Download Ribotyping

Using molecular methods the grouping of bacterium allow us to
determine if a collection of isolates are: part of an outbreak or
part of the background which occurs each year
Why do we want to know if a group of bacteria which were isolated
over a short time period are related?
Suggests there is a common source of infection (outbreak)
If there is an outbreak, pubic health measures may be able to do
something to remove the source of infection and decrease
future infections
If the isolates are not related; less like to have common source or
represent an outbreak situation- no need for intervention
Southern blot analysis
Transfer nucleic acid to membrane, treat to make single stranded
Apply probe which is labeled, hybridize, and detection
Restriction Fragment Length Polymorphism (RFLP)
DNA restriction enzymes recognize specific 4-8 base pair sequences
and cleave within this recognition site
RFLP= subtyping method that generates DNA fragments of different
lengths using restriction enzymes. The patterns can then be
analyzed isolates to determine if they are related or not
Most enzymes can be purchased
Cut any DNA regardless of source
Cut plasmid and chromosomal DNA
Basic protocol for chromosomal DNA is cut it with a restriction
enzyme, run on gel, make Southern blot and probe blot with a
probe(s) for multi-copy genes/sequences that exists in different
locations throughout the genome
Type of Genes used for RFLP Typing
Repeative sequences in the bacterial chromosome used to distinguish
related [outbreak] vs unrelated isolates [ not outbreak]
Chromosome cut with restriction enzyme: too many bands to work with
RFLP reduces the # of bands to a manageable level
1. Ribosome genes [Ribotyping]
Most bacteria have > 2 ribosomal gene sets [rrn]
(5S, 16S and 23S rRNA plus spacer regions) in chromosome
which show variability (polymorphisms)
Change in location of the rrn genes is slow so trait is stable
Genes between bacteria closely related so one set of probes allows
use a single set for most bacteria
Patterns are relatively simple
Need to know only that test microbe has > 1 ribosomal genes
Automated machines available with large # of references
Ribotype patterns for comparison; software available
Different programs may give different relationships between
strains
Software programs generate a Dendogram which is a tree for visual
classification so isolates with similar patterns are placed
closer together: can be used for all bacteria, and all other life
Ribotyping requires bacteria to have > 1 ribosomal set of genes
otherwise they CAN NOT BE Ribotyped
M. tuberculosis 1 set of genes
Bacteria with recent evolution CAN NOT BE Ribotyped
E. coli O157:H7 only recently developed so no differences in genes
locations
Ribotyping can be done for other E. coli isolates because they have not
recently evolved
2. IS sequence typing
First useable method for typing M. tuberculosis
Each species of Mycobacterium has unique IS sequences
Need to know what IS sequences present to use with species
Change in location of the IS sequences is slow so trait is stable
Patterns are relatively simple
Separate probes because each species IS sequence is different
so need to know the sequence of the IS element for each
bacteria: multiple probes needed
A few centers do this work and have a large bank of reference IS
patterns for comparison
Software programs available to produce dendogram
3. Phage typing
Requires multiple copies of phage in genome
Need to know information to do phage typing
Phage carried toxin genes often used as probes
Need to know about the phage genes to be used
4. Any other sequences for typing
Potentially any sequence that is repeated and inserted in varying
locations of the genome in different isolates could be used
for typing
Antibiotic resistance gene(s), virulence gene(s)
5. Plasmid RFLP
Instead of chromosome plasmid DNA can be purified and cut
specific antibiotic genes probes could then be used
Look for related genes in multiple different plasmids
6. PCR generated bands
PCR bands can be cut to and the resulting patterns compared
[more discussion with PCR lecture]
RFLP
1. RFLP can be done on plasmids, viruses, or whole eukaryotic
genomes
2. In small genomes [plasmids, viruses] look at banding pattern
directly
3. Large genomes [bacterial chromosomes] it is difficult to look
directly at banding pattern because of the large number of
bands: use probes to reduce the pattern complexity {ribotyping,
phage typing, IS typing, specific gene typing}
4. Probes used must different between isolates, is relatively stable
and chromosomal, often multiple copies are required for typing
5. Ribotyping is good for any bacteria that have > 2 ribosomal genes
6. Probes can be widely used or very specialized
RFLP Advantages
Ribotyping can be widely used with same primers, automation available,
reference patterns available, software available to make dendograms
1) Need to know limited amount of information about the microbe to be
typed
2) Same system used for some eukaryotics
3) With automations can handle large number of isolates
IS typing has worked well for Mycobacterium, able to send strains for typing to
a center which has reference patterns available, software available to make
dendograms, need to only know the species
Other probes need more information about the microbe to be typed
Patterns are usually very reproducible between laboratories
RFLP Disadvantages
Ribotyping not good for bacteria with 1 rrn set, or specific strains that
are relatively new, or has limited diversity in rrn
Automated equipment is relatively expensive and not worthwhile
unless doing large # of ribotypes
Different software can give different relationship patterns
Mycobacterium centers often do work retrospectively, answers may
take months-limited help with current situation
If DNA is not totally cut could give false pattern which is not
reproducible- see 1 band instead of 2
Phage typing-only works on bacteria that carry phage and there must be
variation in the location and number of phage in the species
Interpretation of data various by microbe and laboratory- are two strains
related only if they have identical patterns?
Pulsed-field gel electrophoresis (PFGE)
PFGE originally developed to look at multiple chromosomes of
parasites, yeast and some viruses
Does not require knowledge of the organism
Looks at the entire genome
Patterns can be analyzed with computer software-different programs
may give different groupings
LITTLE CONSENSUS OUTSIDE PULSENET & FOODNET ON HOW TO
DISTINQUISH BETWEEN RELATED AND NONRELATED
REQUIRES GROWTH OF THE ORGANISM
Need highly trained technicians
Time required for between 1-3 days
Used extensively for epidemiology
S. aureus cut with SmaI
PFGE Stability
My laboratory found that if you transfer a strain for 50 passages in the
laboratory and compare with parental isolate see no PFGE band
differences
In vivo: Had N. gonorrhoeae isolates for partners pairs over
75 days found two band change between the first and
last isolate in one of three enzymes used: indicates PFGE is
stable enough for typing
For PULSENET , MRSA typing 1 enzyme used due to cost: may call
two strains related when they are not
Personal work uses 2-3 restriction enzymes because one enzyme often
is not adequate to determine related from unrelated isolates
MRSA look at 70% related which can by 5-7 band differences
In my laboratory we use > 3 band differences needed with at least 2
different enzymes to call two isolates unrelated
PFGE can be used for all DNA carrying organisms: thus very
versatile
PFGE Advantages
1. Use for both prokaryotes and eukaryotes which can be grown
in the laboratory
2. PulseNet & FoodNet has standardized protocol, nomenclature
and large reference data bank for reference
3. Has been used to construct physical maps now PCR assay is
replacing PFGE
4. Used extensively for a wide range of microbes:
Generally considered Gold Standard for typing
5. As long as you can grow it you do not need to know much
else about the microbe
PFGE Disadvantages
1. Equipment costs $15,000-30,000
2. Enzymes and reagents are expensive
3. Requires highly trained technicians
4. Other than PulseNet, FoodNet there are no standards or agreement
on how to interpret results or nomenclature
5. Assay is very sensitive to small changes in conditions and
different machines from different manufactures can look
different
6. Can not be used with non-culturable microbes
7. PulseNet can suggest molecular linkage between 2 isolates
when there is no epidemiological data to support it