Antibiotics are a boon to human civilization for saving millions of human and animal lives and alleviating infection-inflicted miseries (1, 2). Despite their undisputable health benefits, antibiotics exert unintended collateral effects, including microbiota perturbation or dysbiosis and impaired host immunity to infections and vaccines in humans and mice (3–7). In infants, since the microbiota is immature and the immune system is not fully functional, the consequences inflicted due to antibiotic treatment are more severe and long-lasting than in adults (8–10). Despite these differences in the microbiota and immune landscape, there is a paucity of information on the role and mechanism of antibiotics in regulating immune phenotype and function in infants. Human infants, especially underweight and preterm, are vulnerable to bacterial infections, which cause significant mortality and morbidity worldwide, particularly in low- and lower-middle income countries (11–13). Therefore, infants are subjected to prophylactic and/or therapeutic antibiotic treatments against bacterial infections (14–18). In fact, empiric antibiotic use is a common practice in neonatal intensive care units. Additionally, prepartum antibiotic treatment in mothers is not seldom recommended to reduce the risk of postpartum infections in newborns (19). Around 40% of pregnant mothers and infants receive antibiotics for the prevention and control of infections around the globe (20, 21). Unfortunately, inadequate and inappropriate use of antibiotics in neonates still exists, particularly in developing countries (22, 23). Emerging evidence underscores that antibiotic exposures exert detrimental effects on the infant microbiota and immune system, which render them susceptible to several diseases, including infections, in the times to come (17, 18). This unmasks the dark side of antibiotics, thus warranting in-depth and timely attention of physicians, scientists, and policy makers.

In this review article, we discuss recent findings that stem from human and animal studies focusing on the impact of antibiotics on the infant innate and adaptive immune responses to infections. We also focus on how early life exposure to antibiotics can make the infants prone to infection in later life. A better understanding of the antibiotic effects on the infant immune defense is crucial for developing a rational strategy for disease control and management in infants and adults.

The human microbiota consists of commensal microbes, such as bacteria, fungi, and viruses. Out of these, bacterial microbes are the most widely studied and include the dominant phyla – Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia (24, 25). They exert a profound impact on the phenotype and function of our body by playing a critical role in the synthesis and absorption of various nutrients and metabolites (e.g., vitamins and short-chain fatty acids), promoting immunity (e.g., pathogen defense), and regulating the brain function and behavior (26, 27). Although still controversial, it is possible that infants are exposed to the maternal microbiota and its metabolites that cross the placenta and enter the amniotic fluid already in the fetal stage (28). However, more recent human and animal studies that have taken in consideration the contamination of laboratory reagents, equipment, and tissue samples with bacterial DNA suggest that fetal and placental tissues are sterile (29–33). Around the time of child labor and birth, a major intergenerational microbiota transfer and colonization occurs, and continues up to 3 years of age (28). This contributes to the development and education of the neonates' immune system, often termed as immunological imprinting, which prepares them to optimally respond to “danger signals” in later phases of life (9). Compositional and functional perturbations of the microbiota, which are possible and usually termed as dysbiosis, can have disproportionate consequences at this stage. With the help of remarkable advances in microbiological techniques, such as metagenomics, and systems biology, it is easier to characterize dysbiosis in terms of alterations in microbial composition, diversity, and function (34). The important factors causing dysbiosis include, but are not limited to, dietary habits, stress, mode of child delivery (cesarean vs. vaginal) and antibiotics (35–37). Antibiotic-driven dysbiosis in particular stands as a major clinically relevant phenomenon that correlates with a myriad number of diseases. It generally leads to decreased microbial diversity in both children and adults, although the nature of the effects depends upon multiple factors, such as antibiotic types and doses (38, 39). The effects of antibiotics on the microbiota can last long after infancy and have long-term consequences for health and disease (40).

Antibiotics have attracted much attention as one of the most important factors that causes microbial dysbiosis during infancy and are linked to a higher risk of diseases with immune involvement in later life, including infectious diseases (10, 41–44). In case control retrospective studies, prolonged exposure to antibiotic therapy was found to be associated with an increased risk of necrotizing enterocolitis, late-onset sepsis, or death among very low birth weight infants (41, 45–47). By analyzing the stool microbiota and metabolites of preterm infants with seven days of empirical antibiotic treatment, Zhu et al. showed a significant reduction in bacterial diversity and enrichment of pathogens, such as Streptococcus and Pseudomonas (48). A Danish cohort study demonstrated that maternal antibiotics prescribed before or during pregnancy is associated with an increased risk of infant infection-related hospitalization (49). Epidemiological studies further shed light on how antibiotic use predisposes human infants to susceptibility to diarrhea and respiratory tract infections (42, 43). Although these studies provide crucial indications on increased susceptibility to infection, more convincing and direct evidence originates from studies involving animal models.

