The effect of breastmilk and saliva combinations on the in vitro growth of oral pathogenic and commensal microorganisms

E L Sweeney,J A Duley,S S Al-Shehri,H G Liley,D M Cowley,N Bansal,P N Shaw,C L Knox,B G Charles

doi:10.1038/s41598-018-33519-3

Abstract

Neonates are exposed to microbes in utero and at birth, thereby establishing their microbiota (healthy microbial colonisers). Previously, we reported significant differences in the neonatal oral microbiota of breast-fed and formula-fed babies after first discovering a primal metabolic mechanism that occurs when breastmilk (containing the enzyme xanthine oxidase) and neonatal saliva (containing highly elevated concentrations of the substrates for xanthine oxidase: xanthine and hypoxanthine). The interaction of neonatal saliva and breast milk releases antibacterial compounds including hydrogen peroxide, and regulates the growth of bacteria. Using a novel in vitro experimental approach, the current study compared the effects of this unique metabolic pathway on a range of bacterial species and determined the period of time that microbial growth was affected. We demonstrated that microbial growth was inhibited predominately, immediately and for up to 24 hr following breastmilk and saliva mixing; however, some microorganisms were able to recover and continue to grow following exposure to these micromolar amounts of hydrogen peroxide. Interestingly, growth inhibition was independent of whether the organisms possessed a catalase enzyme. This study further confirms that this is one mechanism that contributes to the significant differences in the neonatal oral microbiota of breast-fed and formula-fed babies.

Highlights

The infant is first exposed to bacteria in utero from the placenta during gestation[1], at the time of birth, is colonised with rich and diverse microorganisms from maternal sites[2], including the maternal lower genital tract, faeces and/or skin[3,4], from milk feeds as well as from the general environment[5]
We found that the numbers of viable microorganisms were not initially affected when exposed to the control (CON) saliva and breastmilk mixture, and subsequently, growth ensued in most cases except for methicillin resistant Staphylococcus aureus (MRSA), 107 colony-forming units (CFU) (Fig. 1)
The numbers of viable microorganisms in each experiment decreased immediately when added to the purines and pyrimidines (PP) + HX saliva-breastmilk mixture supplemented with hypoxanthine (H) and xanthine (X), and in many cases the growth did not recover to control levels following 24 hr incubation

Summary

Introduction

The infant is first exposed to bacteria in utero from the placenta during gestation[1], at the time of birth, is colonised with rich and diverse microorganisms from maternal sites[2], including the maternal lower genital tract, faeces and/or skin[3,4], from milk feeds as well as from the general environment[5]. When neonatal saliva combines with breastmilk during feeding, hydrogen peroxide (H2O2) at concentrations up to 100 micromolar are generated, and this in turn activates the ‘lactoperoxidase system’ (LPO) in milk to further produce other reactive oxygen species (ROS) and reactive nitrogen species (RNS)[8]. The production of these ROS and RNS was confirmed to have in vitro antibacterial activity: we previously demonstrated that the growth of Staphylococcus aureus and Salmonella spp. was inhibited during in vitro experiments when these pathogenic bacteria were exposed to physiological mixtures of simulated neonatal saliva and human breastmilk, whereas the commensal bacteria Lactobacillus spp. and Escherichia coli were unaffected[8]. We examined the effects of varying hydrogen peroxide concentrations on catalase-positive versus catalase-negative microorganisms, which were incubated for 24 hr in nutrient broth, a non-defined medium

Methods

Results

Conclusion