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Association Between CRISPR Phage DNA in Streptococcus mutans and Caries
Myra RanaAlabama School of Fine Arts
Dental Caries● Dental caries (tooth decay) is a multifactorial
widespread disease
● Low socioeconomic families and those with limited access have a higher prevalence
● Caries can do two things to the teeth:
○ Causes plaque- sticky biofilm
○ Lowers the pH, which creates a very acidic
environment, leading to demineralization
of the tooth
h t tp : / /w w w . ju n io r d e n t is t .c o m /d i f fe r e n c e s -b e tw e e n -n u r s in g - b o t t le - c a r ie s - a n d - r a m p a n t-
c a r ie s .h tm l
Bacteriophage (phage)
● Phage- a virus that injects DNA and replicates
within the bacterial cell
● Causes the bacterial cell membrane to break
down
https://www.news-medical.net/news/20151202/Bacteriophage-therapy-an-alternative-to-antibiotics-An-interview-Professor-Clokie.aspx
Streptococcus mutans and CRISPRS● The primary bacteria associated with caries is
Streptococcus mutans (S. mutans)● There are CRISPRs within this type of bacteria and
contribute to prokaryotic acquired immunity
● The bacteria obtain spacers from the genome of the
phage● These spacers are then placed in the CRISPR loci
○ Next time the same phage attacks, the CRISPR
system will recognize and degrade the phage
h t tp : / / r n a .b e r k e le y .e d u /c r is p r .h tm l
Objective and Hypotheses
The purpose of this research is to determine whether CRISPRs containing phage DNA within S. mutans are associated with caries experience
Hypothesis for Whole Genome Sequencing AnalysisThere will be an association between S. mutans with CRISPR containing phage DNA and cariesexperience
Hypothesis for Virulence Biofilm AnalysisThe biomass will be increased and pH will be decreased in Phage associated strains as compared to
No Phage
Introduction● A large scale epidemiological study evaluating S. mutans and dental caries was
conducted in Uniontown, Alabama
● Using bacterial-typing methods, the UAB Department of Pediatric Dentistry found
13,906 S. mutans strains from children and their household family members.
● In total, 34 S. mutans representative strain types were identified; these strains were
then assigned dmfs/DMFS (caries) scores
● A score of four or greater is considered high and a score less than four is considered
low for early childhood caries
Methodology
➢ Uniontown, Alabama, High Risk Caries, Limited
Access to care
➢ 40 S. mutans draft genomes
○ 34 representative of rep-PCR S. mutansclinical strains type
○ 6 strains were special cases
○ 33 Children
○ 7 Adult
➢ 988 Spacers
U niontow n, AL rep-PC R
Methodology-Whole Genome Sequencing Analysis
Draft Genomes
obtained by Illumina MiSeq
Sequences checked for
quality, trimmed
de novo assembly
using AbySS
dmfs/DMFS score are
assigned to strains
CrisprCompr Program
(CRISPR and Spacer Identification)
DNA sequence evaluated through NCBI
BLAST
Statistical Analysis
using Fishers Exact Test
Methodology-Virulence Biofilm Analysis
● Two assays:
○ Crystal Violet Assay- measures biofilm
○ pH rodo red/Cascade Blue Assay-measures pH (fluorescence of this dye increases as pH
decreases)
● Read assays using spectrometer (measures turbidity/cell density of biofilm)
● Image fluorescence using ImageJ
● T-test for statistical analysis
NCBI Blast Analysis
➢ Hypothesis= There will be an associationbetween S. mutans with CRISPR containingphage DNA and caries experience
➢ Two tailed Fisher's Test performed withp=0.05
➢ Fisher’s Exact test statistic = 0.0491Fail to Reject Hypothesis
➢ Hypothesis= There will be an associationbetween S. mutans with CRISPR containingphage DNA and caries severity
➢ Two tailed Fisher's Test performed withp=0.05
➢ Fisher’s Exact test statistic = 0.3476Reject Hypothesis
Crystal Violet Assay and pH rodo/Cascade Blue AssayM ean B iom ass of Phage vs. N o Phage S. m utans Strains
(Crystal V io let A ssay)
This data suggest that no phage strains have sign ificantly h igher b iom ass (p=0.006, re ject hypothesis) due to increased dextran (F ig . 3 ). The m ean pH w as
sign ificantly low er in Phage (p= 0 .011, fail to re ject hypothesis) (ind icated by h igher m ean fluorescence F ig . 4 )
M ean B iofilm pH of P hage vs. N o P hage S. m utans strain B iofilm s
(pH Rodo/Cascade B lue A ssay)
Representative Biofilm pH and Cell Density using Dextran Conjugated Probes
U A B -5 Phage U A B -24 N o Phage
Syto
9pH
rodo
Red
Mer
ged
U A B -5 Phage U A B -24 N o Phage
Syto
9M
erge
d
B iofilm pH by pH R odo R ed B iofilm D extran by C ascade B lue (contro l)
Casc
ade
Blue
M ean pH as indicated by pH rodo red intensity w as significantly low er in Phage stra ins.
M ean dextran as indicated by cascade blue w as significantly h igher in N o Phage stra ins.
Conclusion
➢ This data supports that there is a statistically significant association between S. mutanswith CRISPR containing phageDNAand caries experience
➢ Phage associated S. mutans strains may decrease biofilm pH and thus may haveincreased virulence potential in the strains
➢ Phage therapy for this high-risk caries population may not be an effective treatment
option
Future Research
➢ Future research will include choosing a larger sample size
➢ Further study is needed to determine how susceptible these S. mutans strains are to phage
infection
➢ Associations between CRISPRs and other origins such as food, animals, plants, and
chromosomes will be studied
➢ With these improvements, the study can be generalized and provide more insight to the
research of CRISPRs and oral bacteria
Acknowledgements● Dr. Noel Childers, UAB School of Dentistry, Department of Pediatric Dentistry
● Dr. Stephanie Momeni, UAB School of Dentistry, Department of Pediatric Dentistry
● Ms. Hungsin Chin, Alabama School of Fine Arts, Chair of Math and Science
● Ms. Jessica Mayne, Alabama School of Fine Arts
● Ms. Rebecca Thrash, Alabama School of Fine Arts
● Ms. Carol Yarbrough, Alabama School of Fine Arts