Application of OrthoANI combined with MALDI-TOF MS in identification and typing of Campylobacter fetus

Abstract

Objective Establishing mass spectrum library of a Campylobacter- C.fetus subsp.testudinum for rapid species identification in clinical microbiology laboratory. Methods Illumina second generation sequencing platform 2000/miSeq was used to carry out high flux genome sequencing for strains which were collected to establish mass spectrum library.The analysis oforthologous average nucleotide identity (OrthoANI) between collected strains and reference strains was performed at JAVA 8 operation environment. Then, mass spectrums ofcollected strains andreference strains were acquired using MALDI-TOF MS. And mass spectrum library of C. fetus subsp.testudinum. were established and verified. Results The OrthoANI analysis showed that OrthoANI value of collected strains and reference strain C. fetus subsp.testudinum03-427 was 99.30%-99.96%, while OrthoANI values of collected strains and C. fetus subsp.venerealisNCTC10354 orC.fetus subsp.fetus82-40 were 91.05%-92.26%. With reference to OrthoANI ≥ 95% as basis for determination of same strain, strains which collected to establish mass spectrum library was finally identified as C. fetus subsp.testudinum . The identification accuracy rate of mass spectrum library was 100% (consistent with gene sequencing), and confidence interval was 82.3%-99.9%, identification of same strain is 100% reproducible. Conclusions The new gold standard based on high throughput sequencing and total genome analysis has provided ideal reference value for establishment of mass spectrum library.And accurate and objective reference spectrum of the C.fetus subsp.testudinum provides a platform for rapid diagnosis of fetal Campylobacter infection. (Chin J Lab Med, 2018, 41: 583-588) Key words: Campylobacter fetus; Bacteremia; Genomics; High-throughput nucleotide sequencing; Spectrometry, mass, matrix-assisted laser desorption-ionizatino; Bacterial typing techniques