAds DNA

DyeDye

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DNA-DNA hybridizationDNA-DNA hybridization

100% Homology

++

HeatHeat

0% Homology

Strain 1Strain 1

Strain 2Strain 2

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ProfilesProfiles• Long chain Long chain fatty acidsfatty acids

- structural (e.g. cell membrane)- structural (e.g. cell membrane)

• Short chainShort chain- metabolic- metabolic- volatiles - volatiles

- Fatty acids/alcohols - Fatty acids/alcohols

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• Protein profiling: defining a species by Protein profiling: defining a species by characteristic (low molecular weight) characteristic (low molecular weight) proteinsproteins

• Proteomics: defining Proteomics: defining allall proteins proteins expressed by a species under specific expressed by a species under specific growth conditionsgrowth conditions

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Bacterial DNA sequences can be Bacterial DNA sequences can be amplified directly from human body amplified directly from human body

fluidsfluids

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Laboratory diagnosis without Laboratory diagnosis without cultureculture

In general, when speed is of essence

Of additional importance:• The organism grows poorly/slowly• The organism can’t be cultured

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MicroscopyMicroscopy• spinal fluids (meningitis) spinal fluids (meningitis) • sputum (tuberculosis)sputum (tuberculosis)

• sensitivity poorsensitivity poor

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Streptococcal Agglutination Test

Latex beadsLatex beads

StreptococcalStreptococcalantigenic extract antigenic extract

AntibodyAntibody

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Serologic identificationSerologic identification

• antibody response to the infecting agent

• several weeks after an infection has occurred

EpidemicsEpidemics

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• associated with particular strains/types

• state or federal laboratory system