Abstract
Besides Campylobacter jejuni, Campylobacter coli is the most common bacterial cause of gastroenteritis worldwide. C. coli is subdivided into three clades, which are associated with sample source. Clade 1 isolates are associated with acute diarrhea in humans whereas clade 2 and 3 isolates are more commonly obtained from environmental waters. The phylogenetic classification of an isolate is commonly done using laborious multilocus sequence typing (MLST). The aim of this study was to establish a proteotyping scheme using MALDI-TOF MS to offer an alternative to sequence-based methods. A total of 97 clade-representative C. coli isolates were analyzed by MALDI-TOF-based intact cell mass spectrometry (ICMS) and evaluated to establish a C. coli proteotyping scheme. MLST was used as reference method. Different isoforms of the detectable biomarkers, resulting in biomarker mass shifts, were associated with their amino acid sequences and included into the C. coli proteotyping scheme. In total, we identified 16 biomarkers to differentiate C. coli into the three clades and three additional sub-clades of clade 1. In this study, proteotyping has been successfully adapted to C. coli. The established C. coli clades and sub-clades can be discriminated using this method. Especially the clinically relevant clade 1 isolates can be differentiated clearly.
Highlights
Intact cell mass spectrometry (ICMS) emerged as the standard method for the identification of microbial species in clinical microbiological laboratories[1,2,3]
Clade 1 isolates of C. coli are most frequently isolated from farm animals and clinical stool samples of humans suffering from acute diarrhea, whereas clade 2 and clade 3 strains, which are more closely related to each other, are mainly found in environmental waters and samples from waterfowl[32,33,34,35]
The clustered regularly interspaced short palindromic repeat (CRISPR) locus, which is considered to serve as prokaryotic immune system and protection against invasion of alien genetic elements is present in all C. coli clades, its genomic location differs[41,42]
Summary
Intact cell mass spectrometry (ICMS) emerged as the standard method for the identification of microbial species in clinical microbiological laboratories[1,2,3]. These strain-specific characteristics form the basis for the development of a novel microbial typing method that we initially named Mass Spectrometry-based PhyloProteomics (MSPP)[20,21], which we will, in accordance with the terminology used in the scientific community[8], refer to as proteotyping, as our method refers to a limited number of biomarkers and www.nature.com/scientificreports/. Even well-established whole genome sequencing-based MLST schemes are very expensive and time-consuming[25,26,27] These methods are not used in everyday clinical routine diagnostics and subtyping of microorganisms is currently restricted to a limited cohort, mostly in epidemiological surveys. C. coli isolates from different sources were MLST-typed and therewith it was shown that our test cohort included isolates of all three established clades and subclades These isolates were typed by ICMS/proteotyping and their phyloproteomic relatedness was deduced. Comparison of the obtained phyloproteomic proteotyping-based unweighted pair group method with arithmetic mean (UPGMA) tree with the corresponding MLST-based UPGMA dendrogram demonstrated that proteotyping is able to differentiate the clinically relevant clade 1 isolates from clade 2 and 3 isolates
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