β-lactam antibiotics form the fundamental basis of treatment for the majority of neonatal infections(14). Among them cefotaxime and ampicillin/sulbactam are two of the most prescribed antibiotics for neonatal infection(15, 16). The use of antibiotics is a double-edged sword. While it helps eliminate harmful pathogens, it comes at the expense of eradicating beneficial bacteria, disrupting the normal developmental trajectory of the gut microbiota. This, in turn, can lead to the overgrowth of opportunistic pathogens, ultimately increasing the risk for the onset of disease(17–19). The disruptive effect of antibiotics on the gut microbiota is more pronounced in neonates, especially within the first week(20). Furthermore, the different types of antibiotics(20) and their durations of use(21) will have varying degrees of impact on the gut microbiota. Hence, the primary finding of this study revolves around our investigation into the effects of β-lactam antibiotics, specifically cefotaxime and ampicillin-sulbactam, on the gut microbiota of neonates. We also delved into understanding how these effects varied among neonates with different ages and were influenced by distinct treatment durations, types of antibiotics, delivery mode, and feeding mode.
First, we observed that β-lactam antibiotic administration significantly affected the composition of the gut microbiota, including β-diversity (Fig. 1C and 1D) and the abundance of phylum (Fig. 2 and Figure S1) and genera (Fig. 3, 4) of the neonates. Specifically, the top 5 genus was significantly alteration after β-lactam antibiotic use, characterized by strongly increased proportions of Klebsiella, Enterococcus and Streptococcus and dramatically decreased Escherichia-Shigella, Clostridium sensu stricto 1, Bifidobacterium (Fig. 4). Our findings supported an earlier investigation, which found that newborns exposed to antibiotics in their first week of life had higher levels of Klebsiella and Enterococcus than the control group(20).
Klebsiella and Enterococcus, potential pathogens colonizing in neonatal gut, are important organisms for neonatal late onset sepsis(22). A recent global neonatal sepsis observational cohort study involving 3,195 infants (90.4% neonates aged < 28 days) revealed that of the 17.7% blood culture pathogen positive, the most common pathogen was Klebsiella pneumoniae, accounting 4.1%(23). In addition, Klebsiella spp are reported frequently enriched in the gut microbiota of preterm neonates, and overgrowth is associated with necrotizing enterocolitis (NEC), nosocomial infections and late-onset sepsis(24). Also, Klebsiella/Enterococcus-dominated faecal microbiota is associated with an increased risk of developing NEC in preterm infants(25). Klebsiella spp, through Toll-like receptor-4 activation, induce the recruitment of pro-inflammatory T helper 17 cells, resulting in the release of pro-inflammatory cytokines (IL-17, IL-22), which can lead to erythrocyte death, mucosal injury, and bacterial translocation to the microvasculature beneath the intestinal epithelium(25, 26). Therefore, β-lactam antibiotic administration induced increased abundance of the Klebsiella and Enterococcus genera may potentially predispose individuals to late infections. On the other hand, the underlying pathogens of Streptococcus was also significantly increased after β-lactam antibiotic treatment. However, study indicated that intravenous antibiotics decreased the relative abundance of Escherichia-Shigella and Streptococcus, while increased the relative abundance of Enterococcus and Lactobacillus species during the first two postnatal weeks(27). Our results are largely consistent with the findings, however, one inconsistency is that in our study, the use of antibiotics led to an increase in Streptococcus abundance, whereas in previous research, it resulted in a decrease. However, consistent with our results, in preterm infants, one-week empirical antibiotic therapy increased harmful bacteria such as Streptococcus and Pseudomonas(12). The variations observed in Streptococcus susceptibility to different β-lactam antibiotics may be associated with differences in their sensitivity to antibacterial agents or resistance mechanisms within Streptococcus. The specific mechanisms await further research. Streptococcus, a group of Gram-positive bacteria, has the potential to lead to severe infections associated with significant morbidity and mortality(12). It has been reported that late-onset neonatal bloodstream infections can be caused by the enteric habitat of bacteria(28), including Streptococcus, which commonly resides within the mucosal lining of the intestinal tract and has the capacity to disseminate to various organs, resulting in severe infections(29). Therefore, the overgrowth of Streptococcus in the intestine after β-lactam antibiotic treatment may increase the risk for subsequent infection, such as late-onset sepsis. In combination, Klebsiella, Enterococcus, and Streptococcus, accounting for more than half (approximately 53.3%) of the genera in the β-lactam antibiotic group, may significantly increase the risk of these neonates’ developing illnesses. Reversely, many