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Conventional and molecular biology methodsNational Institute of Infectious Disease Lecture 2: Overview of laboratory methods used in molecular epidemiology investigations January 16, 201

Conventional and molecular biology methods

National Institute of Infectious Disease

Lecture 2: Overview of laboratory methods used in molecular epidemiology investigations

January 16, 2017

Principles of Molecular Epidemiology

1

Epidemiologic investigations

Application of laboratory techniques to epidemiologic investigations

Laboratory steps used to perform molecular epidemiology investigations—bacterial infections

 Isolation in pure culture and identification

 Culture-independent diagnostic tests (CIDT)

 Limitations:

 quantitatively distinguishing the etiologic agent from other microbes present in a clinical specimen,

 performing quantitative drug-susceptibility tests

 unequivocally or quantitatively distinguishing pathogenic or drug-resistant variants among mixed bacterial populations

 transporting isolates to other laboratories for additional testing (e.g., WHO Collaborating Center-directed quality assurance and standardization tests), and

 storing isolates for future use and analyses (e.g., for developing and validating new

diagnostic tests, identifying new drug targets, designing new vaccines)

Laboratory steps used to perform molecular epidemiology investigations—viral infections

Separation from complex biological samples

Filtration, nuclease treatment, ultracentrifugation

Isolation by cell culture

Culture-independent diagnostic tests

Speciation

Further phenotypic subtyping

Serotyping

5

Laboratory-based data stratification—core principle in molecular

non-sterile niche Separation

Pure culture Identification to

the species level

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Conventional laboratory techniques used to subtype

organisms

 Growth and morphologic characteristics

 Biochemical characteristics

 Serologic or immunologic characteristics

 Functional or physiologic characteristics

 antibiotic susceptibility tests

 phage typing

 colicin typing

 cell culture assays

 toxigenicity assays

 in vitro survival characteristics

 Multilocus enzyme electrophoresis (MLEE)

Shigella spp. Salmonella typhimurium Salmonella typhi

Triple sugar iron (TSI) slants (alkaline/acid/gas)

7

API 20E system (identifies Gram negative bacteria)

From de la Maza et al, Color Atlas of

Medical Bacteriology, 2004

Biochemical tests—automated systems

http://www.biomerieux-diagnostics.com/vitek-2

From de la Maza et al, Color Atlas of Medical Bacteriology, 2004

Acid fast stain (AFB)

Gram-negative rods Gram-positive cocci

Gram negative Gram positiveMicroscopy

9

Serologic tests

e.g., Gram-negative bacteria

 Serogrouping—O-polysaccharide (O) antigens

 Serotyping—O and flagellar (H) antigens

CCBC Faculty Web

H antigen: flagella

11

Strain typing by functional or physiologic characteristics

 antibiotic susceptibility tests antibiograms

 phage typing—phage types

 colicin typing—colicin (or bacteriocin) types

 cell culture assays—virulence types (pathotypes)

 toxigenicity assays—virulence types (pathotypes)

 in vitro survival characteristics—virulence types

13

Susceptibility testing

 Antimicrobial susceptibility testing (AST)

Disk diffusion test

E-test

Broth microdilution assay

Tissue culture assay:

E coli K12 and HeLa cells

Localized adherence (LA) of EPEC on HeLa cells

Diffuse-adherent

E coli (DAEC)

Transmission EM of enteroinvasive E coli (EIEC)

Laboratory methods used to stratify data related to infectious diseases

• Examine observable characteristics expressed by a cell or an organism (e.g., drug resistance, virulence, morphology)

Phenotypic

methods

• Examine genetic characteristics of a cell or an organism according to its genome or specific genetic loci.

Genotypic

methods

17

Laboratory-based data stratification—core principle in molecular

Phenotypic subtyping -serotype

-drug resistance

Genotyping

Clone or clonal complex

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What is a “clone” or “clonal complex”?

Standard definition: any isolate or a group of isolates

descending from a common precursor strain by non-sexual

reproduction.

Loose definition: a group of bacterial strain belonging to a

same species that are indistinguishable by a genotyping test (e.g., MLST, PFGE) used.

Clonal complex: based on consensus definition used to include variants of a genotype

19

Molecular biology laboeratory methods applied to epidemiology

Genotyping methods used in epidemiologic investigations

Electrophoretic banding patterns

Nucleic acid hybridization Nucleic acid sequencing

21

Genotyping techniques used in epidemiologic investigations

 Plasmid profile analysis

 Genomic “fingerprinting”

-chromosomal digest

-restriction fragment length polymorphism (RFLP) -pulse field gel electrophoresis (PFGE)

-segmented RNA gel electrophoresis

-ribosomal RNA gel electrophoresis

-PCR-based methods

 Nucleic acid hybridization

 Nucleic acid sequencing

Genotyping techniques

• Analyzes changes that occur

in part of the genome of a microorganism

Genotyping method to be discussed

Electrophoretic band pattern analyses PCR-based methods

Nucleic acid sequenced methods

Gel electrophoresis: principles

Conventional DNA electrophoresis: can separate

DNA <20 kb (0.02% of human genome)

• Negative charge allows DNA to move in an electrical field applied in one direction

• Molecular size affects its drag in the gel matrix.

