Microbial Sequences in Multiple Sclerosis Brain Specimens John D. Kriesel, Preetida Bhetariya, Cheryl Palmer, and Kael Fischer University of Utah School of Medicine, Salt Lake City, Utah, USA Session: 243. Host-Pathogen Interactions Presentation Number 2206 ABSTRACT RESULTS Table 2. MS Microbial Candidate List Background: Our group has developed RNA sequencing and analysis for Figure 2 Normalized Hit Rate Sum MS Group the identification of microbes in diseased brain tissue. Hierarchical Cluster 3.0 Analysis. Phylum1 Contributing Families2 Raw Hits3 # Specimens Increased4 Growth Conditions Methods: Twelve primary demyelination (MS) biopsy and 15 control Log2 transformed normalized Euryarchaeota Methanobacteriaceae 162 3 Anaerobic epilepsy surgical human brain specimens were studied. RNA was hit rates (in pairs per million (archeae) Methanosarcinaceae 96 1 Anaerobic extracted and quantified from the formalin-fixed paraffin-embedded reads, PPM) were centered for all Unclassified (NC10) Unclassified 66 4 n/a specimens, subjected to quality control, and then deep sequenced. The microbial families where at least Acidobacteria Acidobacteriaceae 1266 5 Aerobic reads were filtered and aligned against a panmicrobial database of 1.3 one MS samples was significantly Atopobiaceae 1588 2 Anaerobic Coriobacteriaceae 562 3 Anaerobic million (M) sequence records representing 10,654 species. Microbial overrepresented (FDR, q < 0.05) relative to the set of controls. The Actinobacteria Eggerthellaceae 578 6 Anaerobic reads and human gene expression were compared between the groups. Rubrobacteraceae 472 3 Aerobic, 60˚C. MS (N=12) and epilepsy control Results:100-300M high-quality 125 bp paired-end reads were derived Unclassified 76 4 n/a (N=15) sample values (columns) from each brain specimen. 6.4M read pairs (0.1% of total), from both the Porphyromonadaceae 4330 4 Anaerobic MS and control specimens, aligned to the panmicrobial database. Outlier were used to center the data Bacteroidetes Rikenellaceae 3574 2 Anaerobic analysis, corrected for multiple comparisons, identified significantly for display. The mean HR value Flammeovirgaceae 218 2 Aerobic increased microbial sequence in 11/12 MS brain specimens. 43 genera for each microbial family was Flavobacteriaceae 84 2 Anaerobic and 36 families from 11 microbial phyla (archaea 2, unclassified 1, subtracted from each cell in the Cyanobacteria Gomontiellaceae 284 2 Aerobic Leuconostocaceae 194 2 Aerobic bacteria 11, fungi 2, viruses 1) were overrepresented in at least one of row to provide a visual display of distance from the mean, expressed Clostridiales Family XVII 94 4 Anaerobic the MS brain samples. Many of the MS candidate microbes are anaerobic Firmicutes Lachnospiraceae 866 2 Anaerobic (Alistipes, Akkermansia), noncultivable (Candidatus), or extremophiles as log2 increases (yellow) or decreases (blue) from the mean Peptococcaceae 480 1 Anaerobic (Lawsonia). Gene expression analysis showed that several immune Thermoanaerobacterales 90 5 Anaerobic, 55˚C value (black). A clustering of system pathways are significantly increased in the MS brain specimens. Erysipelotrichaceae 996 2 Aerobic candidate MS microbial families Conclusions: The sequencing data shows that most of the MS brain Fusobacteria Fusobacteriaceae 230 1 Anaerobic among 6 MS brain specimens can Nitrospirae Nitrospiraceae 230 2 Aerobic with nitrite specimens studied contain a set of microbial sequences that are be seen in the upper right corner. significantly different than the controls. This suggests that MS lesions Planctomycetes Planctomycetaceae 156 2 Aerobic Rhodospirillaceae 412 2 Aerobic or anaerobic may be related to the invasion of a diverse set of microbes into the brain, Anaplasmataceae 166 3 Chicken embryo or to a disturbance of preexisting microbes within brain tissue. Human Figure 3. Bacterial Antigen in Human Brain Specimens. Brain specimens were stained with the mouse mAb M995 against bacterial Proteobacteria Desulfovibrionaceae 126 2 Anaerobic gene expression in these brain specimens supports a role for a bacterial Cystobacteraceae 498 1 Aerobic pathogenesis of MS. Studies are in progress to visualize specific MS peptidoglycan, or the macrophage/microglia anti-CD68 Ab, or no primary Ab (control). Magnification is 125x. Tenericutes Acholeplasmataceae 206 2 n/a candidate microbes within the brain tissue specimens. Mycoplasmataceae 152 1 Aerobic MS-019 anti-PG C-40 anti-PG Brain Abscess anti-PG Verrucomicrobia Akkermansiaceae 25094 1 Anaerobic Ascomycota Saccharomycetaceae 242 1 Aerobic Figure 1. Sequencing and Alignments. Basidiomycota Ustilaginaceae 1080 3 Aerobic Viruses Bromoviridae 486 2 n/a Table 2 Footnotes 1 As classified in NCBI Taxonomy. 2 Genera within the phylum that had the HR from one or more MS samples significantly increased over the set of controls (N=15). To be listed here, the sum of normalized HRs over the entire MS group (N=12) had to exceed 1.0 pair per million read-pairs. 3 Concordant read pairs aligning to this genus summed over the entire MS group (N=12) 4 Number of MS brain specimens that had HRs significantly greater (one-tailed q<.05) than the set of controls (N=15). CONCLUSIONS MS-019 control C-40 control MS019 anti-CD68 1. Certain microbial sequences, representing microbial RNA, are significantly enriched in many of the MS brain specimens compared to a set of 15 epilepsy control brain specimens. 2. No single microbial family, genus, or species was specific to MS. However, a cluster analysis shows that a set of diverse anaerobic and extremophile microbes are associated with 6 of the 12 MS cases we studied. 3. The presence of bacterial antigen was confirmed by immunohistochemistry in all the MS brain specimens where there was sufficient specimen for analysis (N=4). 4. The human gene expression analysis revealed overexpression of immune system related genes in the MS group compared to controls. Some of the overexpressed genes are consistent with an anti-bacterial immune response (e.g. TLR2, TLR4). 5. Additional studies are in progress to determine the significance of these findings..