The effect of early life antibiotic exposure on the development of the gut microbiota and enteric nervous system

© 2019 Dr. Lin Yung HungEarly postnatal life is a critical stage of microbiota establishment and ENS development. While the initial postnatal stage from birth is fundamental for the development of the gut microbiota and ENS, weaning is another key developmental period where there are major changes in diet, behaviour and physiology, and notably, microbiota. Antibiotics are frequently administered to infants and young children, however, recent studies have identified prospective long-term health consequences of early life antibiotic exposure on the developing gut microbiota. Yet, how antibiotic influences short and long-term ENS development remains unclear. Vancomycin is given as a prophylactic to preterm babies and paediatric patients to treat and prevent infections. It is also one of the most commonly used antibiotics on its own or as part of a cocktail in research to induce dysbiosis in mice. The aim of my PhD was to examine how early life exposure to vancomycin during two critical developmental periods affects microbiota and ENS development and whether changes observed during early postnatal life have long-term repercussions. In chapter 3, I investigated if acute administration of vancomycin, during the early postnatal period, influenced gut microbiota and ENS development. A single regimented dose of either water or vancomycin was administered daily to Wnt1-Cre;R26R-GCaMP3 mouse pups from postnatal (P) day 0 to P10/11. These mice contain a genetically-encoded fluorescent Ca2+ indicator in all enteric neurons and glia. At P10/11, vancomycin-fed pups showed significant dysbiosis, reduced myenteric neuron density and altered nNOS and calbindin neuronal subtype proportions compared to water-fed littermates. Using Ca2+ imaging, I showed that vancomycin-fed pups had more neurons responding to electrical stimulation applied to interganglionic connectives and larger amplitudes of train-evoked [Ca2+]i transients. These changes in the ENS contributed to dysmotility of the colon of vancomycin-fed pups. In contrast to the colon, the structure of the ENS and motility patterns of the duodenum were not affected by vancomycin, ruling out drug toxicity effects. P10/11 vancomycin-fed pups also had lower numbers of serotonin (5-HT) positive cells in the colonic mucosa. Altered 5-HT metabolism in these animals were confirmed by performing mass spectrometry on 5-HT biosynthesis intermediates, showing reduced concentrations of the 5-HT metabolite, 5-HIAA and droplet digital PCR (ddPCR) revealing increased gene expression of the 5-HT transporter, SERT. Bypassing tryptophan hydroxylase, by supplementing vancomycin-fed pups with 5-HTP, restored 5-HIAA levels in the colonic mucosa and prevented some of the vancomycin-induced effects on myenteric neurons, colonic motility and gut microbiota. Therefore, vancomycin exposure during the neonatal period induced significant developmental changes to both the gut microbiota and ENS. Some of these changes could be mediated by altered mucosal serotonergic signalling. In Chapter 4, I examined if vancomycin-induced changes on the gut microbiota and ENS observed at P10 were long-lasting. Newborn mouse pups were only treated with water and vancomycin till P10, then pups were left to grow to adulthood. 6-week-old mice given neonatal vancomycin had enlarged caeca, which is an indication of dysbiosis. This suggests that the gut microbiota of vancomycin-fed mice was not fully recovered despite cessation of antibiotic treatment. Adult mice treated with neonatal vancomycin had sustained reduction in myenteric neuron density. However, alterations in the proportions of nNOS+ and calbindin+ neurons observed during the neonatal periods was now restored. In contrast to the heightened [Ca2+]i activity at P10s, adult mice given neonatal vancomycin had lower numbers of neurons responding to electrical stimulation and no change in the amplitudes of electrically-evoked [Ca2+]i transients in their myenteric neurons compared to water-fed controls. Furthermore, there were no treatment-induced changes in colonic motility. Interestingly, faecal water content, which was unaffected in vancomycin-fed pups at P10, was lower in adult mice given neonatal vancomycin compared to controls. These findings indicate that although vancomycin treatment is terminated, the gut microbiota is not fully recovered and significant re-modelling of the ENS occurs, some of which are distinct to changes observed during the neonatal period. In Chapter 5, I explored the effects of vancomycin exposure between weaning and adulthood. From the day of weaning, mice were administered vancomycin or sterile water in their drinking bottles for three weeks. At 6-weeks of age, vancomycin-treated mice had dysbiosis accompanied with enlarged caeca. Similar to vancomycin-treated neonates in Chapter 3, increased synaptic activity exhibited by enteric neurons were mainly observed by larger amplitudes of train-evoked [Ca2+]i transients and increased number of neurons responding to electrical stimulation. However, in contrast to antibiotic exposure during the neonatal period, vancomycin-treated mice displayed significantly slower colonic motility, increased faecal water content and a decrease in the proportions of ChAT+ cholinergic neurons including calbindin and neurofilament-M subtypes in the myenteric plexus of the colon. Moreover, vancomycin treatment between weaning and adulthood had no effects on the serotonergic system in the colonic mucosa. Collectively, these findings suggest that vancomycin exposure from weaning had differential effects on the gut microbiota and ENS compared to administration of the antibiotic during the neonatal period. Together, my study is the first to identify and compare effects of antibiotic exposure on the gut microbiota and ENS during two critical stages of development. While vancomycin did not deplete bacterial diversity and abundance, it caused profound shifts in microbial composition in both developmental periods. Additionally, acute vancomycin exposure in both periods, resulted in dysmotility and alterations of the neuronal circuitry. Although the effects on colonic motility for mice given neonatal antibiotic treatment did not appear to be long-lasting, changes in the ENS and disrupted faecal and caeca weights, which manifested only in adulthood, suggests that early life exposure to antibiotics can have other long-term consequences on microbiota and host gut physiology