Today, phylogenetic methods like MLST  and flaA-SVR sequencing  are considered to be the standard typing methods for C. jejuni isolates. Thus, every new classification technique must be compared with those genomic classifications . However, the genomic methods reflect some phenotypic aspects only insufficiently.
In this context, MALDI-TOF MS-based ICMS has recently advanced to be a widely used routine species identification tool for cultured bacteria and fungi [20–22]. In contrast to species identification by ICMS, subtyping within a single species (or differentiation between extremely close related species) is a more subtle process. Nevertheless, several examples already do exist proving the applicability of this method for isolate differentiation at the subspecies level, for example it was shown that methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains can be discriminated by ICMS . ICMS can also be used to differentiate between the Lancefield groups A, B, C, and G of Streptococci[29, 30]. Other examples are the subtyping of Listeria monocytogenes, Salmonella enterica[26, 32, 33], Yersinia enterocolitica, and Stenotrophomonas spp. .
The discrimination between the different Campylobacter and closely related species is well established and species-specific mass spectra are integrated in routine databases [23, 36–39]. It has also been demonstrated that shifts in biomarker masses, which are observable in MALDI-TOF spectra due to amino acid substitutions caused by nonsynonomous mutations in the biomarker gene, can be used to discriminate between the C. jejuni subspecies C. jejuni subsp. jejuni and C. jejuni subsp. doylei[37, 40].
As noted above the C. jejuni population is divided into two major isolate groups, which differ significantly from each other in stress response, transmission route, host tropism, temporal distribution, and pathogenic potential for humans. These two (including related C. jejuni subgroups) are associated with specific genetic markers. CC 21 isolates as well as the vast majority of other C. jejuni isolates are positive for cj1365c (cjj81176-1367/1371), cj1585c, cj1321-cj1326, fucP, cj0178, and cj0755/cfrA (Additional file 2: Table S2) [18, 19].
In contrast to that, MLST-CC 45 isolates and the related isolates of the MLST-CC 22, 42, and 283 are predominantly negative for these marker genes; with the exception that MLST-CC 22 and 42 isolates harbor cj1365c. In these isolates the oxidoreductase gene cj1585c is replaced by the tripartite anaerobic dimethyl sulfoxide oxidoreductase dmsA to –D facilitating an alternative anaerobic metabolic pathway. Additionally this isolate group has an extended amino acid metabolism and is characterized by the presence of ggt and ansB. The cj1365c-positive isolates of MLST-CC 22 and 42 are also cstII-positive, whereas MLST-CC 45 and 282 isolates have no LOS-sialyltransferase genes [18, 19]. Theses isolates positive for ggt but negative for fucP could be significantly associated with a higher rate of hospitalizations and bloody diarrhea and bear apparently a higher pathogenic potential for humans .
There are also smaller evolutionary intermediate isolate groups, which are for example positive for dmsA, ansB, cj1365c and fucP but not for ggt[18, 19].
Furthermore, MLST-ST 21 isolates have a variation of TLP7, which is expressed as dimer [18, 41]. In this group of isolates the most in vitro hyperinvasive strains can be found . These isolates are mostly responsible for outbreaks associated with cattle .
We have shown in this study that biomarker shifts can be used to discriminate not only between the vast majority of C. jejuni isolates and this C. jejuni subgroup with an extended amino acid metabolism (ggt
+), which was shown to have a higher pathogenic potential for humans , we were even able to discriminate between MLST-CC 45/282 isolates and MLST-CC 22/42 isolates. MLST-CC 22/42 isolates positive for the LOS-sialyltransferase cstII could be associated with GBS and higher host cell invasiveness .
Furthermore, we were able to identify another biomarker ion (m/z = 5303) that differentiates the subset of MLST ST 21 isolates associated with the dimeric TLP7m+c-variant.
It should be noted that the biomarker ions are not based on the expression of the marker genes used, as the proteins encoded in the marker genes are of entirely different sizes than the observed masses, but there is an obvious evolutionary association between the presence of specific marker genes and some of the biomarker ions.