Proteotyping of Clostridioides difficile as Alternate Typing Method to Ribotyping Is Able to Distinguish the Ribotypes RT027 and RT176 From Other Ribotypes

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.

An Outbreak of Clostridium (Clostridioides) difficile Infections within an Acute and Long-Term Care Wards Due to Moxifloxacin-Resistant PCR Ribotype 176 Genotyped as PCR Ribotype 027 by a Commercial Assay

“…Later in the year, between October and December 2016, another 23 cases from five Slovakian hospitals were reported during the participation of Slovakia in the European standardized CDI surveillance [ 16 ], which indicates the possible dissemination of RT176 across healthcare facilities in the country. In addition to Slovakia, the epidemiologically significant occurrence of RT176 has been reported in its neighboring countries, Poland and the Czech Republic [ 18 , 19 ], as well as other countries in Europe: Germany, Croatia, Finland, Hungary and France [ 20 , 21 , 22 , 23 , 24 ] ( Figure 5 ).…”

“… European countries where the PCR ribotype 176 was reported. Sources of information include reports from: Czech Republic [ 19 ], Croatia [ 21 ], Hungary [ 23 ], Germany [ 20 ], Finland [ 22 ], France [ 24 ], Poland [ 18 ] and Slovakia [ 16 ]. …”

Antimicrobial Resistance Patterns in Clostridioides difficile Strains Isolated from Neonates in Germany

“…The highly pathogenic ribotypes 027 and 176 were excluded by proteotyping using the Autoflex III smart beam mass spectrometer (Bruker) as described recently [ 22 ].…”

Rapid Classification of Clostridioides difficile Strains Using MALDI-TOF MS Peak-Based Assay in Comparison with PCR-Ribotyping

“…PCR-ribotyping and PFGE are the most used genotyping methods adopted in Europe and Northern America, respectively. PCR-ribotyping characterizes different C. difficile strains by the amplification of the Intergenic Spacer Region (ISR), located between 16S and 23S ribosomal genes, which has intraspecific high variability in terms of both length and nucleotide sequence; therefore, its variations identify different ribotypes [ 20 , 21 ]. PFGE is based on the catalytic activity of the SmaI restriction enzyme, which cuts C. difficile genomic DNA at specific restriction sites: the long DNA fragments obtained are separated by size, with an agarose gel electrophoresis on an electromagnetic field [ 20 ].…”

“…During the last decade, Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is catching on as a valid support in the workflow for laboratory diagnosis in clinical microbiology, especially for bacteriological identification [ 21 ] and virology [ 25 , 26 ]. More recently, thanks to its rapidity, accuracy and moderate price, MALDI-TOF MS has been employed as an alternative in the detection of antibiotic susceptibility/resistance biomarkers [ 27 , 28 ], in the identification of aminoacidic sequences and chemical structure of protein terminal groups [ 29 ], and as an emerging method in microbial typing [ 29 , 30 ].…”