Microbiome associated with severe caries in Canadian First Nations children

M Agnello3, J Marques1,2, L Cen3, A Huang1,2, B Mittermuller1,2, W Shi3, X He3, RJ Schroth1,2 1 College of Dentistry and College of Medicine, Faculty of Health Sciences, University of Manitoba 2 Children ‘s Hospital Research Institute of Manitoba 3 University of California – Los Angeles

Background • Canadian Aboriginal (First Nations, Metis, and Inuit)

and American Indian and Alaska Native children have high rates of severe Early Childhood Caries (S-ECC)

• QUEST organization: Quantifying, Understanding and Eliminating Severe Tooth Decay in AI/AN Children

• Knowledge gaps in our understanding of S-ECC. Four key topical areas—Epidemiology, Microbiology, Enamel Hypoplasia and New Products

Schroth, Harrison & Moffatt. Pediatriic Clinics of North America 2009.

2015 Symposium on Caries in American Indian and Alaska Native Children

Objectives

• To determine the caries-associated microbiome

among Canadian First Nations children with

severe Early Childhood Caries (S-ECC)

Methods – Study Population

• Medically healthy First Nations (and Métis) children enrolled from community clinics or from surgical facilities.

• Children with S-ECC were recruited at the Misericordia Health Centre

• Caries-free children were enrolled from the community in Winnipeg

• Living in Manitoba, Canada

• U of M Health Research Ethics Board approved & approved by Misericordia Health Centre

Methods Inclusion Criteria:

• Children <72 months of age

• Severe dental caries meeting the case definition for

S-ECC

• Parent or caregiver self-identifying the child as Canadian First Nations or Métis

Exclusion Criteria:

• Children >72 months of age

• Children who have taken antibiotics within the last 3 months

Goal: 20 children with S-ECC

20 Caries-free children

Methods • Questionnaire on general health,

oral health, demographics, and diet.

• Plaque and saliva samples collected following set protocol.

• DNA sequencing – Forsyth HOMINGS (Human Oral

Microbe Identification using Next Generation Sequencing) service was used

– Preliminary data analysis were conducted at UCLA

• Data analysis included descriptive and bivariate analyses (Chi Square and t tests). P ≤ 0.05 was significant

Results – Characteristics of Participants

VARIABLE S-ECC Caries-Free P Value

Sex

Male

Female

19 (63.3%)

11 (36.7%)

9 (45.0%)

11 (55.0%)

0.20

Child's Age

(months) 42.8 ± 12.2

37.4 ± 10.3 0.11

Lives in FN Community

Yes

No

17 (56.7%)

13 (43.3%)

0 (0%)

20 (100%)

< 0.0001

Indigenous Group

First Nations

Métis

30 (100%)

0 (0%)

19 (95.0%)

1 (5.0%)

0.22

Birth Weight (grams) 3421.3 ± 573.2 3529.9 ± 699.0 0.22

Results VARIABLE S-ECC Caries-Free P Value

Child's overall health

Fair

Good

Very Good

1 (3.3%)

17 (56.7%)

12 (40.0%)

0 (0%)

6 (30.0%)

14 (70.0 %)

0.082*

Child was breastfed

Yes

No

12 (40.0%)

18 (60.0%)

13 (65.0%)

7 (35.0%)

0.083

Age when

weaned/stopped

breast (months)

3.3 ± 5.4

12.9 ± 11.4

0.014

Bottle fed

Yes

No

30 (100%)

0 (0%)

16 (80.0%)

4 (20.0%)

0.021*

Results

VARIABLE S-ECC Caries-Free P Value

Age when weaned off

bottle (months)

25.8 ± 12.0

17.9 ± 8.9

0.028

# times of day child

snacks

3.9 ± 1.4

3.7 ± 1.7

0.71

How often are child's

teeth cleaned/brushed

≥ daily

< daily

16 (53.3%)

14 (46.7%)

3 (15.0%)

17 (85.0%)

