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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