Mass spectrometry proteomics data are typically evaluated against publicly available annotated sequences, but the proteogenomics approach is a useful alternative. A single genome is commonly utilized in custom proteomic and proteogenomic data analysis. We pose the question of whether utilizing numerous different genome assemblies in a search database would be beneficial. We reanalyzed raw data from the exoprotein fraction of four reference Enterobacterial Repetitive Intergenic Consensus (ERIC) I-IV genotypes of the honey bee bacterial pathogen Paenibacillus larvae and evaluated them against three reference databases (from NCBI-protein, RefSeq, and UniProt) together with an array of protein sequences generated by six-frame direct translation of 15 genome assemblies from GenBank. The wide search yielded 453 protein hits/groups, which UpSet analysis categorized into 50 groups based on the success of protein identification by the 18 database components. Nine hits that were not identified by a unique peptide were not considered for marker selection, which discarded the only protein that was not identified by the reference databases. We propose that the variability in successful identifications between genome assemblies is useful for marker mining. The results suggest that various strains of P. larvae can exhibit specific traits that set them apart from the established genotypes ERIC I-V.

• MeSH
• bakteriální proteiny * genetika   metabolismus   MeSH
• databáze proteinů MeSH
• faktory virulence * genetika   metabolismus   MeSH
• genom bakteriální * genetika   MeSH
• Paenibacillus larvae * genetika   patogenita   metabolismus   MeSH
• proteogenomika * metody   MeSH
• proteomika metody   MeSH
• včely mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

• MeSH
• Enterococcaceae * MeSH
• larva mikrobiologie   MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• Paenibacillus larvae * genetika   MeSH
• Paenibacillus * genetika   MeSH
• plazmidy genetika   MeSH
• transpozibilní elementy DNA MeSH
• včely genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH