Introduction
Over the last several years, the intestinal microbiota has been well recognised as a major contributor to human health and disease [
3]. Development of the intestinal microbiota from birth to childhood and adulthood is a process that co-occurs with maturation of the immune, digestive and cognitive systems. The interplay between humans and their intestinal microbes could, therefore, greatly influence health, especially during critical developmental stages in early life. Despite its described importance, intestinal microbiota development is not completely understood, as it is a highly dynamic process affected by multiple host and environmental factors, of which gestational age, mode of delivery, diet and antibiotics are perceived as the major influencing factors [
29]. Previous studies showed that antibiotic treatment in early life can lead to short- and long-term alterations of the intestinal microbiota, which has been related to early and later life health outcomes such as asthma and adiposity [
8,
19,
32].
Antibiotic treatment is common practice in the neonatal ward for the prevention and treatment of sepsis, which is one of the leading causes of mortality and morbidity in preterm infants. Antibiotics, such as amoxicillin, ceftazidime, erythromycin and vancomycin, are frequently used as they target a broad spectrum of pathogens. Intrauterine infections are a common cause of preterm birth; thus, many preterm infants are born with suspicion of infection and are, therefore, treated with antibiotics from birth onwards. In preterm infants, group B streptococci and
Escherichia coli are associated with onset of neonatal sepsis [
31]. Since sepsis in preterm infants still has a high mortality and morbidity, it is not possible to wait for test results before starting antibiotic treatment. To reduce the antibiotic load in the neonatal ward, it is common practice to evaluate the need for antibiotics after 48 h and stop antibiotics if the infection is not proven.
The applied antibiotic strategies in neonatology led to decreased mortality and morbidity rates. However, there is a risk of impeding gut microbiota development and increasing antibiotic resistance [
15]. It has been shown that, in infants, intestinal microbiota composition and activity in early life is associated with gestational age and can be related to the degree of perinatal antibiotic administration [
1,
34]. In addition, it has been shown that preterm infants admitted to the neonatal intensive care unit (NICU) are particularly colonised by antibiotic-resistant and virulent bacterial strains during early life, which was restored around two years of age [
24]. Despite increased understanding of how antibiotic treatment affects preterm infant microbiota development, not much is known about the effect of duration of treatment. Previous studies showed that long antibiotic treatment (> 5 days) in preterm infants results in lower diversity of the faecal microbiota than short treatment, but no clear differences in overall microbiota composition were observed [
9,
17]. This could be due to other factors that influence microbiota composition which were not accounted for during stratification of the infants, such as gestational age [
17] or mode of delivery [
9]. As gestational age and mode of delivery by themselves influence early-life microbiota development, the present study accounted for these in order to further understand the effect of antibiotic treatment duration on preterm infant microbiota development.
Discussion
Intravenous antibiotic administration for the prevention and treatment of infection and sepsis occurs frequently in preterm infants during the neonatal period. Therefore, studying the side effects of antibiotic treatment, including the effect on microbiota development, is of great relevance. The idea that short antibiotic use negatively affects clinical success and induces antibiotic resistance is gradually being replaced by the aim to avoid antibiotic overuse [
20]. In this study, we focused on the effect of intravenous antibiotic treatment duration on intestinal microbiota development in preterm infants during the first six postnatal weeks. Our main findings are: (1) both short and long treatment with amoxicillin/ceftazidime during the first postnatal week drastically disturbed the normal colonisation pattern; (2) short, but not long, antibiotic treatment allowed for the recovery of
Bifidobacterium levels within the first six postnatal weeks; and (3) community richness and diversity were not affected by antibiotic treatment, but were associated with postnatal age and with the dominance of specific bacterial taxa, leading to differences in microbial networks.
In the current study, 16S rRNA gene sequencing and qPCR analysis showed that preterm infants’ faecal microbiota was dominated by
Bifidobacterium throughout the first six postnatal weeks.
