TYPING METHODS

WHAT IS TYPING AND WHAT ARE

TYPING METHODS?

• Pathogenic bacteria replicate and persevere in

ecological niches called reservoirs

• Reservoirs may be humans, including (fellow)

patients and healthcare personnel, animals,

plants, water, food and various niches in the

environment

• Transmission of bacteria from any of these sources may generate clusters of colonization or infection among humans

• Such clusters are recognized mostly as outbreaks of infectious diseases

• When these outbreaks are not controlled, major epidemics (due to unrestricted further transmission) may arise

• Bacterial epidemiological typing generates isolate-specific genotypic or phenotypic characters that can be used to elucidate the sources and routes of spread of bacteria

The scope of typing studies

• Clinical : (dissemination of infections from patients,

animals or other sources to non-colonized and

uninfected individuals)

• Environmental: (the presence or spread of organisms

in inanimate surroundings) or even‘industrial’

(identification of organisms that are either valuable or

a menace to bio-industry)

Different levels of Typing

• Locally, at a hospital or other primary laboratory

• Regionally or nationally, in a reference laboratory

to bear upon wider issues of public health and surveillance

• Internationally through collaborative networks, to define or survey the worldwide dissemination of major bacterial clones

TYPING TERMS

• Bacterial epidemiology: The study of the dissemination of human bacterial pathogens, including their transmission patterns, risk factors for and control of infectious disease in human populations

• Clonal complex: A group of bacterial isolates showing a high degree of similarity, ideally based on typing methods. Clonal complexes are identical to clonal groups

Isolate

A population of bacterial cells in

pure culture derived from a single colony. In

clinical microbiology, isolates are usually

derived from the primary culture of a clinical

specimen obtained from an individual patient

Strain

• A strain may be considered an isolate or group of isolates that can be distinguished from other isolates of the same genus and species by phenotypic and genotypic characteristics

• Cultures of a particular microorganism, isolated at the same time from multiple body sites of a patient and indistinguishable by typing, also represent a single strain.

Clone

Bacterial isolates that, although they may have

been cultured independently from different

sources in different locations and perhaps at

different times, still have so many identical

phenotypic and genotypic traits that

the most likely explanation for this identity is a

common origin within a relevant time span

Outbreak

Local, initially small-scale, cluster of disease

generally caused by increased frequency of

infection in a distinct population (may be

caused by single epidemic strains or

combinations of different strains)

Typing services for nosocomial pathogens

Phenotypic and/or genetic analysis of bacterial isolates, below the species/subspecies level, performed in order to generate strain/clone-specific fingerprints or datasets that can be

used, for example, to detect or rule out cross-infections, elucidate bacterial transmission patterns and find reservoirs or sources of infection in humans. ‘Subtyping’, a term

commonly seen in American literature, is often

used as a synonym for typing

REASONS FOR TYPING

• Surveillance of infectious diseases

• Study of pathogenesis and the course of infection

• Outbreak investigation

• Study of bacterial population genetics

• Identify emerging pathogenic strains or clones within a

species, including potential agents of bioterrorism

Performance criteria

• Stability

– This refers to the stability of the markers assessed by the typing method

• Typeability

– This refers to a method’s ability to assign a type to all isolates tested by it.

• Discriminatory power

– This refers to a method’s ability to assign a

– different type to two unrelated strains sampled

– randomly from the population of a given species.

• Epidemiological concordance

• Reproducibility

Convenience criteria

• Flexibility (or spectrum)

• Rapidity

• Accessibility

• Ease of use

• Cost

• Amenability to computerised analysis and

incorporation of typing results in electronic databases

Phenotypic typing methods

• Biotyping

• Antimicrobial susceptibility testing

(antibiogram- based typing)

• Serotyping

• Phage and bacteriocin typing

• SDS-PAGE of cellular and extracellular

components

• Multilocus enzyme electrophoresis (MLEE)

1.Inoclution well

2.L-Arabinose

3.Lactose

4.Melbiose

5.Melezitose

6.Raffinose

7.Inositol

8.Sorbitol

9.Mannitol

10.Gal-lacton

11.Amygdalin

12.Gluconate

PhP typing for screening

PhP typing of MRSA

Genotypic typing methods

• Genotypic typing methods assess variation in the genomes of bacterial isolates with respect to composition

– presence or absence of plasmids

– overall structure (e.g., restriction endonuclease

profiles, number and positions of repetitive elements

– precise nucleotide sequence (of one or more genes or intergenic regions).

