Abstract
Clostridioides difficile, a Gram-positive spore-forming bacterium, is the leading cause of nosocomial diarrhea worldwide and therefore a substantial burden to the healthcare system. During the past decade, hypervirulent PCR-ribotypes (RT) e.g., RT027 or RT176 emerged rapidly all over the world, associated with both, increased severity and mortality rates. It is thus of great importance to identify epidemic strains such as RT027 and RT176 as fast as possible. While commonly used diagnostic methods, e.g., multilocus sequence typing (MLST) or PCR-ribotyping, are time-consuming, proteotyping offers a fast, inexpensive, and reliable alternative solution. In this study, we established a MALDI-TOF-based typing scheme for C. difficile. A total of 109 ribotyped strains representative for five MLST clades were analyzed by MALDI-TOF. MLST, based on whole genome sequences, and PCR-ribotyping were used as reference methods. Isoforms of MS-detectable biomarkers, typically ribosomal proteins, were related with the deduced amino acid sequences and added to the C. difficile proteotyping scheme. In total, we were able to associate nine biomarkers with their encoding genes and include them in our proteotyping scheme. The discriminatory capacity of the C. difficile proteotyping scheme was mainly based on isoforms of L28-M (2 main isoforms), L35-M (4 main isoforms), and S20-M (2 main isoforms) giving rise to at least 16 proteotyping-derived types. In our test population, five of these 16 proteotyping-derived types were detected. These five proteotyping-derived types did not correspond exactly to the included five MLST-based C. difficile clades, nevertheless the subtyping depth of both methods was equivalent. Most importantly, proteotyping-derived clade B contained only isolates of the hypervirulent RT027 and RT176. Proteotyping is a stable and easy-to-perform intraspecies typing method and a promising alternative to currently used molecular techniques. It is possible to distinguish the group of RT027 and RT176 isolates from non-RT027/non-RT176 isolates using proteotyping, providing a valuable diagnostic tool.
Highlights
Clostridioides difficile (Lawson et al, 2016) is a Gram-positive anaerobic spore former and the most frequent cause of antibioticassociated diarrhea (Lo Vecchio and Zacur, 2012; Leffler and Lamont, 2015; Martin et al, 2016; Smits et al, 2016)
For all isolates included in the study, observed mass shifts in comparison to the spectrum of the C. difficile reference strain 630 (= DSM 27543) were noted and the allelic isoforms assigned by comparing observed mass shifts with the established isoform list
A proteotyping-based phyloproteomic tree was calculated from concatenated biomarker amino acid sequences and compared to the respective multi locus sequence typing (MLST) data constructed in an analogous fashion
Summary
Clostridioides difficile (Lawson et al, 2016) is a Gram-positive anaerobic spore former and the most frequent cause of antibioticassociated diarrhea (Lo Vecchio and Zacur, 2012; Leffler and Lamont, 2015; Martin et al, 2016; Smits et al, 2016). The symptoms of a C. difficile infection (CDI) appear in various manifestations: The spectrum comprises rather weak symptoms like mild diarrhea and serious forms like toxic megacolon, pseudomembranous colitis (PMC) or perforation of the colon (Nanwa et al, 2015). Despite the fact that the involvement of the small intestine has been observed, characteristic PMC lesions are usually limited to the colon (Jacobs et al, 2001; Keel and Songer, 2006). Infections outside of the intestine only occur very rarely (Byl et al, 1996)
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