Common critical illnesses, including sepsis, acute respiratory distress syndrome, and multiorgan failure, are a growing economic burden on healthcare worldwide (Adhikari et al., 2010). Pathophysiological changes that occur in critically ill patients can affect the composition of the intestinal flora. For example, both the hypoperfused and reperfused states of the intestinal wall can lead to intense mucosal inflammation, which further leads to changes in the intestinal environment. Increased nitrate concentrations and altered mucosal oxygen concentrations favor the growth of Aspergillus phylum microorganisms, including clinically common gram-negative Bacilli such as Pseudomonas aeruginosa and Escherichia coli. The growth of Firmicutes microorganisms, such as Staphylococcus aureus and Enterococcus, can also be affected (Dickson, 2016). Disturbances in the intestinal flora are associated with disease progression and clinical prognosis in critically ill patients. Ojima et al. (2022) showed that dysbiosis in the ratio of the Bacteroides to the Firmicutes (B/F > 8 or B/F < 1/8) in critically ill patients within 7 days of admission was associated with increased mortality. Andremont et al. (2020) also showed that carrying rectal or pharyngeal ultra-broad-spectrum β-lactamase-producing Enterobacteriaceae was a risk factor for the subsequent development of ventilator-associated pneumonia caused by bacterial infection in critically ill patients. In addition, a strong link exists between the intestinal flora and function of organs such as the brain, lungs, liver, and kidneys. In a study by Dickson et al. (2020) intestine-associated bacteria such as Trichophytonaceae and Enterobacteriaceae were present in the pulmonary flora of critically ill patients, and an increase in these bacteria predicted the number of days a patient would have without being on a ventilator. The loss of endothelial barrier integrity and dysbiosis of the intestinal flora are major pathophysiological alterations in sepsis that play important roles in sepsis-related acute kidney injury (Xu et al., 2022).

Most bacterial communities in the gut have a symbiotic relationship with the human host; however, many conditionally pathogenic bacteria, such as Enterobacteriaceae and Enterococcaceae, are also present in the gut and can cause severe infections in immunocompromised patients (Mcinnes et al., 2020). Antibiotic use is a key factor in the induction of drug-resistant genes. In recent years, infections caused by drug-resistant strains of Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis have been increasing worldwide (Guzman Prieto et al., 2016; Dunn et al., 2019). In addition, some studies have shown that the expression levels of resistance genes in the gut microbiota increase during antibiotic treatment in children; however, not all resistance genes return to baseline levels after antibiotic discontinuation (Yassour et al., 2016).

Therefore, gut microbiota has gathered increasing attention from clinicians as an important element in the management of critically ill patients. Despite decades of medical and technological advances, therapies targeting the gut flora for critical illnesses have not been developed on a large scale. Metagenomics next generation sequencing (mNGS) is a new method of high-throughput RNA/DNA sequencing that enables the detection of all pathogenic bacteria in a sample without bias (Diao et al., 2022; Gökdemir et al., 2022). This method allows further exploration of the composition, diversity, gene function, and drug resistance genes of the gut microbiota.

This study aimed to describe the changes in the composition, gene function, and drug resistance genes of gut microbiota in critically ill children after receiving short-term PICU treatment and to provide a theoretical basis for the rational use of medication and reduction of the incidence of adverse events in PICU patients.

The human gastrointestinal tract stores a large number of microorganisms, and close communication occurs between them and their hosts. Disturbances in the structure and function of gut microbiota are associated with numerous pathological processes in the human body, including inflammatory bowel disease, type II diabetes, and colorectal cancer (Manor et al., 2020). In recent years, with increasing research, more studies have demonstrated that the gut is a key factor in the initiation and development of critical illnesses and that intestinal failure is associated with poor prognosis (Szychowiak et al., 2022). Therefore, understanding the structure and function of gut microbiota and their role in critical illnesses is crucial.