genera, such as Escherichia-Shigella, Clostridium sensu stricto 1, and Bifidobacterium significantly reduced after β-lactam antibiotic administration. In term infants, an initially aerobic environment primarily hosts aerobes and facultative anaerobes, such as Escherichia, enterococci, Enterobacteriaceae, Staphylococcus, and Streptococcus species(30, 31). Subsequently, a rapid fall in gut luminal oxygen levels due to consumption by these bacteria and secretory immunoglobulin A, allows the proliferation of strict anaerobic bacteria, such as Bifidobacterium spp., Clostridium(32). However, antibiotic use significantly decreases the abundance of enteric anaerobic bacteria, including Bifidobacterium, enterobacteria and clostridia(20, 33). Cefotaxime and ampicillin/sulbactam are broad-spectrum β-lactam antibiotics, targeting Gram-positive and -negative bacteria. It has been shown that Bifidobacterium species are sensitive to β-lactam antibiotics and that treatment with amoxicillin can greatly influence the composition of Bifidobacterium species in infant intestinal microbiota(4, 34). Moreover, intravenous antibiotic combinations, namely penicillin + gentamicin, co-amoxiclav + gentamicin or amoxicillin + cefotaxime, significantly decreased the abundance of Bifidobacterium, and speculated that antibiotic treatment may directly eliminate these genera(20). Therefore, the deceased proportion of Bifidobacterium after cefotaxime or ampicillin/sulbactam treatment in the present study may attribute to their directly eliminate these kinds of genera. However, which species of the genera were influenced by cefotaxime or ampicillin/sulbactam treatment remains unknown, and deserves further investigation. On the other hand, we found that the overgrowth Klebsiella, Enterococcus and Streptococcus may also inhibit the growth of Escherichia-Shigella, as relative abundance of Klebsiella, Enterococcus and Streptococcus was negatively related with abundance of Escherichia-Shigella (Fig. 5A, C, D). Conversely, the growth of certain bacteria can lead to synergistic effects. Examples of such pairs include Klebsiella and Enterobacter, Enterococcus and Streptococcus, as well as Aeromonas and Streptococcus, which exhibit a positively correlated abundance (Fig. 5B, E, F). Bacteria can inhibit the growth of each other by complex mechanisms involved of direct mechanisms, including spatial competition, nutritional competition and other mechanisms(35). Moreover, a particular Klebsiella species, like Klebsiella pneumoniae, can produce various bacteriocins that exhibit antimicrobial effects against closely related species.(36) Furthermore, through secretion and injecting toxic proteins by type VI secretion system, Klebsiella can kill the surrounding bacteria and contributes to colonization across the gastrointestinal tract(37). On the other hand, the mechanisms that may lead to a positive correlation between microbiota could include competition for shared resources and nutrients in similar ecological niches, as well as the possibility of a mutualistic relationship that promotes their growth(38). Certainly, further research is necessary to clarify the potential mechanisms of interactions between microbial communities.
The composition of neonatal gut microbiota, influenced by antibiotic treatment, is determined by various factors, including the timing, duration, and the specific type of antibiotics administered(10). Our study focused on assessing the effects of β-lactam antibiotic treatment on the neonatal intestinal microbiota within specific age ranges, which included the following periods: ≤ 7 days, 8–14 days, 15–21 days, and 22–28 days. Remarkably, we observed a significant increase in the abundance of Klebsiella and Enterococcus following β-lactam antibiotic treatment at all four distinct stages compared to healthy group. Our results was consistent with previous study where 147 infants born at ≥ 36 weeks of gestational age received intravenous antibiotic treatment including penicillin + gentamicin, co-amoxiclav + gentamicin or amoxicillin + cefotaxime, in the first week of life, significantly increased abundance of Klebsiella and Enterococcus spp and decreased abundance of Bifidobacterium spp.(20) During the first days of life, the gut microbiota primarily consists of aerobic/facultative anaerobic bacteria belonging to the phyla Proteobacteria (e.g., Enterococcus spp) and Firmicutes (Staphylococcus, and Streptococcus)(32, 39). The abundance of these facultative bacterial taxa decreases rapidly because of the consumption of oxygen and intestinal secretory immunoglobulin A, along with the expansion of anaerobic bacteria Bifidobacterium and Clostridium during the first months of life21. However, the use of antibiotics, which is one the most disruption factor for neonatal gut microbiota development(40), strongly interfere the normal gut microbiota development at every neonatal period. Our research provides more detailed insights into the influence of antibiotic usage on neonates of varying age groups, indicating that the use of antibiotics in neonates at any age can have a substantial impact on their gut microbiota.
