Changes in intestinal bacterial composition caused by antibiotics vary from antibiotic to antibiotic. Gentamicin is a broad-spectrum aminoglycoside with strong antibacterial activity against gram-negative bacteria. Cefradine, a broad-spectrum cephalosporin belongs to β-lactam, has a bactericidal effect on both gram-positive and gram-negative bacteria. In the previous study, we found the numbers of bacteria and colibacillus decreased rapidly as the use of gentamicin and cefradine. The number of yeast and mold presented an increasing trend. Similar changes were found in other AAD models. Theriot et al. treated mice with a variety of antibiotics to create distinct microbial and metabolic (bile acid) environments and found that susceptibility to C. difficile in the large intestine was observed only after specific broad-spectrum antibiotic treatment (cefoperazone, clindamycin, and vancomycin). These changes were correlated to the loss of members of families Lachnospiraceae and Ruminococcaceae. Larcombe et al. established an S. aureus infection model in mice pre-treated with kanamycin, gentamicin, colistin, metronidazole, vancomycin, and cefaclor. The results showed that colonization of various S. aureus strains could be achieved after antibiotic pre-treated. Antibiotics lead to an alteration in bacteria composition resulting in changed metabolism and diminished anti-colonization.
The results, as direct evidence, suggested that the mice developed AAD was associated with alteration of the normal gut microbiota, which was mainly manifested as fewer beneficial bacteria and more potential pathogens. As shown in figure 3, after treatment with antibiotics, the number of bacterial species in intestinal contents decreased to different degrees at the levels of phylum, class, order, family, and genus. This result indicated that antibiotics changed the structure and density of normal intestinal flora, resulting in the disorder of intestinal flora. Specifically, to the level of phylum, the gut microbiota of normal mice was comprised of three dominant phyla, namely Firmicutes (63.52%), Bacteroidetes (17.27%), and Proteobacteria (13.07%). As for AAD mice, it was transformed into Firmicutes (52.63%) and Proteobacteria (46.37%). There was a significantly higher F/B ratio in AAD mice. Similar shifts occurred in the intestinal mucosal bacteria of AAD mice. Massive data identified Proteobacteria as a possible microbial signature of diseases which are sustained by various degrees of inflammation. Notably, inflammation is demonstrated to be implicated in the development of metabolic disorders. Thus, an increased abundance of Proteobacteria implies the risk of infection and metabolic disorder in a pathological state.
Furthermore, significant alterations in the gut microbiota composition were found at the genus levels. The dominant genera of healthy mice were Bacillus and Lactobacillus, which were beneficial to maintaining healthy intestinal flora and reducing the colonization of pathogenic organisms[26, 27]. However, with the administration of antibiotics, bacteria that were sensitive to gentamicin and/or cefradine were suppressed or killed, and bacteria that were resistant to them have the opportunity to invade and multiply. As a consequence, the abundance of Prevotella, Bacteroides, and Adlercreutzia showed a significant downward trend, while Ruminococcus, Blautia, Enterococcus, Eubacterium, Clostridium, Coprococcus, Aerococcus, and Pseudomonas showed a statistically upward trend in AAD mice (Figure 6). Among these bacteria, the changes of Enterococcus and Eubacterium were especially prominent (Figure 6, 7). In the AAD mice, the abundance of Enterococcus and Clostridium showed a significant increase, which was similar to the changes in the intestinal mucosa of AAD mice. In our previous studies, we found a significant reduction in Lactobacillus in the intestinal mucosa of AAD mice, and the main genera in the intestinal mucosa of AAD mice were Enterococcus, Stenotrophomonas, Glutamicibacter, Citrobacter, and Pseudomonas. The main genera in the intestinal contents of the same AAD model mice were Lactobacillus, Enterococcus, Blautia, [Ruminococcus], and Bacillus. Previous studies also showed that the role of intestinal microbiota in the development of AAD from the perspective of intestinal microbial function enzyme (lactase) gene. The main lactase-producing strains differed in the intestinal content and mucosa. The main lactase-producing strain in the intestinal contents is Pseudomonas fluorescens, while the main lactase-producing strain in the intestinal mucosa is Stenotrophomonas. Besides, antibiotics reduced the diversity of bacterial lactase genes in the intestinal contents but increased it in the intestinal mucosa[28, 29]. These dissimilarities in the composition and function of intestinal mucosal microbiota and intestinal contents microbiota provide new evidence for the spatial heterogeneity along the cross-section of the digestive tract (from lumen to mucosa). Factors known to drive this spatial heterogeneity along the longitudinal and transverse axes include chemical gradients (e.g., pH), oxygen levels, nutrient availability, immune effectors, and functional heterogeneity of each gastrointestinal tract segment[12, 30].
Enterococcus are important opportunistic pathogens, with E. faecalis and E. faecium as the most representative species, causing a wide variety of infections. Many Enterococci have plasmid-encoded resistance genes which cause less susceptible to several antimicrobial agents intrinsically including gentamicin and cefradine[31-33]. Biofilm formation has been identified as an essential factor in the evasion of the host’s immune response, the inhibitory or killing effects of antibiotics, and the pathogenesis of enterococcal infections[32, 34]. In addition, Enterococci are recognized as possessing a variety of virulence factors, which contribute to the mediation of adhesion, colonization, and invasion into the host tissues, modulation of the host immunity, and extra-cellular production of enzymes and toxins. In this study, Enterococcus exhibited its intrinsic resistance to gentamicin and cefradine. Instead of being inhibited or killed, they proliferated in large quantities. A comparative genomic analysis discovered that the core-genome of Enterococcus obtains many genes related to carbohydrate metabolism and mannose, fructose, lactose, and galactose were the principal energy sources of Enterococcus. Based on previous studies and the results presented here, we propose that the overgrown Enterococcus may cause colonic infection and homeostasis disorder by forming a biofilm, possessing virulence factors, and adjusting carbohydrate metabolism. The specific mechanism needs further verification.
Another noteworthy bacterium was Clostridium. As known to all, C. difficile and C. perfringens are high-risk infectious origins of AAD. The former can produce an enterotoxin (toxin A) and a cytotoxin (toxin B), which cause mucosal injury and colonic inflammation. The later can produce potent protein toxins (α-toxin, β-toxin, ε-toxin, and ɩ-toxin), which cause many different histotoxic and enterotoxic diseases in humans and animals. There was no direct taxonomic evidence for C. difficile and C. perfringens in our data, but a significantly increased abundance of Clostridium also attracted our attention. We blasted the original sequence pairs that were classified into the genus Clostridium to the NCBI database separately. Then, a suspected strain of C. difficile and a suspected strain of C. perfringens were found. However, the identity of the specific strain remains to be further confirmed.