Characterisation of strains serves to describe the population structure of a species and has been mostly used to research the epidemiology of disease, phylogenetics or
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pathogenicity of microorganisms. Genotyping is the preferred method for both identification and subtyping of microorganisms rather than phenotyping (49). As such, early Campylobacter phenotyping schemes such as those based on antibiotic sensitivity profiles, serotyping, or biotyping have been superseded by schemes based on the genetic makeup of the organisms, genotyping (17, 47) hence phenotyping has been omitted in this review. Genotypic methods are divided in three main categories: (1) DNA banding pattern-based methods, which classify bacteria according to the size of fragments generated by amplification and/or enzymatic digestion of genomic DNA, (2) DNA sequencing-based methods, which study the polymorphism of DNA sequences, and (3) DNA hybridisation-based methods using nucleotide probes (49).
There is a large number of restriction enzymes that can be used to cut (digest) DNA at specific sequence. Digestion by restriction enzymes and amplification of DNA produces millions of copies of fragments available for analysis (229). Pulse-field gel electrophoresis (PFGE) is an enzymatic restriction-based method that separates large DNA molecules in a flat agarose gel by applying alternating electric fields at different angles. PFGE has a high discriminatory power and is considered a “gold standard” for typing of many bacteria (230). Restriction enzymes most commonly used with C. jejuni are SmaI, SalI, KpnI, ApaI, and BssHII (229) whereas XhoI appears to be the most useful for C. upsaliensis (231). PFGE has also been successfully applied to C. coli (232), C. fetus (233) and C. hyointestinalis (234). The main limitation of PFGE is the time and labour consuming aspect of the method and the lack of inter-laboratory comparability due to considerable variations in the restriction enzymes and electrophoretic conditions (229).
Similar issues with inter-laboratory comparability can affect ribotyping. Ribotyping is a method based on genotyping of rRNA genes using agarose gel electrophoresis of digested genomic DNA followed by Southern blot hybridisation with a probe specific for rRNA genes (5S, 16S and 23S). Unlike PFGE, the discriminatory power of ribotyping is limited because most Campylobacter spp. contain only three rRNA gene copies (229). Ribotyping was successfully applied to investigate C. upsaliensis
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outbreak in a children’s daycare centre in Belgium (235) and, in combination with plasmid profiling, for comparisons of human and canine C. upsaliensis isolates (236). Restriction fragment length polymorphism (RFLP) is similar to ribotyping except that hundreds of short fragments are generated. This poses difficulties in separating fragments by agarose gel electrophoresis but this can be mitigated using Southern blotting with radioactively labelled probes (237). Another way to overcome this shortcoming is to apply RFLP to a specific locus amplified by PCR, thus enzymatic digestion is applied following DNA amplification. Typing of Campylobacter spp. using this technique was applied to flagellar genes flaA and flgE which are a highly conserved yet variable region (238) but with variable success partly due to at least seven different procedures reported (229). It was also shown to be applicable to C. coli, C. lari, and C. helveticus (239). By applying a multiplex PCR to more than one locus, the PCR-RFLP method with C. jejuni genes gyrA and pflA reached the discriminatory power of PFGE (240).
Amplified fragment length polymorphism (AFLP) is another highly discriminatory technique using restriction enzyme digestion following amplification by PCR. The technique is based on two restriction enzymes with recognition sites of variable length that guide PCR amplification so that only those fragments flanked by both restriction sites are amplified (229). AFLP was successfully applied to characterise C. jejuni (241), C. coli (242), C. lari (243) and C. upsaliensis (244). Comparative studies in C. jejuni showed AFLP to have higher discriminatory power than both PFGE and PCR-RFLP (245), and was comparable to multi-locus sequence typing (MLST) and sequence analysis of clustered regularly interspaced short palindromic repeats (CRISPRs) (246). However, the disadvantages are that the AFLP technique is complex (comparable to PFGE) and requires major capital investment (an automated DNA sequencer and appropriate software) (229).
Typing methods based on fragment analysis may also be based on DNA amplification without the use of restriction enzymes. The afore mentioned CRISPR analysis is such a method based on typing of near-perfect direct repeat sequences (usually 24-48 bp) that are interspersed with (similarly sized) non-repetitive spacer
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sequences (49, 246). High-resolution melting (HRM) uses PCR amplification and, through melting curve analysis, enables discrimination of DNA alleles to the level of single nucleotide polymorphisms (SNPs) (49). These methods can be combined, such as HRM analysis of hypervariable CRISPR regions of C. jejuni and binary gene typing (247) in order to give equivalent or better performance than the “gold standard” of PFGE (230). Another method called Rapid Amplified Polymorphic DNA (RAPD) analysis is based on the use of arbitrary short single primers to amplify genomic DNA at multiple loci and has been used to characterise Campylobacter spp. (49). However, while the RAPD method is inexpensive and with a fast turnaround time, the poor reproducibility of results between laboratories is a major disadvantage (248). DNA banding differences due to the influence of subjective interpretation of RAPD data were observed between strains from an outbreak of C. jejuni (249) and between duplicate samples (68). A slightly different approach for amplifying random genomic DNA fragments involves using primers specific for enterobacterial repetitive intergenic consensus (ERIC) sequences but this method is still limited by low reproducibility (229).
As each organism is uniquely defined by its DNA sequence, typing methods based on DNA sequencing have perhaps been the most successful due to the resolution of the data obtained and provision of the broadest range of applications (17, 49). Molecular cloning, breeding, species identification, genetic and genomic comparative studies as well as phylogenetic and evolutionary studies are available, to name a few. The major advantage of sequencing methods is the reproducibility between laboratories and the ease of sharing data that enables a ready use within research communities (49). Taken together, these are the reasons that over the last decades the sequencing technologies had the largest development of all typing methods. Of the public databases, GenBank is currently the largest online DNA sequence database (http://www.ncbi.nlm.nlh.gov/genbank).