In the present study, children received broad-spectrum antibiotics intravenously after enrollment, and different proportions of children received proton pump inhibitors, vasoactive drugs, invasive mechanical ventilation, parenteral nutritional support, and probiotics, in addition to exhibiting varying conditions such as underlying disease, site of infection, pathogenic bacteria, and immune status. The microbial environment in the gut of critically ill patients, often influenced by genetics, diseases, and therapeutic factors, is often disturbed (Wozniak et al., 2022), characterized by reduced abundance and diversity and an increase in conditionally pathogenic bacteria (e.g., Clostridium difficile, multi-drug-resistant bacteria) (Szychowiak et al., 2022). For example, intestinal emptying can be reduced in critically ill patients with decreased bacterial excretion, leading to excessive growth of pathogenic bacteria in the intestine (Dickson, 2016; Ladopoulos et al., 2018). Antibiotic use has been associated with decreased gut microbiota diversity (Ramirez et al., 2020). In addition, a study in adults showed that the combination of meropenem, gentamicin, and vancomycin led to an increase in Enterobacter and other pathogenic bacteria in the intestine, whereas Bifidobacteria and butyrate-producing flora were reduced (Palleja et al., 2018). Drugs such as non-steroidal anti-inflammatory drugs, proton pump inhibitors, and β-blockers can affect bacterial growth by altering the pH of the intestine (Wozniak et al., 2022), and continuous parenteral nutrition has been associated with significant disturbances in the flora (Dahlgren et al., 2019).

In this study, the diversity of the gut microbiota was investigated. The ACE and Chao1 indices of the children in the D7 group were significantly lower than those in the D1 group. The ACE and Chao1 indices are two indicators that describe the richness of the gut microbiota (Szychowiak et al., 2022), which implies that a significant decrease in the number of bacterial species in the gut microbiota occurred after 7 days of treatment in the PICU. While the Shannon and Simpson indices, describing both richness and evenness (Szychowiak et al., 2022), did not change significantly in our study.

Studies performed on the gut flora of critically ill children, using follow-up data, remain insufficient. Rogers et al. found that the Shannon diversity index of the gut flora was decreased significantly compared with healthy children, and it was significantly negatively correlated with the time spent in the PICU (Rogers et al., 2016). This differed from our results, which showed that the Shannon index did not change significantly after short-term PICU treatment. One of the reasons for this discrepancy might be that we followed-up with the patients for different periods of time. Our study further investigated changes in the composition of the gut microbiota in critically ill children after short-term PICU treatment, while Rogers et al. followed-up with patients for 30 days. The results showed that on both days 1 and 7 of PICU admission, the composition of the children’s gut microbiota at the phylum level was dominated by the Firmicutes, Bacteroides, Aspergillus, and Actinomycetes, and a majority of patients had a predominant proportion of the Firmicutes and Bacteroides. In 2005, Eckburg et al. found that the sum of the relative abundances of Firmicutes and Bacteroidetes exceeded 90% in the human gut microbiota (Eckburg et al., 2005). We further compared changes in flora at the phylum level, where the relative abundance of Bacillus decreased. β-hexosaminidase in the Methanobacterium has been shown to be a driver of lymphocyte-specific differentiation and that β-hexosaminidase-specific lymphocytes are protective against intestinal inflammation in a mouse model of colitis (Bousbaine et al., 2022). A study conducted on 115 critically ill patients revealed similar results, with patients leaving the ICU showing a significant decrease in the relative abundance of the Firmicutes and Bacteroidetes of the gut microbiota, a significant increase in the Proteobacteria, and an increase in pathogenic colonization with Enterobacter spp. and Staphylococcus spp., compared with those at the time of admission to the ICU (Mcdonald et al., 2016).

Notably, there were differences in the composition of the flora among individual patients after ICU treatment (Figure 3). Ojima et al. (2016) conducted a longitudinal observational study of the gut microbiota of 12 mechanically ventilated patients in a large tertiary care hospital; they found that the percentages of the Bacteroidetes and Firmicutes changed significantly during the follow-up period and that an extreme imbalance of flora might be associated with poor patient prognosis. Disturbances in the flora have been shown to be associated with increased susceptibility to nosocomial infections, sepsis, organ failure, and severe COVID-19 (Lynch and Pedersen, 2016; Ojima et al., 2016; Yeoh et al., 2021). In the present study, children treated in the PICU showed significant changes in C-reactive protein, calcitonin, white blood cells, platelets, creatinine, and lactate levels. In addition, the relative abundance of colonies such as Bacilli, Lactobacillales, and Campylobacter hominis changed significantly. Chao1 and ACE indices also decreased significantly, and the random forest analysis revealed that the species levels of Prevotella corporis and Enterbacter cloacae were specific markers before and after PICU treatment in critically ill children. This suggests that future studies should correlate changes in the gut microbiota of patients with changes in clinical indicators to provide novel reference indicators for assisting in the assessment of changes in patient conditions.

