There are several methods which could be used for molecular typing [356]. For this project, the data are produced by one molecular typing technique: multilocus sequence typing (MLST). A major advantage of MLST that is the results are com- parable among dierent laboratories [237], and information can therefore be shared worldwide.
Although MLST was originally designed for identifying lineages of pathogens, many other applications have shown a signicant usefulness of this method in a wide range of bacterial related study areas [237, 267].
2.3.1 Selection of MLST
As MLST provides data that can be used in long-term or short-term studies, it was selected for this study on Campylobacter. MLST is now a universally accepted system to characterize many bacterial species, including Campylobacter. MLST has many advantages over other methods for this research project, for example: a large number of Campylobacter are not typeable by serotyping [325]. Although the operation of AFLP is easier, faster and cheaper, AFLP is dicult to compare the results between laboratories due to variation of DNA sequence data caused by experimental dierences [325].
MLST and multilocus enzyme electrophoresis (MLEE) operate on the same prin- ciple, as both of them sequence fragments of multiple housekeeping genes selected across the genome [89]. MLEE is a band-based typing method, and it measures electrophoretic mobilities of selected metabolic enzymes. MLST is a sequence-based method. The advantage of MLST over MLEE is that the results from MLST are comparable between laboratories. Compared to MLEE, MLST is more precise and convenient [72, 237, 329]. Compared to a-RFLP, MLST has more discriminating power [75]. The combination of MLST and agellin A short variable region (aA SVR) can discriminate C. jejuni in outbreak investigations as well as PFGE [315]. Thus the introduction of MLST provided a useful tool for population genetic analysis [74, 237].
For evolutionary studies the sequence typing technique needs the following properties [237, 369]:
suitable for evolutionary modelling a highly discriminatory method the results should be unambiguous
the process can be repeatable across laboratories
MLST has the above properties, and it can be applied widely, and the results can be compared worldwide.
How MLST works
The aim of MLST is to provide an accurate and highly discriminating typing system that can be used for most bacteria and is particularly helpful for the typing of bacterial pathogens. This unambiguous procedure characterizes isolates of bacterial species using the DNA sequences of internal fragments of multiple (usually seven) housekeeping genes. This method uses approximately 450-500 bp internal gene fragments, which can be accurately sequenced on both strands using an automated DNA sequencer. At the gene level, each unique housekeeping gene sequence is assigned a distinct allele number within a bacterial species. At the isolate level, the alleles (usually seven) at each of the loci dene the allelic prole or sequence type (ST) [74, 237].
Data from many MLST analyses have been stored and can be accessed from the pub- lic database PubMLST [72]. Seven loci are chosen for Campylobacter MLST stud- ies: aspA (aspartase A), glnA (glutamine synthetase), gltA (citrate synthase), glyA (serine hydroxymethyl-transferase), pgm (phosphoglucomutase), tkt (transketolase), and uncA (ATP synthase a subunit). The reason for choosing seven housekeeping genes is because the information from these seven housekeeping genes provides dis- criminatory information equivalent to the 15 to 20 loci required by multi locus enzyme electrophoresis analyses [74, 237]. Because the positions of these seven housekeeping genes on the chromosome are far enough apart, it is unlikely that two of them will be changed in one recombination event (Figure 2.2) [74].
How MLST has been applied
MLST has been successfully applied to a wide range of bacteria [2, 88, 343, 358] and was rst used to analyse Campylobacter in 2001 [74]. Schouls et al. [2003] commen- ted that the typing of Campylobacter strains only works well when identifying an outbreak but may fail in source tracing and global epidemiology due to the enorm- ous variation in strains and the carriage of multiple types in animals. But they mentioned several future research areas, such as analysis of the Campylobacter host source. Later studies [72, 243, 260, 335] have indicated MLST's ability to identify dierent sources of infections of human diseases.
In 2004, MLST was applied to C. coli [72], which allowed C. jejuni and C. coli to be compared. Compared to C. jejuni, C. coli does not have a large diversity, but this may be due to the limited sample size for C. coli [72]. It is crucial to understand the molecular evolution of C. jejuni and C. coli, since both of them are responsible for a large percentage of gastroenteritis worldwide.
Figure 2.2: The positions of MLST loci on the chromosome of one C. jej strain NCTC 11168 (GenBank accession number NC002163) [286].
Because MLST can be repeated across laboratories, the results are comparable worldwide. MLST has been applied in dierent countries, such as Canada [49], Australia [66, 151], the UK [74], the US [237] and NZ [260]. Extended MLST (10- locus typing scheme) [73] represents a highly discriminatory typing scheme, which combines MLST, aA-SVR typing, aB-SVR, and porA typing systems. This ex- tended typing sequence method can also be useful in both long-term epidemiology and outbreak analysis [73].
PubMLST is a publicly accessible dataset which stores MLST typing results for several bacteria species, including C. jejuni and C. coli. In the PubMLST dataset for C. jejuni and C. coli, the genes, such as aA, aB and porA, related to cell surface antigens have been integrated.