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