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Proteotyping as alternate typing method to differentiate Campylobacter coli clades.

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.

SUBMITTER: Emele MF

PROVIDER: S-EPMC6414644 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost- and time-efficient routine monitoring.

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Proteotyping as alternate typing method to differentiate Campylobacter coli clades.

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