• Agarose has large pores: separates DNA of 500-20 kb size

• Acrylamide has small pores: 1-700 bp

DNA separation in gel depends on its size

25

Strain typing methods based on electrophoretic analysis of

nucleic acid fragments

 Restriction fragment length polymorphism (RFLP)

 Pulsed field gel electrophoresis (PFGE)

 RFLP-Southern blot hybridization

 Ribosomal RNA gel electrophoresis (ribotyping)

 Segmented RNA gel electrophoresis

 PCR-based methods

All of these techniques were developed to overcome the problem of ease of interpretation of the pattern of nucleic acid restriction fragments

resolved by electrophoresis.

Endonucleases used to cut DNA

5’ overhand

Blunt end

27

PvuII cutting site

PvuII site:

29

Restriction fragment length polymorphism (RFLP) analysis of M tuberculosis

DNA (Ferrazoli et al)

IS6110 Southern blot hybridization of RFLP analysis

(Ferrazoli et al)

31

Gel electrophoresis: principles

• movement depends on DNA’s plasticity in electrical field

applied in different directions

• separation depends on DNA reorienting itself in the gel matrix as the electrical field orientation changes

Pulse field gel

Obstacles that had to be overcome to do PFGE

 DNA fragments of >25,000 are poorly resolved by agarose gel electrophoresis

 Autodigestion of large pieces of DNA generated by cutter” endonucleases.

“rare-33

Conventional gel electrophoresis PFGE

Principles of gel

electrophoresis Pulse field gel electrophoresis (PFGE): pattern interpretation

(Tenover et al J Clin Microbiol 1995; 33:2233)

35

Criteria for interpreting PFGE patterns (Tenover et al, J Clin Microbiol 1995;33:2233)

Criteria for interpreting PFGE patterns —

cont.

Important to identify an

appropriate reference strain first;

comparison of PFGE patterns must

be made against such a reference

strain.

Comparison should not be

made among subtype strains

themselves.

• Recognized epidemic or outbreak strain

• Predominant strain in a limited geographic and temporal setting identified by a surveillance system

• Well-documented newly introduced strain (e.g., in

a hospital or community)

Reference strain:

37

PCR and sequence-based genotyping methods

PCR-based strain-typing methods

Single amplified product having different MW:

E coli pathovar detection

ETEC (LT)

EIEC

EPEC

ETEC (ST)

PCR-based strain-typing methods —cont.

Repetitive elements

ERIC (Enterobacterial Repetitive Intergenic Consensus) sequences

REP (Repetitive Extragenic

Palindromic) sequences

BOX (boxA, boxB, boxC)

IS elements (e.g.,

IS6110 in M

tuberculosis)

PGRS (polymorphic GC-rich repetitive sequences)

DRE-PCR (double repetitive element PCR)

ITS-PCR (intergenic transcribed spacer) Multiple locus VNTR analysis (MLVA)

41

Repetitive element

PCR assays based on repetitive elements

Distribution of ERIC sequences among members of Enterobacteriaceae

43

ERIC PCR of selected E coli isolates

(from Amee Manges, UCB)

BOX PCR analysis of serotype 14 Pneumococcal isolates from meningitis cases, Bahia, Brazil 1996-98

(Ko et al, Clin Infect Dis;2000;30:191-95)

45

PCR-based method:

Multiple loci VNTR analysis (MLVA)

 VNTR: variable number tandem repeats

 Short, repeated segments found in a specific locus

 Each locus has direct tandem repeats of variable number

 The number of repeats per locus varies between strains

 Amplification of these loci (using primers specific to flanking

regions) will produce amplicons of different MW that vary between strains

Strain A

Strain B

Strain typing methods based on nucleic acid sequencing

• Chain-termination (Sanger) sequencing (capillary electrophoresis)

• New generation sequencing methods

Whole genome

sequence (NGS)

comparison

• Multilocus sequence typing (MLST)

• Single nucleotide polymorphism (SNP)

Multilocus sequence typing (MLST)

Compares allelic differences

among a selected set of gene

segments, usually <500 bp in

length.

Each set of allele sequences

comprises a sequence type

(ST), which can be compared

across different labs.

• arcC, aroE, glpF, gmk, pta, tpi, yquL

• Each of these genes has 18-33 alleles

In Staphylococcus aureus, as an

example:

ST1 ST2 ST3 ST4 MLST

designation

 Steps:

PCR-amplify target sequences using the MLST database protocol.

Sequence the amplified products.

Make sure the consensus sequence used to designate the allele type (allotype) is accurate.

Submit strain information and sequence results (including ABI tracings) to the MLST database site

( http://www.mlst.net/submissions/default.asp )

Sequence type (ST) designation will be assigned by the MLST

database curator; based on combination of allotypes.

Multilocus sequence typing (MLST)—cont.

49

Multilocus sequence typing (MLST)—cont.

SNP analysis can be performed by concatenating all the allelic sequences.

Concatenated sequences can be aligned to create a dendrogram.

Group assignments made by the dendrogram can be compared to ST designations.

Dendrogram (tree) construction

ST1 ST2 ST3 ST4

MLST

designation

SNP analysis

53

 Tenover, FC et al Interpreting Chromosomal DNA Restriction Patterns Produced

by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing J Clin Micro 1995;33:2233–2239

 Kamerbeek, J et al Simultaneous Detection and Strain Differentiation of

Mycobacterium tuberculosis for Diagnosis and Epidemiology J Clin Micro