Since the first independent development work by the Sanger (the dideoxy method), and Maxam and Gilbert (the chain-termination method) teams (for which they shared the Nobel Prize in 1980), today there are three generations of methodological design (49). The second-generation methods are characterised by various approaches that
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rely on PCR amplification such as sequencing by ligation (SOLiD), by synthesis (Roche 454 Pyrosequencer, Illumina) or semiconductor-based detection of hydrogen release during DNA polymerisation (Ion Torrent). The third generation methods (e.g., the Pacific Biosciences system and nanopore sequencing) are characterised by removing the reliance on PCR amplification and by signal detection during the enzymatic reaction of adding nucleotides to the complementary strand in real time.
Early DNA sequencing methods were limited to one or a few genes due to constraints on cost, time, and availability. Genes highly conserved between bacteria are useful for identification and phylogenetic analyses, for instance the 16S rRNA gene essential for bacterial survival (250, 251). The RpoB gene (252) that encodes the ß subunit of RNA polymerase, which is presented in Fig. 2.4, and the groEL gene (253) encoding a universal 60-kDa chaperonin involved with heat-shock response, and the flagellar gene fla (238) have also been used for identification and/or typing of
Campylobacter species. However, the multi-locus sequence-typing (MLST) scheme based on sequencing of multiple loci, all housekeeping genes, has been one of the most widely adopted methods (254, 255) and to date the schema exists for many different species (http://pubmlst.net/databases/default.asp). The allelic profile for each locus, a fragment of a gene in the scheme, is assigned a unique number in order of its discovery and isolates with identical sequences are assigned the same allele number. In the C. jejuni/coli MLST scheme, distinct allelic profiles of seven loci characterise the isolate and the sequence type (ST) is defined by the combination of alleles at each locus (256). The clonal complex (CC) groups are formed by two or more isolates that share identical allelic profiles for at least four loci and is named after the ST identified as the putative founder of the group. MLST schema exist for C. coli, C. lari, C. upsaliensis, C. helveticus (257), C. fetus (258), C. sputorum, C. hyointestinalis, C. curvus, and C. concisus (259). The use of MLST in Campylobacter
research has been applied to source attribution studies in New Zealand (260) and worldwide (261-264), niche adaptation (265), investigations of origin of antibiotic- resistance (266), genetic diversity (267) or distributions of a specific clone (268), and phylogenetic studies (269, 270).
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Fig. 2.4. Neighbour-joining phylogenetic tree of the genus Campylobacter based on partial rpoB gene sequences. E. coli was used as an outgroup. Bootstrap values of 500 simulations are indicated at major branches. Bar, 2% divergence.
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As the development of sequencing methods has progressed and became more accessible and less cost-prohibitive, more complex typing schema have been developed too. For instance, unlike MLST, the whole-genome needs to be sequenced for a ribosomal MLST (rMLST). The rMLST scheme uses 53 housekeeping genes that encode the bacterial ribosome protein subunits (271). The extension of sequencing of multiple loci may be applied to whole genomes too (272, 273). Whole genome sequencing has the highest discriminatory power that can differentiate isolates down to the meroclone (variants within the colony forming unit) and clone level, whereas rMLST is suitable for differentiating from the species to strain level, and MLST can to the genus, species and the lineage or clonal complex while 16S rRNA can only discriminate to the genus level (274).
The sequencing of the C. jejuni genome (150) was a significant landmark and large- scale comparative genomic studies revealed extensive inter- and intra-species diversity (275) and introduced new concepts such as the core and pan-genome to species computational biology. The pan-genome of a species is a sum of all of the genes present in all strains of the respective species, whereas the core genome are those genes that are exclusively present in each and every strain of that species. The difference between these two sets of genes is a dispensable or accessory genome that represents genes present in some but not all of the species’ strains (276). Therefore, the core genome is considered to represent genes involved with major genotypic (and accordingly phenotypic) traits of a species while the accessory genome contributes to the species’ diversity and may confer differential features between strains such as antibiotic resistance, niche adaptation and the ability to colonise new hosts (276). Species may differ in the proportions of their core genomes in their pan-genomes (277). Species with a smaller core genome are associated with living in a highly variable environment with a sympatric lifestyle to which the large accessory pool has a greater ability to respond to. In contrast, species with a large proportion of the genome represented in the core genome are associated with a stable, or isolated environment and allopatric lifestyle (277, 278). The pathogenicity of a species has been shown to be associated with genome reduction due to gene loss and gene degradation, resulting in pseudogenes, a
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pattern that was observed in comparison between facultative and obligate pathogens (279).
Whole-genome analyses in Campylobacter research have been mostly focused on
C. jejuni and C. coli. One study showed how the pan-genome of C. jejuni and C. coli
combined is around 3,000 genes but each species has a pan-genome size of around 2,600 genes. This demonstrated that the gene repertoire of the two sister species are largely overlapping (280). Another study showed evidence of a convergence of C. jejuni and C. coli species, that is, the clade 1 of the C. coli population was “despeciating” toward C. jejuni (269). However, there are debates over this phenomenon, with suggestions that interspecies genetic exchange is rare and limited, and biased by only a few housekeeping genes and the boundary between the two species is unlikely to be eroded (281). An important note is that genome association studies are in the relatively early stages, and the observed differences in results between studies can be due to sampling and analytical methods (282). Nevertheless, this is an active research area and is likely to be significantly expanded in the coming years and further combined within the genome-wide association studies framework with other “~omics” techniques such as analysis of RNA (transcriptomics), protein (proteomics), metabolites (metabolomics) and other phenotypic methods such as phenotype microarray systems (283 , 284).