Using a mouse model of perinatal antibiotic exposure, Gonzalez-Perez et al. studied the role of antibiotics in causing dysbiosis and regulating immune defense against a systemic vaccinia virus infection (50). Antibiotic treatment of dams led to a significant alteration in the composition of the infant gut microbiota with the predominance of Enterococcus faecalis (50). Furthermore, infant mice born from antibiotic-treated, but not sham-treated, dams succumbed to death following intraperitoneal injection with a higher vaccinia virus inoculum, suggesting that antibiotic-driven dysbiosis reduces resistance to infection (50). Even at the lower viral inoculum, control mice exhibited decreased viral titers compared with antibiotic-exposed mice (50). Interestingly, under hygienic environmental conditions, infant mice, which were not exposed to antibiotics, revealed increased susceptibility to vaccinia virus infection (50). This underscores the relationship between the hygiene conditions under which infants are raised and the development of immunity against infection. Recent studies have also assessed the antibiotic-mediated effects on host susceptibility to infections with Gram-positive and Gram-negative bacteria. In a mouse model of bacterial infection, nursing dams were treated with antibiotics in drinking water, and their infants were then exposed via mother's milk (51). The exposed infants, compared with naïve controls, when challenged with Citrobacter rodentium 80 days later, showed enhanced pathology (colitis and fecal occult blood), body weight loss, and bacterial burden, which indicates the adverse effects of early life antibiotic usage on pathogen defense in later life (51). Furthermore, the transfer of the antibiotic-perturbed microbiota to germ-free mice led to worsened disease pathologies, suggesting that antibiotic-driven microbiota dysbiosis was responsible for the development of disease phenotype (51). Similarly, antibiotic exposure disturbed the microbial homeostasis in the gut of newborn mice and increased susceptibility to Streptococcus pneumoniae infection (52). In addition, neonatal mice exposed to a cocktail of antibiotics demonstrated reduced survivability against challenge infection with Escherichia coli serotype K1 or Klebsiella pneumoniae compared to control mice (53). Overall, these studies suggest that antibiotics significantly perturb the microbiota composition that leads to long-lasting consequences on host susceptibility to viral and bacterial infections. This highlights the crucial roles played by the commensal microbiota in promoting the infant's resistance to infection (54).

Animal models are particularly relevant to establish cause relationships that cannot be directly tested in humans in controlled settings. Although there are limitations in extrapolating findings from animal models, designing experiments that closely resemble the use of antibiotics in humans, including antibiotic combinations, doses, and route of administration, would have the potential to offer more relevant information. For instance, the common practice of extrapolation of antibiotic dose from humans to mice, which is based on the body weight (mg/kg) alone, does not stand appropriate due to physiological and biochemical differences between these two animal species that influence pharmacodynamics and pharmacokinetics of antibiotics. Hence, better approaches, such as allometric scaling based on normalization of dose to body surface area, are needed to calibrate the drug dose (55). While mice are the preferred animal models for antibiotic-related studies in neonates, the use of large animals like pigs may provide a better alternative due to (1) anatomical and physiological similarities with humans, and (2) large-sized neonates allow easier therapeutic manipulations like intramuscular and intravenous injections.

Antibiotics are often unavoidable drug of neonatal clinical care to prevent and treat bacterial infections that inflict considerable socioeconomic burden on society. This is mainly due to challenges posed in accurate diagnosis of neonatal sepsis. There is an increasing awareness about serious consequences of antibiotic use in infants. Antibiotics promote changes in microbial ecology, which have been implicated in altered immune responses against pathogens and vaccines, and increased susceptibility to infections in later life. Understanding how antibiotic treatments during infancy shape the microbiota and immunity is crucial for designing better prophylactic and therapeutic strategies. It remains unclear whether antibiotics can directly modulate the infant immune function. Recent studies involving germ-free adult mice have provided interesting findings that demonstrate how antibiotics can regulate host immunity, including pathogen defense, in a microbiota-independent fashion (68, 69). But these findings await confirmation in infants. Future studies need to address the following questions: 1. What are the underlying mechanisms by which antibiotic-driven dysbiosis in infants control the immune responses to vaccines and infections? 2. Do antibiotics directly act on the infant immune system without involvement of the microbiota? 3. How can the negative effects of antibiotic use in infants be neutralized in order to strengthen antibiotic stewardship programs? 4. How to develop better animal models to study the antibiotic effect on the microbiota and immunity?

SS and FP wrote the manuscript, assisted in the conception of the manuscript, and gave approval of the last version to be published.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.