Childhood acute leukemia has become the second mortality disease in China. Currently, chemotherapy is the main treatment, and the long-term survival rate of this disease after chemotherapy can amount to 85%(17, 18). However, children with leukemia after chemotherapy inevitably suffer from bone marrow suppression and neutropenia. At the same time, because of the abnormal white blood cell function and followed with lose of normal human resistance, thus increases the chance of infection. Intestinal mucosal barrier mainly includes mechanical barriers, chemical barriers, immune barriers, and biological barriers. The principal function is to prevent bacteria translocation and endotoxin absorption(19). Stringer, A. Met al found that chemotherapy can induce the synthesis of reactive oxygen species (ROS), free radicals and ceramides, which can lead to cellular DNA damage, intestinal mucositis, and activate nuclear transcription factor (NFκ B ). It can also produce many pro-inflammatory factors to increase intestinal inflammation, break the intestinal microecological balance, damage the intestinal mucosal barrier function, which leads to mass propagation and translocation of opportunistic pathogens, harmful bacteria invading, and eventually causing infection(20).
Most studies have shown that most of the infections in patients with hematological malignancies originate from intestinal flora. However, few studies have been conducted on the composition and structure of intestinal flora in childhood acute leukemia and the relationship between infection and infection outcomes. In this study, a total of 66 fecal specimens of childhood acute leukemia were collected, of which 16 were neutropenia and 50 were non-neutropenia. Aarne Kolonen’s study found that bloodstream infection affected the prognosis of childhood acute leukemia receiving intensive treatment. The results showed that there were 977 courses of blood culture data, of which 503 cases of bloodstream infection (51%), and 20 cases died of infection (5.6%) ), of which 16 were bloodstream infection-related deaths (80%) (21). A total of 29 infection events occurred in our study, the infection rate was 43.9%, and no death occurred, which was lower than the infection rate and mortality rate of Aarne Kolonen’s study. Previous studies have found that neutropenia in childhood acute leukemia has a higher incidence of infection(5). In this study, the infection rate of childhood acute leukemia with neutropenia was 87.5% (14/16), while the infection rate of that without neutropenia was only 30% (15/50). By comparing the structural characteristics and differences of the intestinal flora of the two groups, this study is expected to find potential infectious pathogens and use targeted antibiotics early, thereby shortening the length of hospital stay, reducing the progression of severe infections to systemic inflammatory response syndrome, and even death happened. Our results showed that Alpha diversity index (Chao1 index P = 0.03, Shannon index P = 0.15) in neutropenic with childhood acute leukemia was significantly lower than that in non-neutrophilic with childhood acute leukemia children. Previous studies have also found that the microbial diversity of intestinal flora in children with ALL, AML or non-Hodgkin's lymphoma (NHL) decreased during chemotherapy, and gradually recovered with the extension of chemotherapy (13, 22, 23). Early studies found that the diversity of intestinal flora in childhood acute lymphoblastic leukemia was significantly lower than that in healthy controls at baseline(24). There were also significant differences in intestinal flora diversity among childhood acute lymphoblastic leukemia who had recently used antibiotics or not(25). We can expand the sample size, track the changes in the diversity of intestinal flora during the chemotherapy-induced remission, consolidation, and maintenance phases of children, and predict the likelihood of infection based on their baseline, which is our next goal.
Previous studies have shown that Firmicutes, Bacteroidetes, Actinomycetes, and Proteobacteria constitute the vast majority of intestinal flora, of which the former two account for more than 90%(26). In addition to the Firmicutes and Bacteroidetes, the proportion of Proteobacteria is also large, in our research, and the combined proportion of the three is greater than 95%. Simultaneously, our study observed that the abundance of Proteobacteria in the intestinal flora of children with neutropenia is higher than that of children with non-neutropenia, which is consistent with the study by Hana Hakim(13), suggesting that the relatively high abundance of phylum Proteobacteria may predict febrile neutropenia. Research by Taur, Ying(27) found that during allogeneic hematopoietic stem cell transplantation, Firmicutes accounts for more than 30%, which can increase the risk of Gram-negative bacilli bacteremia by 5 times. In terms of ecological imbalances, the relative abundance ratio (F/B) of Firmicutes and Bacteroides is a commonly used evaluation index and represents the composition structure of intestinal flora. The Phylum Firmicutes helps the body absorb and store energy from food, while the Phylum Bacteroidetes helps the body consume energy. In some hematological malignancies, such as multiple myeloma(28, 29), Hodgkin’s disease(30), the proportion of Bacteroides in the intestine tracts of patients was significantly increased compared with the healthy control group, suggesting that the increase of phylum Bacteroides is related to the high energy consumption state of immune disorder. Most Bacteroidetes are Gram-negative bacteria with an outer membrane containing endotoxin. Related studies have shown that intestinal permeability of patients with multiple myeloma with high-abundant Bacteroides increased(30), which is beneficial to the intestinal flora and toxin product translocation. In this study, the abundance of Firmicum decreased and Bacteroides increased in neutropenic acute leukemia children, and the F/B ratio was lower than that in non-neutrophilic acute leukemia children. It is suggested that neutrophil deficient with childhood acute leukemia have reduced energy intake and storage, and maybe in a state of high energy consumption due to immune disorder, resulting in poor body resistance during bacterial invasion and more prone to infection.
