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