Additionally, different duration of antibiotic treatment significantly affects the structure of enteric microbiota, we compare the impact of different duration of antibiotic treatment (3 days, 5 days and 7 days, respectively) on the neonatal gut microbiota. The 7 days group showed the highest abundance of Lactobacillales_Unclassified, Ruminococcaceae_Unclassified, and Lawsonella compared to the 3 days and 5 days groups. Reversely, the abundance of Serratia was gradually reduced with the increased duration of antibiotic use, with the lowest in the 7 days group compared to the 3 days and 5 days groups (Fig. 7A). Rooney et al. suggested that 1 week of discontinuation of antibiotic treatment, each additional day of antibiotics was associated with lower richness of obligate anaerobes(21). Zwittink et al. observed a significant reduction in Bifidobacterium levels in preterm infants (35 ± 1 week’s gestation) following short (≤ 3 days) or long (≥ 5 days) antibiotic treatment, which persisted until the third week after birth (P = 0.028). In cases of long antibiotic treatment, this reduction continued until the sixth postnatal week (P = 0.009)(41). These studies, including our own research findings, suggest that the longer the duration of antibiotic use, the greater the impact on the gut microbiota. Simultaneously, long antibiotic treatment led to the emergence and even overgrowth of antibiotic-resistant microbes (17, 42). Therefore, it is important to minimize the duration of antibiotic use as much as possible while treating neonatal infections.
Moreover, the choice of antibiotics administered to neonates can exert distinct effects on the composition of their intestinal microbiota. We found that ampicillin/sulbactam led to a notable increase in the richness of Enterobacter, Citrobacter, Lachnospiraceae_Unclassified, and Staphylococcales_Unclassified when compared to cefotaxime, indicating that the impact of each antibiotic on each neonate is not uniform, leading to variability in post-antibiotic microbiome compositions(43), and that ampicillin/sulbactam may be more harmful for neonatal microbiota as opportunistic pathogen enrichment was increased. Previous study revealed that broad-spectrum antibiotics for treatment of suspected early-onset neonatal sepsis, amoxicillin + cefotaxime shows the largest effects on both microbial community composition and antimicrobial resistance gene profile, whereas penicillin + gentamicin exhibits the least effects(20). Antibiotic treatment – especially treatment with broad-spectrum antibiotics – kills members of the gut microbiota and disrupts colonization resistance(44). The use of antimicrobials for neonatal infection, depends on the most frequent causative microorganisms, and their choice will be empirical until culture results and antibiograms are available(45). Ampicillin and gentamicin were the WHO first line regimen for empiric antibiotic combinations and cefotaxime was WHO second-line regimen(23). Adding a β-lactamase inhibitor, such as sulbactam to β-lactam antibiotic, such as ampicillin, can broaden the spectrum of activity sufficient to cover gram-negative extended-spectrum β-lactamase producers(46). However, we found the contrary effect as ampicillin/sulbactam led a notable increase in the richness of Enterobacter, Citrobacter. We speculated that its effect may be associated with the increased antibiotic resistance microbiota. The exact mechanism await needs further classification.
Delivery mode significantly affect the colonization and development of neonatal intestinal microbiota(9, 47). In comparison with vaginally delivered infants, infants born by cesarean section showed decreased relative abundance of Bacteroides and Parabacteroides and enrichment of Clostridium_sensu_stricto_1, Enterococcus, Klebsiella, Clostridioides, and Veillonella(9). We further found that compared Vaginal delivery, β-lactam antibiotic group treatment with cesarean delivery further increased the abundance of Klebsiella genus, Enterobacteriaceae_Unclassified, Lactobacillales_Unclassified and Pectobacterium (Fig. 7C), indicating that β-lactam antibiotic treatment seems to further worsen intestinal flora disturbance caused by cesarean section. In addition, feeding mode also significantly affect neonatal intestinal composition(48, 49), however, the effect did not change the overall adverse effects of antibiotic use on intestinal flora, as the abundance of the main influenced genera by β-lactam antibiotic treatment, such as Klebsiella, Enterococcus and Streptococcus, displayed no significant difference among artificial feeding, breast feeding, and mixed feeding groups and only differences in small proportion of genera (Fig. 7D). These results suggested that regardless of previous feeding patterns, β-lactam antibiotic treatment will significantly impact the compositions of neonatal microbiota. Van Daele et al. indicated that antibiotic exposure in the first week perturbated the term infant’s fecal microbiota, and this perturbation was still notable at 1 month in formula-fed infant, but only until 2 weeks in breast-fed infants(50). The results indicated breast-fed can promote restoring dysbiosis. Mechanically, breastmilk is abundant in bioactive components, including human milk oligosaccharides, immune cells, lactoferrin, cytokines, antibodies, antimicrobial proteins and peptides, which aids restoration by stimulating the growth of bifidobacteria and reducing (potential) pathogens(30, 51). A recent exciting study showed that breastfeeding and antibiotics have opposing effects on the infant microbiome and that breastfeeding enrichment of Bifidobacterium longum subsp. infants is associated with reduced antibiotic-associated asthma risk(52). Taken together, breastfeeding for neonates may do not prevent the adverse effects of antibiotic on the gut microbiota, but can restore gut microbiota after antibiotic treatment.