Nitrate reductase activity of bacteria in saliva of term and preterm infants

Abstract

The salivary glands of adults concentrate nitrate from plasma into saliva where it is converted to nitrite by bacterial nitrate reductases. Nitrite can play a beneficial role in adult gastrointestinal and cardiovascular physiology. When nitrite is swallowed, some of it is converted to nitric oxide (NO) in the stomach and may then exert protective effects in the gastrointestinal tract and throughout the body. It has yet to be determined either when newborn infants acquire oral nitrate reducing bacteria or what the effects of antimicrobial therapy or premature birth may be on the bacterial processing of nitrate to nitrite. We measured nitrate and nitrite levels in the saliva of adults and both preterm and term human infants in the early weeks of life. We also measured oral bacterial reductase activity in the saliva of both infants and adults, and characterized the species of nitrate reducing bacteria present. Oral bacterial conversion of nitrate to nitrite in infants was either undetectable or markedly lower than the conversion rates of adults. No measurable reductase activity was found in infants within the first two weeks of life, despite the presence of oral nitrate reducing bacteria such as Actinomyces odontolyticus, Veillonella atypica, and Rothia mucilaginosa. We conclude that relatively little nitrite reaches the infant gastrointestinal tract due to the lack of oral bacterial nitrate reductase activity. Given the importance of the nitrate-nitrite-NO axis in adults, the lack of oral nitrate-reducing bacteria in infants may be relevant to the vulnerability of newborns to hypoxic stress and gastrointestinal tract pathologies.

Figure 1

Effect of antiseptic mouthwash on the assay for oral nitrate-reducing bacteria. Following six treatments with antimicrobial mouth rinse at twelve hour intervals, oral nitrate reductase activity in four adult subjects was decreased to unmeasurable levels compared to baseline values. Nitrate reductase activity returned to normal within 48 hours following the last antimicrobial mouth rinse treatment.

Figure 2

Nitrite and nitrate concentrations in saliva of infants and adults. A) Nitrite concentrations in the saliva of infants (8 ± 5 μM) were less than adult saliva (55 ± 22 μM, ** = p<0.01). B) Salivary nitrate levels were not significantly lower in the saliva of infants (328 ± 97 μM) than in the saliva of adults (538 ± 125 μM).

Figure 3

A) Time course of nitrite production by nitrate-reducing bacteria cultured from saliva samples. After introduction of nitrate substrate into cultures [NO₂⁻] was measured in samples from adults (◆), infants greater than two weeks of age (◇), newborn infants less than five days old (○), and newborn infants less than five days old receiving antibiotics (▲). B) Summary of kinetic results are shown for adults and each of the infant groups based on average rates of change of nitrite concentration with time, as shown in A. There was no significant nitrite production in the saliva of infants <5 days old, regardless of whether they were receiving antibiotics (abx). There was significant nitrite production in the infants older than two weeks (+ = p<0.01), and nitrite production was significantly greater in the adult saliva compared to infants (*** = p<0.001).

Fig. 4

Real time PCR detection of nitrate-reducing bacteria in newborn mouths. The presence of Veillonella atypica, Rothia mucilaginosa, Actinomyces odontolyticus, and Staphylococcus epidermidis in bacterial cultures collected from the mouths of term (●) and preterm (○) infants and adults (◆) was confirmed using primers specific to the respective bacteria. Horizontal gray bars represent the cutoff Ct value, below which the presence of the bacteria is confirmed. The data indicate the presence of most or all of these nitrate reducing bacteria in the infant mouth within the first few days of life, suggesting the lower rates of nitrate reducing activity in infants is not due to lack of inoculation.

Fig. 5

Effect of antiseptic mouth rinse on adult blood nitrite concentrations. Blood nitrite concentrations in healthy adult volunteers were significantly decreased by ~19% compared to baseline levels following six treatments with antiseptic mouth rinse (chlorhexidine) at 12-hour intervals (*=p<0.04). No significant change was observed in a parallel group of placebo (saline) control subjects.

Fig 6

Schema showing the pathways and interconversions of nitrite and nitrate. 1) Nitrate enters the mouth from diet and from the plasma by a concentrating action of the salivary glands. 2) In the mouth nitrate is converted to nitrite by commensal bacteria on the tongue [21]. 3) In the acid milieu of the stomach nitrite is converted to NO by disproportionation [11] or absorbed into the circulation. Nitrite may also be converted to NO in blood and tissues by the action of metalloproteinases [1]. NO can be converted to 4) nitrate by reaction with oxyhemoglobin [41] or 5) nitrite by ceruloplasmin [42].