Patients undergoing allo-HSCT are subjected to chemotherapy, radiation, and antibiotics within a short time frame. In this study we observed extreme shifts in the intestinal microbiota during transplant. In many instances, domination by a single bacterial taxon occurred. These perturbations correlated with subsequent development of a corresponding bloodstream infection with either VRE or gram-negative bacteria.
The intestinal microbiota is engaged in a complex relationship with the mucosal epithelium. Intestinal epithelial cells, underlying immune tissues, and the microbiota establish a state of equilibrium that optimizes resistance to infection and facilitates the absorption of nutrients [26
]. Allo-HSCT disrupts this equilibrium, resulting in dramatic compositional fluctuations that can result in domination by bacteria that invade the bloodstream. Radiation and chemotherapy-mediated destruction of gut epithelial stem cells, inhibition of microbial populations by antibiotic administration, and suspension of innate immune defenses each affect intestinal environment and host-microbe interactions.
We find that microbial diversity is generally not re-established during allo-HSCT hospitalization, perhaps due in part to continued antibiotic pressure. Other studies of the intestinal microbiota have shown the resilience of bacterial populations following antibiotic therapy [12
], and experimental studies in mice demonstrate that decreased diversity increases susceptibility to dense colonization by VRE [15
]. Although the benefits of diversity remain unproven, strategies to reintroduce complex microbial populations into the gut following allo-HSCT may be of benefit.
How the complex microbiota prevents domination by rogue bacterial species such as VRE or Enterobacteriaceae remains unclear. Antibiotic administration results in expansion of Enterobacteriaceae and enterococci in the gut and increases susceptibility to infection by these organisms. The suppression of newly introduced bacteria by the intestinal microbiota is referred to as colonization resistance [28
]. Although commensal microbes may outcompete VRE by depleting nutrients or occupying spatial niches, other mechanisms such as production of inhibitory short-chain fatty acids may play a more important role [31
In this and our previous studies [15
], bloodstream infections during allo-HSCT are frequently preceded by intestinal domination by the same organism. In this study, two-thirds of patients developed either enterococcal or streptococcal domination during the course of transplant. Interestingly, viridians-group streptococci and VRE were historically the most frequently encountered bloodstream infections in allo-HSCT patients during the pre-engraftment period [4
]. Prior to 2002, viridans streptococci were an important cause of bacteremia at our center, with significant morbidity [4
]. Prophylactic vancomycin administration during transplant has virtually eliminated viridans-group Streptococcus
bacteremia in our center [7
]. Because streptococcal domination remained common in this study, vancomycin prophylaxis may have served to prevent bloodstream invasion but not intestinal domination.
In contrast to Streptococcus
domination, enterococcal domination was highly associated with VRE bacteremia. Prior studies of allo-HSCT patients have demonstrated associations between VRE colonization and the development of VRE bacteremia [7
], leading many centers to routinely test for VRE colonization by means of rectal swab cultures prior to transplant. Compared with routine surveillance culture findings, enterococcal domination was detected in a greater percentage of subjects (40% vs 21%, respectively), and we identified more patients with VRE bacteremia (8 of 9 instead of 6). It is possible that bacteremia occurred with greater frequency than was documented, because blood cultures were largely obtained in the setting of fever or other evidence of septicemia. Indeed, a study involving surveillance blood cultures of steroid-treated allo-HSCT patients revealed bacteremia in more than one-third of patients [34
Our finding that metronidazole administration is strongly associated with the development of Enterococcus
domination is consistent with prior studies demonstrating that antibiotics with anaerobic activity promote VRE colonization [35
] and supports the notion that anaerobic bacteria contribute to colonization resistance [30
]. Because vanA
was present in most cases of enterococcal domination, and because most cases of enterococcal bloodstream infections were vancomycin resistant, we presume antibiotic resistance contributes to microbial shifts within the microbiota; however, indirect effects of antibiotic treatment resulting from interdependencies of bacterial taxa in the gut are likely important as well [37
]. The Shannon diversity index was also lower in recipients of metronidazole, presumably reflecting our observed association with enterococcal domination. However, beta-lactam recipients also lost diversity but were not associated with enterococcal domination. Although this disparity may reflect the ability of beta-lactams to inhibit enterococcal growth in the intestine, an alternative explanation is that beta-lactams were administered later during the course of transplant, as empiric therapy for fever in the setting of neutropenia, and thus were largely unevaluated in the Cox model as a preceding risk factor for domination.
We found that fluoroquinolones protected against proteobacterial domination, which in turn protected against gram-negative bloodstream infections. This finding supports prior studies of fluoroquinolone prophylaxis in neutropenic patients [38
] and provides insight into the mechanism of protection. The routine practice of fluoroquinolone prophylaxis in neutropenia has been controversial, in part due to concerns about resistance or disturbance of the microbiota. Other studies show fluoroquinolones having effects of varying duration in healthy humans [12
]. In those subjects, there were no clinically apparent consequences resulting from these microbial disturbances. Whether fluoroquinolone prophylaxis in patients undergoing allo-HSCT has any deleterious consequences remains unclear.
Patients with leukemia were more likely to develop enterococcal domination than those with other underlying diseases. The reason for this association is unclear but is likely multifactorial, including greater pre-transplant exposure to antibiotics, more intense conditioning prior to transplant, and/or greater exposure to VRE in conjunction with prior treatment for leukemia. However, our analysis did not identify associations between these factors and enterococcal domination.
Our study has several limitations. Specimens were collected with variable frequency of approximately 1 week apart. Thus, our microbiologic data could be viewed as interval-censored since we do know the state of the microbiota between successive specimens. At times we observed rapid transitions, and thus there may have been transient microbial states which were not captured. For example, episodes of domination, perhaps preceding some episodes of bloodstream infection, may have occurred but went undetected. To obviate this, we used a cutoff definition of 30% for intestinal domination, even though in many instances the relative abundance was much higher. We chose this cutoff primarily on the basis of resultant grouping of microbial states in our hierarchical cluster analysis, but this cutoff likely enhanced the identification of domination states, given the frequency of collection. The number of 16S sequences obtained per specimen was lower than that of some other studies [37
]. Thus, the depth of coverage may have been insufficient to determine whether certain infrequent bacterial taxons were present. Since we focused on dominating (ie, highly abundant) microbes, we expect that our results would be unaffected by deeper sequencing. Furthermore, Good's coverage was ≥98% in >96% of specimens in this study.
Although our studies are limited to allo-HSCT patients at one medical center, we suspect similar microbiota shifts are also occurring at other centers, and for that matter in other populations of patients who might be at risk for bloodstream infections due to neutropenia, such as those receiving cytotoxic chemotherapy for hematologic malignancies. Based on evidence from prior studies, it is also likely that disturbances of the microbiota are implicated in the pathogenesis of other complications of allo-HSCT, such as such as graft-versus-host disease of the gut and C. difficile
associated diarrhea [40
]. Further study is needed in order to fully grasp the clinical implications of microbial derangements in these patient populations.
Our study demonstrates that intestinal domination in a subset of allo-HSCT patients precedes bloodstream infection by a median of 7 days, suggesting that determination of the fecal microbiota composition can identify patients at highest risk for bacteremia. This longitudinal study provides an example of how analysis of the intestinal microbiota can have relevance in clinical disease. Although current deep sequencing platforms for the analysis of the microbiota require substantial time for specimen preparation, amplification, sequencing, and analysis, it is likely that these platforms will evolve in the near future to provide microbiota analyses within a clinically meaningful period. With this progress, microbiota analyses such as that presented in our study will increasingly guide the treatment of pertinent populations such as allo-HSCT recipients.