Fusobacterium nucleatum Subspecies Identification by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

hWe explored the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification of Fusobacterium nucleatum subspecies. MALDI-TOF MS spectra of five F. nucleatum subspecies (animalis, fusiforme, nucleatum, polymorphum, and vincentii) were analyzed and divided into four distinct clusters, including subsp. animalis, nucleatum, polymorphum, and fusiforme/vincentii. MALDI-TOF MS with the modified SARAMIS database further correctly identified 28 of 34 F. nucleatum clinical isolates to the subspecies level. Fusobacterium nucleatum is an opportunistic pathogen, associated with various forms of periodontal diseases and extraoral infections, as well as colorectal cancer (1, 2). F. nucleatum is a highly heterogeneous species and was classified into five subspecies: animalis, nucleatum, polymorphum, vincentii, and fusiforme (3-5). Recent studies based on phylogenetic analysis of the nucleic acid sequences of 16S rRNA, rpoB, zinc protease, and 22 other housekeeping genes suggested that F. nucleatum subsp. fusiforme and vincentii be classified into a single subspecies, F. nucleatum subsp. fusiforme/vincentii (4, 5).Different subspecies may vary in pathogenesis relating to different levels of disease activity (6-8). Fusobacterium nucleatum subsp. nucleatum is isolated mostly in periodontal diseased sites, whereas F. nucleatum subsp. fusiforme/vincentii is often isolated from healthy sites as normal flora (9). F. nucleatum subsp. animalis and polymorphum are associated with pregnancy complications (7), and F. nucleatum subsp. animalis is also associated with inflammatory bowel disease (10). Until now, molecular technologies have been the most effective and widely accepted tools for subspecies identification (3,4,11). At the subspecies level of F. nucleatum, the sequence divergences of 16S rRNA genes were only 0.6% to 1.9%, so full-length sequencing of 16S rRNA was desirable (4).This study aimed to explore the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of F. nucleatum at the subspecies level. A commercially available database was amended using 15 F. nucleatum isolates comprising the type strains and other wellcharacterized clinical isolates of the five subspecies and then tested for rapid identification of F. nucleatum subspecies.(This study was presented in part at the 113th (10). A total of 34 F. nucleatum clinical isolates were collected from Case Western Reserve University, Memorial Sloan-Kettering Cancer Center, Harvard University School of Public Health, and bioMérieux, Inc. The isolates were stored in Trypticase soy broth with glycerol at Ϫ80°C, cultivated on Columbia agar (Becton, Dickinson and Company, Sparks, MD) supplemented with 5% sheep blood, and incubated at 37°C for 48 h in an anaerobic chamber, as previously described (10).Full-length 16S rRNA gene sequencing. Bacterial DNA was extracted from a pure colony with a Pure Link genomic DNA minikit (Invitrogen, Carlsbad, CA). The 16S ...

Fusobacterium�nucleatum promotes the progression of colorectal cancer by interacting with E‑cadherin

“…Previous studies have demonstrated the significant overabundance of F. nucleatum in colorectal adenomas and cancer. However, it remains unclear how F. nucleatum is involved in the CRC microenvironment (24,25). In the present study, F. nucleatum enhanced the growth and proliferation of NCM460 cells, suggesting that F. nucleatum may contribute to the development of CRC.…”

Fusobacterium nucleatum — symbiont, opportunist and oncobacterium

“…in this model, F. nucleatum evolved as a lineage with Fusobacterium periodonticum , and these species share not just a niche but also similar functions that are associated with invasion of host cells. F. nucleatum itself can be further delineated into four subspecies 97 — nucleatum , animalis , vincentii (inclusive of fusiforme ) and polymorphum — although it has been argued that these subspecies are sufficiently divergent at a DNa level as to be considered separate species 9,98 . traditional consideration of these subspecies as largely either commensal ( polymorphum and vincentii ) or disease-associated ( nucleatum and animalis ) merits re-evaluation as fusobacterial isolates from colorectal tumours encompass all of these subspecies 45 (Supplementary table 1).…”

Immunological Pathways Triggered byPorphyromonas gingivalisandFusobacterium nucleatum: Therapeutic Possibilities?

“…F. nucleatum is a human pathogen that is filamentous, Gram-negative, non-spore-forming, nonmotile, and anaerobic [13]. It is a heterogeneous species that belongs to the family Fusobacteriaceae and includes five proposed subspecies ( ss ): ss animalis , ss fusiforme , ss nucleatum , ss polymorphum , and ss vincentii [13, 39–41]. Despite the fact that F. nucleatum has been found in various tissues, the most common anatomical site in humans is the oral cavity [13].…”