Background
The benefits and/or risks of short term and long term breastfeeding on infant oral health is still unclear. However, there seems to be minimal cariogenic capability of human milk alone as findings from primitive childhood skulls show “extremely low rates of caries in children”1. The question remains: What is the role that highly refined sugar plays on the caries development in breastfed children? We know that adding 10% sucrose to human milk2, and bovine milk3 greatly increased its cariogenic ability and that infant formula has increased cariogenic activity4. To our knowledge, no study has compared the impact that adding refined carbohydrates to human milk, bovine milk and infant formula may have on caries in infants. Our in vitro study attempts to investigate how sugar impacts the cariogenic capabilities of these three infant food sources. Hypothesis: Human milk has minimal biofilm formation in comparison to bovine milk and infant formula

Methods
Control Groups: - Human milk, sterile distilled water and BHI medium added. - Bovine milk, sterile distilled water and BHI medium added. - Infant formula, sterile distilled water and BHI medium added. - Human milk, 10% sucrose and BHI added. - Bovine milk, 10% sucrose and BHI added. - Infant formula, 10% sucrose and BHI added. Experimental Groups: - Human milk, sterile distilled water and 1X107 CFUs ml-1 S. mutans in BHI medium added2,3 - Bovine milk, sterile distilled water 1X107 CFUs ml-1 S. mutans in BHI medium added. - Infant formula, sterile distilled water and 1X107 CFUs ml-1 S. mutans in BHI medium added. - Human milk, 10% sucrose and 1X107 CFUs ml-1 S. mutans in BHI medium added. - Bovine milk, 10% sucrose and 1X107 CFUs ml-1 S. mutans in BHI medium added. - Infant formula, 10% sucrose and 1X107 CFUs ml-1 S. mutans in BHI medium added. Determination of the ability of S. mutans to form biofilms The ability of S. mutans to form biofilms was measured in 96-well polystyrene tissue culture plates, and growth in the presence of bovine plasma. An overnight culture of S. mutans was diluted in fresh BHI or medium to get an optical density at OD600 of 0.2. Similar dilutions of the overnight grown S. mutans were made in breast milk, bovine milk, or infant formula with and without 10% added sucrose. 200 μl of these dilutions were added to the wells of a 96-well polystyrene tissue culture plate. After a 48 h incubation at 37°C, culture medium and free-floating bacteria were removed by aspiration. The wells were washed twice with phosphate buffered saline (PBS). The biofilms were stained with 0.05% crystal violet dye (100 μl) for 10 min and wells washed twice with PBS to remove unbound crystal violet dye. Wells were dried for 2 h at 37°C. After adding 100 μl of 95% (V/V) ethanol to each well, the plate was shaken for 10 min to release the stain from the biofilms, and the absorbance at 490 nm was determined to quantify biofilm formation.

Discussion
- Human milk had the least biofilm formation. - Bovine milk and infant formula had comparative biofilm formation. - Adding 10% sucrose and 1X107 CFUs ml-1 Srep mutans increased biofilm formation in all three sources. - This study provides additional information for clinicians that struggle with nutritional and oral health advice for infant feeding. - Clear evidence-based guidelines concerning infant foods and their association with childhood caries are vital for effective and consistent communication between mothers and both dental and primary care providers.

Conclusions/Clinical Application
In 2012 the American Academy of Pediatrics (AAP) reaffirmed its breastfeeding guidelines reinforcing the health benefits of breastfeeding for both mother and child.7 The American Academy of Pediatric Dentistry (AAPD) also released policy statements concerning breastfeeding regarding its association with Early Childhood Caries (ECC). The AAP states breastfeeding is recommended for general health and decreases risk for diseases but adds, “Ad libitum breastfeeding after introduction of other dietary carbohydrates and inadequate oral hygiene are risk factors for ECC.”8 This statement may seem ambiguous for recommendations to parents and unparallel with the AAP breastfeeding guidelines. An overview of breastfeeding and oral health research in JADA states “scientifically rigorous research is needed to elucidate whether associations exist between breastfeeding and ECC, and such evidence is lacking.”9