Molecular Basis of Bacterial Infections Evelien Floor
Molecular epidemiology of bacterial
infections
Introduction: goal of molecular typing
An increase in cases of multidrug-resistant pathogens in a particular hospital can be due to
endogenous selection or an outbreak (transmission). In case of transmission you will try to increase
hospital hygiene measurements. In case of selection the antibiotic used needs to be changed. We can
distinguish between both types because in case of transmission the strains will be identical and in
case of selection the strains are diverse between patients.
Determining genetic relationships can be done with (molecular) typing and phylogenetic trees.
Typing is looking at genetic relatedness of strains, so at the (sub)species (= strain) level.
The goal of (molecular) typing is to provide laboratory evidence that epidemiologically related
isolates collected during an outbreak of disease are also genetically related and thus represent the
same strain, or to exclude this. Typing is helpful for understanding and controlling the spread of a
disease.
Assumptions made with typing:
Isolates representing the outbreak strain are the recent progeny of a single (or common)
precursor
Such isolates will have the same genotype
Epidemiologically unrelated isolates will have different genotypes
By chance, some epidemiologically unrelated isolates may have similar or indistinguishable
genotypes, particularly if the used typing method lacks resolution
Criteria for typing methods:
Typeability
Reproducibility
Discriminatory power / resolution
Ease of performance and data interpretation
Ease of data exchange
Costs
Conventional typing techniques
Different conventional, non-molecular typing techniques are:
Biotyping
o Based on reaction to biochemical tests: causes this sugar a colour change
Antimicrobial susceptibility typing
o Based on resistance to antibiotics
Phage typing
o To which phage is the strain resistant
Serotyping
o Use a set of sera and look at agglutination
Limitations of phenotypic methods:
Rely on expressed characteristics
Affected by isolation/culture/test conditions, thus “unstable”
Poorly reproducible
High-percentage non-typeability
Labour-intensive, impractical, slow
1
, Molecular Basis of Bacterial Infections Evelien Floor
Lack of discriminatory power
Molecular typing techniques
Fingerprint-based methods
Fingerprint-based methods are still seen in literature. Different fingerprint-based methods are:
restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE) and
amplification fragment length polymorphism (AFLP).
Restriction fragment length polymorphism
In this method DNA is digested with
restriction enzymes. Restriction enzymes
recognize and cut DNA at specific sites
which causes multiple double strand
breaks. Fragments of DNA will be left
over. Mutations and insertions or
deletions cause loss of restriction
enzyme specificity. This causes different
fragments in different strains. Those
fragments are separated in agarose gel
and compared. With the bands the
relatedness of the strains can be
determined.
AFLP is the same technique as RFLP but
with that technique the fragments are ligated to an adapter and amplificated.
Pulsed-field gel electrophoresis
In this method DNA is also digested
with restriction enzymes and
separated with gel electrophoresis.
Separation of larger fragments is
possible because of an alteration of
the current: vertical net migration. A
problem with PFGE is that the
patterns generated may be not
reproducible and difficult to interpret.
Some bands for instance appear to be on the same height but aren’t due to differences in
concentrations.
PCR-based methods
PCR-based typing methods are: random amplified polymorphic DNA (RAPD), repetitive element
palindromic PCR (Rep-PCR) and
multiple-locus variable-number
tandem repeat analysis (MLVA).
Multiple-locus variable-
number tandem repeat
analysis
MLVA is also still seen in today’s
literature. This technique uses
differences in tandem repeats by
looking at multiple loci. Tandem
2
Molecular epidemiology of bacterial
infections
Introduction: goal of molecular typing
An increase in cases of multidrug-resistant pathogens in a particular hospital can be due to
endogenous selection or an outbreak (transmission). In case of transmission you will try to increase
hospital hygiene measurements. In case of selection the antibiotic used needs to be changed. We can
distinguish between both types because in case of transmission the strains will be identical and in
case of selection the strains are diverse between patients.
Determining genetic relationships can be done with (molecular) typing and phylogenetic trees.
Typing is looking at genetic relatedness of strains, so at the (sub)species (= strain) level.
The goal of (molecular) typing is to provide laboratory evidence that epidemiologically related
isolates collected during an outbreak of disease are also genetically related and thus represent the
same strain, or to exclude this. Typing is helpful for understanding and controlling the spread of a
disease.
Assumptions made with typing:
Isolates representing the outbreak strain are the recent progeny of a single (or common)
precursor
Such isolates will have the same genotype
Epidemiologically unrelated isolates will have different genotypes
By chance, some epidemiologically unrelated isolates may have similar or indistinguishable
genotypes, particularly if the used typing method lacks resolution
Criteria for typing methods:
Typeability
Reproducibility
Discriminatory power / resolution
Ease of performance and data interpretation
Ease of data exchange
Costs
Conventional typing techniques
Different conventional, non-molecular typing techniques are:
Biotyping
o Based on reaction to biochemical tests: causes this sugar a colour change
Antimicrobial susceptibility typing
o Based on resistance to antibiotics
Phage typing
o To which phage is the strain resistant
Serotyping
o Use a set of sera and look at agglutination
Limitations of phenotypic methods:
Rely on expressed characteristics
Affected by isolation/culture/test conditions, thus “unstable”
Poorly reproducible
High-percentage non-typeability
Labour-intensive, impractical, slow
1
, Molecular Basis of Bacterial Infections Evelien Floor
Lack of discriminatory power
Molecular typing techniques
Fingerprint-based methods
Fingerprint-based methods are still seen in literature. Different fingerprint-based methods are:
restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE) and
amplification fragment length polymorphism (AFLP).
Restriction fragment length polymorphism
In this method DNA is digested with
restriction enzymes. Restriction enzymes
recognize and cut DNA at specific sites
which causes multiple double strand
breaks. Fragments of DNA will be left
over. Mutations and insertions or
deletions cause loss of restriction
enzyme specificity. This causes different
fragments in different strains. Those
fragments are separated in agarose gel
and compared. With the bands the
relatedness of the strains can be
determined.
AFLP is the same technique as RFLP but
with that technique the fragments are ligated to an adapter and amplificated.
Pulsed-field gel electrophoresis
In this method DNA is also digested
with restriction enzymes and
separated with gel electrophoresis.
Separation of larger fragments is
possible because of an alteration of
the current: vertical net migration. A
problem with PFGE is that the
patterns generated may be not
reproducible and difficult to interpret.
Some bands for instance appear to be on the same height but aren’t due to differences in
concentrations.
PCR-based methods
PCR-based typing methods are: random amplified polymorphic DNA (RAPD), repetitive element
palindromic PCR (Rep-PCR) and
multiple-locus variable-number
tandem repeat analysis (MLVA).
Multiple-locus variable-
number tandem repeat
analysis
MLVA is also still seen in today’s
literature. This technique uses
differences in tandem repeats by
looking at multiple loci. Tandem
2