Our study found that after a short period of PICU treatment, the gene function of the gut microbiota of critically ill children can undergo significant changes, mainly including the processes of metabolism, DNA degradation, and transmembrane transport. However, it should be noted that our results are derived from computerized predictions, and their exact roles in human physiological activities remain unclear. Nevertheless, several studies have confirmed the involvement of metabolites of the gut microbiota in the regulation of vital organ functions. Hayakawa et al. (2011) found that changes in the composition of the flora, including increases in Enterococcus faecalis and Pseudomonas aeruginosa, accompanied by decreases in the three major short-chain fatty acids, butyric acid, propionic acid, and acetate, could occur within 6 h of the onset of critical illness. Short-chain fatty acids are products of the glycolytic process of dietary fibers by intestinal microorganisms, including acetate and butyrate; they are involved in regulating human immune functions, including promoting the differentiation and expansion of T cells, forming a complete mucosal immune system, and influencing the phagocytosis of macrophages (Furusawa et al., 2013). Butyrate also plays a role in regulating the transcription factor HIF-1, which is involved in maintaining the stable function of the intestinal barrier by decreasing the oxygen concentration in tissues (Lopez-Siles et al., 2017). Fatty acids may also be involved in the physiological functions of the brain, lungs, and cardiovascular system by activating the vagus nerve, reducing lung inflammation, and affecting renin secretion (Wozniak et al., 2022). Short-chain fatty acid levels have been shown to be negatively correlated with the severity of portal hypertension and systemic inflammation, emphasizing the role of gut microbiota in enterohepatic interactions and the progression of liver pathologies such as cirrhosis (Juanola et al., 2019). Short-chain fatty acids also play a role in acute kidney injury (AKI) by modulating the inflammatory response, and thus improving AKI outcomes (Andrade-Oliveira et al., 2015). Therefore, gut microbiota disorders can cause a decrease in beneficial metabolites, further causing adverse outcomes such as immune and organ dysfunction and increasing susceptibility to disease. Moreover, GO analysis in our study revealed that bacterial cell wall synthesis and the transmembrane transport of macromolecules were affected in the gut microbiota of critically ill children after a short period of ICU treatment. We hypothesized that this may be related to patients receiving antibiotics, proton pump inhibitors, and inadequate organ perfusion, causing a disturbance of the intestinal microenvironment and further affecting the physiological activity of the flora.

The intestine is a major site of drug-resistant bacteria. A healthy gut microbiota is a stable and diverse community that protects the host from invasion by pathogenic bacteria. Antibiotics can disrupt the stable ecosystem of the gut, providing conditions for colonization by drug-resistant bacteria, increasing resistance gene load, and further spreading resistant bacteria to other sites, causing infection (Anthony et al., 2021). Norgaard et al. found that the use of broad-spectrum β-lactam antibiotics was most significantly associated with increased microbial destruction and resistance characteristics in patients treated with hematopoietic stem cell transplantation (Nørgaard et al., 2023). The increasing prevalence of carbapenem-resistant Enterobacteriaceae poses a major global health threat (Jean et al., 2022). Studies have shown that the risk of infection with carbapenem-resistant Acinetobacter baumannii can increase by four-fold with exposure to carbapenems, and a new meta-analysis confirmed an association between carbapenem-resistant P. aeruginosa and increased mortality (Brink, 2019).

Our study has some limitations. First, while the purpose of this study was to describe the composition and function of the flora of critically ill children after PICU treatment, the degree of influence of factors was not evaluated. Second, owing to the observational nature of our study, it was not possible to control for the variables that might have affected the intestinal flora. Third, this study was a single-center study, and there was a certain selection bias.

After short-term treatment in the PICU, the richness of the gut microbiota in critically ill children was significantly decreased, while the bacterial diversity and the community structure between groups remained stable to some extent. The composition of some colonies was also altered significantly, with a significant increase in the relative abundances of Bacilli and Lactobacillales and a significant decrease in the relative abundance of C. hominis. GO and KEGG analyses showed that gene functions of the gut microbiota were also altered, mainly in genes responsible for metabolism, DNA catabolism, and transmembrane transport. In addition, the expression of resistance genes in critically ill children was changed significantly after short-term treatment in the PICU. The top 10 up-regulated genes were Erm(A), ErmX, LptD, eptB, SAT-4, tetO, adeJ, adeF, APH(3′)-IIIa, and tetM.

The data presented in this study are deposited in the NCBI SRA repository, accession number PRJNA1033539 (http://www.ncbi.nlm.nih.gov/bioproject/1033539).

The studies involving humans were approved by Ethics Committee of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

JX and YZ: study design. HM and YZ: data collection. JX and XK: statistical analysis. YX: data interpretation. JX, YX, and YZ: manuscript preparation. JL and JX: literature search. All authors contributed to the article and approved the submitted version.