Blautia is a Gram-positive, non motile, spherical, or oval anaerobic bacteria. It produces acetic acid, lactic acid, succinic acid, ethanol, and hydrogen as the main metabolites of glucose metabolism. These metabolic end products, especially the fermentation product butyrate, can enhance the immune function of intestinal mucosa(31, 32). In a cohort study, increased bacterial abundance in Blautia was closely associated with decreased lethal acute graft-versus-host disease (GVHD) and improved overall survival, suggesting that Blautia may result in suppressing immune rejection(33). Blautia is part of the most important markers to improve the prognosis of various clinical diseases such as visceral fat accumulation(34) and Crohn's disease(35). Roseburia bacteria are also a part of symbiotic bacteria that produce short-chain fatty acids (especially butyric acid), which maintain immunity and have anti-inflammatory properties. Flagellin of R intestinalis may up-regulate the expression of tight junction protein through toll like receptor 5 (TLR5), thus restoring the integrity of intestinal barrier, and helping to restore intestinal flora by increasing the expression of IL-22 and REG3γ(36). Our research found that the abundance of Blautia and Roseburia bacteria in acute leukemia children with neutropenia was significantly reduced, which was negatively correlated with the incidence of infection. The loss of Clostridia members (including Roseburia, E. faecalis, Rumenococcus, and Blautia) is associated with poor prognosis in a variety of diseases. In addition to the aforementioned bacteria of genera Roseburia and Blautia, the abundance of the genus Faecalibacterium and Rumenococcus of acute leukemia children with neutropenia is less than that of non- neutropenia group. These two genera are associated with anti-inflammatory effects and may be involved in maintaining the integrity of the intestinal epithelium(36). There is no significant differences between the two bacteria in statistics, which may be due to the insufficient sample size, which needs to be verified by expanding the sample size.
Enterococcus is a lactate-producing bacteria. When the content of this flora is abnormally increased, the accumulation of lactate can increase the permeability of the intestinal mucosa, which can cause endotoxemia, ulcerative colitis, and even lead to bowel resection(37). Studies have shown that enterococci or Streptococcus are predictors of infection after chemotherapy regardless of the stage of chemotherapy(13). A study by the American Society for transplantation and cell therapy found that 89.3% of patients survived two years after allogeneic hematopoietic stem cell transplantation (HSCT) had less than 1% enterococci abundance, suggesting a significant correlation between low abundance enterococci and high survival rate(38). Our study showed that the abundance of Enterococcus and Streptococcus in neutrophilic with childhood acute leukemia was significantly higher than that in patients with non-neutropenia. It is suggested that the intestinal mucosal barrier of children with neutropenia is more permeable, and it is easy for bacterial flora to shift and absorb endotoxin, resulting in infection outcomes. Bacteroides fragilis belongs to the normal intestinal flora, mainly in the colon. Bacteroides fragilis ranks first in clinical anaerobic bacteria isolation, accounts for up to 20%. When the intestinal mucosa of the host is damaged, Bacteroides fragilis heterotopic becomes an opportunistic pathogen, causing purulent infection of various organs of the body, such as intestine, abdominal cavity, liver, lung, and brain, accompanied by abscess and acute and chronic diarrhea(39). Bacteroides fragilis can secrete enterotoxins, make the adhesion and connection between the cells of human colon epithelial cell line HT29 / C1 disappear, round, and swell the cells. At the same time, it can increase the secretion of chloride ions and inhibit the absorption of sodium ions, thereby weakening the intestinal mucosal barrier and resulting in Bacteria translocation(40, 41). In recent years, Bacteroides fragilis has developed resistance to a variety of commonly used antibiotics, especially imipenem and metronidazole(42, 43). Our study has observed that the abundance of Bacteroides fragilis in neutropenic with childhood acute leukemia is significantly higher than that in children with non-neutropenia. This explains why the infection rate of the former is significantly higher than that of the latter.
In conclusion, the infection rate of acute leukemia with agranulocytosis after chemotherapy was significantly greater than that of non-agranulocytosis group. In the agranulocytosis group, Enterococcus, Streptococcus, and Bacteroides fragilis increased, while Clostridium, Blautia and Roseburia decreased. However, according to the binary regression analysis, only relatively high abundance of Bacteroides fragilis in the agranulocytosis group can predict the occurrence of infection, and the risk factor is not high, which is considered to be caused by too few samples. We need to further expand the sample size and conduct multi center research, to find out the intestinal flora which can accurately predict the infection in the early stage, and early targeted anti-infection treatment, so as to reduce the infection rate and mortality rate after chemotherapy.