Bifidobacterium species are considered beneficial early-life colonisers, and are found in high abundance in term, vaginally delivered, breast-fed infants [
27]. Short and long treatment with a combination of amoxicillin and ceftazidime during the first postnatal week drastically disturbed the normal colonisation pattern. Antibiotic treatment was effective against members of the Enterobacteriaceae family, but also negatively affected
Bifidobacterium abundance and allowed
Enterococcus to thrive. It must be noted that
Bifidobacterium abundance was already lower in meconium samples of ST and LT infants compared to control infants, most likely a result of the relatively late (postnatal day 2–4) defecation of meconium samples by most preterm infants.
Enterococcus remained dominant for up to two weeks after antibiotic treatment discontinuation. This might possess a health risk for the infants, as some
Enterococcus species emerged from gut commensals to nosocomial pathogens via the acquisition of multi-drug resistance and other virulence determinants [
2,
16]. Short, but not long, antibiotic treatment allowed for the recovery of
Bifidobacterium levels within the first six postnatal weeks. Although the differences in average
Bifidobacterium abundance between ST and LT infants was less apparent using qPCR instead of sequencing, it did show that
Bifidobacterium levels recovered in 4/5 ST and in only 2/5 LT infants. In addition, both methods indicate that long antibiotic treatment results in increased abundance of members of the Enterobacteriaceae family at postnatal week six.
Antibiotic treatment did not affect community richness and diversity. However, richness and diversity were affected by postnatal age and by the dominance of specific bacterial taxa. Dominance of
Bifidobacterium was negatively associated with abundance of other bacterial genera. Its dominance, however, allowed for higher community richness and diversity compared to dominance by other bacterial genera such as
Enterococcus. We speculate that
Bifidobacterium species control, but not outcompete, other bacterial species and that the microbial networks associated with
Bifidobacterium species can, therefore, play an important role in early-life tolerance induction and immune system maturation. Microbiota profiles associated with antibiotic treatment could negatively influence immune system maturation via disturbance of the normal colonisation pattern. Indeed, previous studies showed that early-life antibiotic exposure increased susceptibility to immune-related diseases such as asthma and allergy, and associated this with perturbations in microbial composition [
14,
33].
In addition to antibiotic treatment, our findings show that postnatal age, gestational age, pre-eclampsia and maternal antibiotics influenced microbiota composition. The latter two highlight the importance of maternal health status on infant microbiota development. Previous studies showed that microbes can be vertically transmitted, and that maternal health status, such as bodyweight and antibiotic use, affect infant microbiota development [
7,
13,
21]. Maternal antibiotics could affect infant microbiota composition via prenatal exposure of the foetus to antibiotics, via alteration of the mother’s microbiota and, therefore, the inoculum at birth, and via transfer of antibiotics through breastfeeding. In the study described herein, the use of perinatal antibiotics was unevenly distributed among the study groups. This, in addition to the relatively small sample size, hindered to unravel the true impact of maternal antibiotics on infant microbiota development. Pre-eclampsia is a condition characterised by high blood pressure and proteinuria, and is associated with maternal and neonatal morbidity and mortality, preterm birth and intrauterine growth restriction [
18]. The aetiology of pre-eclampsia is unknown, but this disorder could be linked to genetic factors, obesity, abnormal formation of placental blood vessels and autoimmune disorders [
18]. Our findings suggest that pre-eclampsia or its accompanying conditions are associated with infant microbiota composition. However, the relation between pre-eclampsia and infant microbiota development needs to be further elucidated, as this study was not designed for studying this matter.
Overall, our findings show that intravenous antibiotic administration during the first postnatal week greatly affects the gastrointestinal microbiota community structure in preterm infants. However, quick cessation of antibiotic treatment allows for recovery of the microbiota. Disturbances in microbiota development caused by short and, more extensively, by long antibiotic treatment could affect healthy development of the infant via interference with maturation of the immune system and gastrointestinal tract. Clinicians should be aware of the disturbances that antibiotic treatment can cause and be strict in discontinuing antibiotic treatment as soon as possible to allow for a fast recovery of the microbiota community structure.