the use of molecular methods in source attribution...

Istituto Zooprofilattico Sperimentale delle Venezie · OIE and National Reference Laboratory for salmonellosis

Zaragoza, 10 june 2014

The use of molecular methods in source attribution, outbreak investigations and surveillance

The use of molecular methods in source attribution, The use of molecular methods in source attribution, outbreak investigations and surveillance outbreak investigations and surveillance

Antonia Ricci



Terms of referenceTerms of reference



Molecular typing can be defined as the classification of microorganisms on the basis of variation in the genotype, and/orthe presence or absence of specific genes (such as those which may contribute to the pathogenicity of the organism or to its ability to survive in less favourable environments) (Hallin et al., 2012).

According to the European Centre for Disease Prevention and Control (ECDC), molecular typing refers to the application of laboratory methods capable of characterising, discriminating and indexing subtypes of microorganisms.

Some definitionsSome definitions……....



Molecular typing of pathogens complements traditional

epidemiological surveillance by providing appropriate discriminatory

analyses:

to allow the rapid and early detection of outbreaks;

to detect and investigate transmission chains and the

relatedness of strains;

to detect the emergence of antimicrobial resistance and new

evolving pathogenic strains;

to support studies to trace-back the source of an outbreak and

identify new risk factors, by linking isolates more accurately to

epidemiological and clinical data



Example of taxonomic nomenclature and general molecular typing nomenclature based on level of discrimination between isolates achieved when employing multi locus sequence typing (MLST)



The significance of genetic structuring for public health is two-fold:

different subgroups of bacteria, even within species, can vary

widely in their phenotypic properties, including those related to

pathogenicity, such as virulence or host association;

the size and diversity of bacterial populations is such that it is

necessary to be able to distinguish variants within isolates for the

purposes of epidemiological analysis and, particularly, in the context

of food-borne infections and source tracing.


Istituto Zooprofilattico Sperimentale delle Venezie · OIE Reference Laboratory for Salmonella



Molecular serotyping Molecular serotyping

The most common methodology uses either one of these two key principles:

1. examination of the genetic loci known to produce the serologically reactive components used in traditional serotyping,

2.examination of variations in the genome, which are indirectly associated with known serovars or serotypes. These variations may include various kinds of polymorphous regions, as long as they show a strong association to the traditional serovars/serotypes.



Low to moderate discriminatory capability, higher than traditional serotyping as sub-types can often be recognised within serotypes.

Reproducibility and repeatability are high.

Internationally harmonised standards for molecular serotyping are not in place except for L. monocytogenes.



Restriction Fragment Length Polymorphism (RFLP) analysis Restriction Fragment Length Polymorphism (RFLP) analysis

In RFLP, a target DNA sequence known to show polymorphism between strains of the species of interest, is cleaved with restriction endonucleases to generate fragments of varying length.

When RFLP analysis is directed at genes encoding ribosomal ribonucleic acid (rRNA) the method is usually referred to as ‘Ribotyping’. Ribotyping has successfully been automated, and fully automated ribotyping is commonly referred to as ‘riboprinting’ after the RiboPrinter® commercial system


Istituto Zooprofilattico Sperimentale delle Venezie · OIE Reference Laboratory for Salmonella

PulsedPulsed--Field Gel Electrophoresis (PFGE) analysis Field Gel Electrophoresis (PFGE) analysis

PFGE was first described in 1984 and is currently the most frequently used DNA-based typing method for food-borne bacterial pathogens. The PFGE-method standardization and rigid quality control introduced by PulseNetInternational has resulted in PFGE becoming the most commonly used method for outbreak identification, surveillance and investigation for a number of important pathogens, in particular Salmonella, STEC and Listeria.

For these pathogens, the performance of new typing methods will be measured against PFGE.



MultipleMultiple--Locus Variable number tandem repeat Analysis Locus Variable number tandem repeat Analysis (MLVA) (MLVA)

All bacterial MLVA-assays simultaneous measure the length of variable number of tandem repeat (VNTR) loci by PCR amplification and electrophoresis, and use this information to create a genotype to distinguish between isolates of the same species.

MLVA has several advantages:

a high index of discriminatory power, which can be easily adjusted by

inclusion or exclusion of loci to be investigated;

handling of pathogenic bacteria is low, which increases laboratory safety;

rapidity, as both PCR and electrophoresis times can now be greatly

reduced due to improved technology.



Multi locus sequence typing (MLST) Multi locus sequence typing (MLST)

MLST indexes sequences variation at a number (usually seven) genetic loci distributed around the chromosome. These are ideally ‘housekeeping’genes, i.e. genes encoding enzymes that are involved in primary metabolism of the organism in question and which are therefore present in all isolates

Introduced in 1998

Schemes exist for Campylobacter coli and C. jejuni, E. coli, Salmonella

enterica, and Listeria monocytogenes.

Accounts for high levels of recombination observed in many bacterial

populations.

Provides sequence data that can be analysed in a variety of ways to study

the population structure and evolution of bacterial pathogens.



Whole Genome Sequence (WGS) analysis Whole Genome Sequence (WGS) analysis

There are four approaches currently in use:

i.pyrosequencing, exemplified by the Roche 454 platform which can generate longer read lengths, but in smaller numbers and with potential miscalling of polynucleotide sequences;ii.Illumina sequencing technology, which produces shorter sequences with very high sequence capacity; iii.IonTorrent, which produces shorter sequences, also with a potential for miscalling polynucleotide tracts;iv.the PacBio SMRT sequencing system, which can produce very long sequences, but with relatively high error rates and cost.