Fragment-based methods

• Plasmid typing

• Restriction fragment length polymorphism

(RFLP) methods

Plasmid analysis

M BM4147 1 2 3 4 5 M

5kb

3kb

2kb

1kb

0.7Kb

Tn1546 like elements digested by ClaI

RFLP

1 2 3 4 5 6

170Kb

78 kb

M 1 2 3 4 5 6

Hybridization-mediated methods

Direct hybridization

16S rRNA 23S rRNA

tARN 5S ARN

Ad rB O4c O16 O24

13909 442 1601 2490

intergenic spacer

E. coli rrnB operon

Ribotyping

Ribotype pattern

Genotyping of Salmonella typhi of Iran

It takes only 8 hr to complete a run of riboprinting

As compare to 4 days of classical typing

Riboprinter

PFGE • The chromosome is the most fundamental component of

identity

• One approach has been to digest chromosomal DNA with restriction enzymes

• Enzymes used to cleave DNA often recognize numerous sites within the bacteria, More recently, restriction enzymes that cleave chromosomal DNA less frequently have been utilized for analysis

• The resulting DNA fragments are too large to be separated by conventional agarose gel electrophoresis

• Two of the most commonly utilized

approaches

– Contour-clamped homogenous electric field

(CHEF)

– Field inversion gel electrophoresis (FISH)

Comparison of Migration: Horizontal vs. CHEF

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Types Of Pulse Field Gel Electrophoresis

Field inversion gel Transverse alternating field

Crossed field

(Reverse)

Contour-clamped

homogeneous electric

field

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Electric Field 1 Electric Field 2 Switch Time

CHEF

PFGE

• Made it possible to separate large DNA fragments in

agarose gels by periodic alternation of the angle of

the electric field’s direction.

• These DNA ‘macrorestriction’ fragments are generated with restriction endonucleases with six or more base pair recognition sites (‘rare cutters’)

• Usually yielding fewer than 30 large fragments, normally ranging in size between 20 and 600 kbp.

Procedure

• Bacterial growth

• Plug preparation

• Cell lysis – Lysis buffer (Tris, EDTA, Brij, Pro K,…)

• Digestion – Enterobaceriacea XbaI

– Enterococci, Staphylococcus, pneumococci SmaI

– Vibrio NotI

– ,……..

– Agarose preparation

– Electrophoresis & staining

PFGE

• Pulse time

• Voltage

• Field Angle

• Temperature

• Agarose concentration

• Buffer concentration

669Kb

398Kb

310Kb

244Kb

217Kb

173Kb

139Kb

105Kb

76Kb

M 1 2 3 4 5 6 7 8 9 10 M

PFGE Pattern

Interpreting DNA fragment patterns

• Analytical Software: Gel Compar

• Thus, genomic pattern similarity values must

be based on a sufficiently large number of

genomic sites/bands for each isolate

Pulsed Field Gel Electrophoresis (PFGE)

• Interpretive Criteria

1. Indistinguishable – banding pattern same number and size

2. Closely related – banding pattern differs due to a single

genetic event (usually, 2 - 3 band difference)

3. Possibly related- banding pattern differs due to 2

independent genetic events (usually, 4 - 6 band

difference)

4. Unrelated – banding pattern differs due to changes

consistent with 3 genetic events.

E. coli

Indistinguishable

○ 2 isolates

Closely related

○ 11 isolates

Possibly related

○ 4 isolates

Unrelated

○ 18 isolates

K. pneumonia

Indistinguishable

○ 4 isolates (A/A, B/B)

Possibly related

○ 3 isolates

Unrelated

○ 9 isolates

P. aeruginosa

Closely related

○ 2 isolates

Possibly related

○ 6 isolates

Unrelated

○ 4 isolates

E. faecalis

Possibly related

○ 2 isolates

Unrelated

○ 9 isolates

PFGE Pattern of VRE Isolates

PFGE Pattern of Clinical Samples

PFGE Pattern of Urban Sewage

PFGE Pattern of Hospital Sewage

PFGE Pattern of Identical Isolates

MRSA

Pulsed Field Gel Electrophoresis (PFGE)

• Advantages of PFGE - Highly discriminatory

• - Reproducible (inter and intra lab)

• - Adapted to most organisms • PFGE has been applied to at least 40 pathogens or pathogen

• groups CHEF systems have also been used for typing of Candida

• species

• - outbreak investigation

• - tracking evolution or long term spread of pathogens

• Disadvantages • - Initial capital outlay expensive

• - Requires skilled technologists

COMPARISON AND SELECTION OF TECHNIQUES

• First depends on application

• The molecular characterization of nosocomial isolates generates data regarding the interrelatedness of isolates.

• In an individual patient, the use of molecular characterization can assist in separating relapse from reinfection or, in the case of bacteremia, whether the organism is from the infection or contamination.

• PFGE is method of choice • Plasmid or transposon analysis of strains is used when

there is suspicion of dissemination of a particular resistance gene or set of genes

• The best method often depends

not only on the specific epidemiologic situation but also on the resources available.

• PFGE analysis provides relatively global chromosomal overview

• scanning more than 90% of the chromosome (the sum of the restriction fragment sizes)

• But minor genetic changes may go undetected

• As PFGE provides the broadest

• genomic overview, it likely remains the method of choice as the initial typing method for most epidemiologic investigation.

• However, on rare occasions, PFGE is unable to resolve discriminatory profiles for certain organisms

• And other typing methods need to be employed to determine the relatedness of the strains

RECENT ADVANCES IN MOLECULAR EPIDEMIOLOGY:

NUCLEOTIDE SEQUENCE-BASED ANALYSIS

Variety of molecular typing approaches that

focus on either single or multiple chromosomal

loci

• SLST

• MLST

Looking into the future