0.032*

Yearly household

income

<$28K

>$28K

Not Sure

25 (86.2%)

3 (10.3%)

1 (3.5%)

12 (65.0%)

7 (35.0%)

1 (5.0%)

0.098*

Results

• Samples

– Saliva samples have poor DNA quality

– Plaque samples have good DNA quality

• Microbiome analyses revealed no unique

pathogens

Car

ies-

free

ECC

0

50

100

Rela

tive A

bu

nd

an

ce (

%)

Streptococcus sanguinis

Streptococcus mutans

Haemophilus parainfluenzae

Veillonella dispar

Lautropia mirabilis

Leptotrichia shahii

Prevotella melaninogenica

Gemella haemolysans

Alloprevotella sp. oral taxon 473

Abiotrophia defectiva

Corynebacterium matruchotii

Granulicatella elegans

Porphyromonas sp. oral taxon 279

Rothia dentocariosa

Neisseria pharyngis

Leptotrichia sp. oral taxon 498

Leptotrichia hongkongensis

Rothia aeria

Veillonella parvula

Corynebacterium durum

Top 20 species present in plaque

Top 10 species present in plaque

Car

ies-

free

ECC

0

50

100R

ela

tive A

bu

nd

an

ce (

%)

Streptococcus sanguinis

Streptococcus mutans

Haemophilus parainfluenzae

Veillonella dispar

Lautropia mirabilis

Leptotrichia shahii

Prevotella melaninogenica

Gemella haemolysan

Alloprevotella sp. oral taxon 473

Abiotrophia defectiva

0 20 40 60 80 100

Streptococcus mutans*

Veillonella dispar

Granulicatella elegans

Prevotella melaninogenica*

Haemophilus parainfluenzae

Streptococcus sanguinis

Rothia aeria**

Corynebacterium durum

Percent of Subjects

Caries-free

S-ECC

Percent of subjects with greater than 2% relative abundance of each species

He

alth

-

asso

cia

ted

Dis

ea

se

-

asso

cia

ted

• Children with 2% or higher relative abundance of

S. mutans were 11x more likely to be in the S-

ECC group than children with < 2%

• With 2% or higher Prevotella melaninogenica

8x more likely to be in S-ECC group

• High levels of Rothia aeria were protective:

Children with 2% or higher abundance were 19x

more likely to be caries-free

Caries-free S-ECC0

10

20

30

40

Nu

mb

er

of

Su

bje

cts

Number of subjects with high abundance of 2 or more

health-associated species

Caries-free S-ECC0

10

20

30

40

Nu

mb

er

of

Su

bje

cts

Number of subjects with high

abundance of 2 or more disease-associated species

High abundance= 2% or greater

**p< 0.01; Fisher’s exact test

• With high abundance of 2 or more ‘disease-

associated’ species: 7x more likely to be in ECC

group

• With high abundance of 2 or more ‘health-

associated’ species: 11x more likely to be in

caries-free group

• First Nations children with S-ECC had significantly higher levels of cariogenic microorganisms than their caries-free counterparts.

• Increased abundance of S. mutans as well as population shifts in oral microbial communities in children with S-ECC.

• This might explain the high prevalence of S-ECC in First Nations and other Indigenous children in Canada and North America.

Acknowledgements • We wish to acknowledge Delta Dental of Wisconsin for funding

this Pilot Project.

Project Team:

• UofM/CHRIM members: RJ Schroth, J Marques, A Huang, E Kliewer, B Mittermuller

• UCLA members: W Shi, X He, M Agnello, J Mclean, L Cen

• Collaborators: Drs. D Robertson, D Marianos, D Drake (QUEST Microbiology Group)

• The Misericordia Health Centre (Winnipeg) and participating pediatric dentists

• QUEST in AI/AN Children is a 501(c)(3) organization whose mission is to convene and focus the expertise and resources necessary to elucidate the etiology of rampant caries in the primary dentition in American Indian and Alaska Native (AI/AN) children, and to identify optimal strategies to prevent and control it.