The discriminatory capability of WGS is very high as it samples the whole genome, including extra-chromosomal DNA.

Reproducibility and repeatability are also high.

Current international harmonisation is lacking except for the availability of data management tools and annotation guidelines – but this does not provide for fully harmonised nomenclature.

The potential for future international harmonisation is unknown, but should be considered high from a technical point ofview.



S. 4,[5],12:i:- - DT 193 - R-type ASSuT emerged in several European countries and it has become one of the most dominant clones. Isolates presenting the PFGE type STYMXB.0131were responsible for two major outbreaks in Luxembourg in 2006.

Strains: S. 4,[5],12:i:-, ASSuT, DT193 STYMXB.0131 strains related to two epidemiologically unrelated outbreaks in Italy (one related to consumption of contaminated food in a rural-guesthouse restaurant – one related to contact with pets)

Aims:

- to confirm the outbreaks

- to investigate whether they were caused by the same clone

Analysis: PFGE (XbaI, BlnI, SpeI), MLVA

A couple of examplesA couple of examples……..



B

A



Barco et al., 2013

B

A



Results

VMST - DT 193 - R-type ASSuT STYMXB.0131 clone is circulating also in Italy and was responsible for two outbreaks

XbaI PFGE did not highlight the full diversity among strains – isolates related to the two outbreaks presented the same pulsotype

BlnI PFGE and MLVA clearly allocated the strains investigated into the twodifferent outbreaks

The subtyping methods used showed a satisfactory discriminatory power to differentiate epidemiological unrelated strains and provide sufficient epidemiologic concordance to be useful in outbreak investigations

When highly clonal strains must be typed it is necessary to use highly discriminatory methods



DT 7a is a quite rare phage-type, differing from DT7 for the clear reaction to phage 29

Strains

S. 4,12:i:- DT 7a STYMXB.0079 isolates obtained from attendees of a wedding reception (16) + from a cooked and highly processed pork product (porchetta) eaten during the weeding party (5)

S. Typhimurium DT7a STYMXB.0079 strains isolated from faeces of pigs raised on the same farm of origin of the pigs slaughtered to produce the contaminated pork

Surprisingly pigs harbored strains indistinguishable from the outbreak strains, all but they were biphasic (S. Typhimurium)

Was the source of this outbreak really the farm of origin of animals used to produce the pork product?



50 S. 4,12:i:- and S. Typhimurium DT7a epidemiologically unrelated (human, animal and environmental origin)

Aims:

- to confirm the outbreak

- to investigate the variability of DT7a strains (both serovars)

-Analysis:

PFGE (XbaI), MLVA

Strains



All strains related to the outbreak shared the same PFGE profile (STYMXB.0079)

Considering also epidemiologically unrelated strains, the same profile was identified for more than 80% of the isolates (both S. Typhimurium and S. 4,[5],12:i ) – more than 90% of the strains showed highly correlated profiles

S. Typhimurium and S. 4, 12:i:- DT7a strains, circulating at national level, are highly clonal



- The high clonality of this phage-type was demonstrated also through the MLVA analysis: all strains were included into the same cluster

- MLVA showed a discriminatory power higher than PFGE

- All but one strains of the outbreak shared the same MLVA profile. The same profile was shown also by strains of different origins (human –veterinary) and different serovar (S. Typhimurium, S. 4,12:i)

Due to this high similaraty between S. Typhimurium and S. 4, 12:i:- DT7a we cannot confirm that the source of this outbreak was the pig farm

To draw conclusions it is essential to know the level of varibility of the strains investigated

VMSTm outbreak

ST outbreak

VMSTm unrelated

VMSTm outbreak isolates



Applicability of typing methods for Salmonella source attribution (SSA) studies

Typing method Usedfor SSA

Applicabilityfor SSA Notes

Serotyping Yes Most common method used, more useful if used in conjunction with other methods

Phage ‐ typing Yes Valuable method for initial evaluation of relatedness of ST and SE strains

Antimicrobial resistance Yes Insability of the targets investigated

PFGE No Gold standard – it potentially represents a valuable method – well standaridized protocols

MLVA NoIt potentially represents a valuable method –serovar specific protocol – standardized protocols only for SE and ST

Plasmid profile No Intsability of the targets investiagated

Ribotyping NoComplexity of manual method – high cost of automated protocol – uncertainty with respct to discriminatory power

MLST No Low discriminatory power when applied within a Salmonella serovar

Barco et al., 2013

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Discriminatory capability

The ‘discriminatory power’ of a typing method is defined as the average probability that the method will assign a different type to two unrelated strains randomly sampled in the microbial population of a given taxon (Hunter, 1990)

To concludeTo conclude……....

What is the degree of discrimination between strains

of different genotype?

What is the consistency of results within and between laboratories, and over time?

Reproducibility and repeatability



Current international harmonisation:

a. Availability and use of Standard Operational Procedures; b. Availability and use of External Quality Assurance systems; c. Presence and use of harmonised nomenclature; d. Availability and use of data management tools.

Potential for future international harmonisation, in situations where any of the above criteria may apply but are not currently harmonised at international level.

What is the current status with regards to the following parameters?

the use of molecular methods in source attribution...

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