Abstract

METHODS

RESULTS

Patient Characteristics

During the study period, fecal specimens were obtained from 113 patients during their transplant hospitalization. Nineteen patients provided insufficient specimens, leaving 94 subjects for analysis. No exclusion was due to early death. Time of discharge following allo-HSCT ranged from 13 to 95 days after transplant (median, 21 days). Clinical characteristics of the 94 patients are listed in Table 1. Most patients engrafted within 14 days after transplant. All patients received antibiotics during the study period; in particular, vancomycin and beta-lactam antibiotics were administered to most patients. Bloodstream infection was detected in 22 (23.4%) patients. Of these, 9 were due to VRE and 10 were due to gram-negative bacteria (Table 1).

Table 1.

Characteristics of Patients and Transplant Course

Parameter	No. (%) of Patients
Age, years
<30	7 (7.4)
30–39	13 (13.8)
40–49	19 (20.2)
50–59	28 (29.8)
≥60	27 (28.7)
Sex
Male	53 (56.4)
Female	41 (43.6)
Underlying diagnosis
Leukemia	44 (46.8)
Lymphoma	26 (27.7)
Multiple myeloma	8 (8.5)
Myelodysplastic syndrome	12 (12.8)
Other	4 (4.3)
Prior antibiotics (within 14 days before study period)
No	52 (55.3)
Yes	42 (44.7)
Conditioning regimen
Non-myeloablative	16 (17)
Reduced intensity	21 (22.3)
Myeloablative	57 (60.6)
T-cell depletion
No	52 (55.3)
Yes	42 (44.7)
Stem cell source
Peripheral blood	71 (75.5)
Umbilical cord	23 (24.5)
Time to engraftment, daysa,b
<14	70 (74.5)
≥14	24 (25.5)
Feverb
No	16 (17)
Yes	78 (83)
Antibiotics administeredb,c
Vancomycin	85 (90.4)
Fluoroquinolone	33 (35.1)
Metronidazole	30 (31.9)
Beta-lactamd	82 (87.2)
Bloodstream infectionb,e
Vancomycin-resistant Enterococcus	9 (9.6)
Gram-negative bacilli, aerobic	10 (10.6)
Other organism	3 (3.2)
None	72 (76.6)
Vital statusb
Alive	92 (97.9)
Dead	2 (2.1)
Total	94 (100)

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^a Engraftment was defined as an absolute neutrophil count of >500 cells/µL for 3 consecutive days.

^b Assessed during inpatient allogeneic hematopoietic stem cell transplantation hospitalization, from 15 days before transplant to 35 days after transplant.

^c Antibiotic variables are not mutually exclusive and do not sum to 100%.

^d Beta-lactams include cephalosporins, beta-lactam–beta-lactamase combinations, and carbapenems.

^e Positive bacteremia values not meeting standard Centers for Disease Control and Prevention definitions of a laboratory-confirmed bloodstream infection (eg, a single positive blood culture for coagulase-negative Staphylococcus) [44] were excluded.

Loss of Microbial Diversity Following Allo-HSCT

There was considerable variation among Shannon diversity index values (Figure 1). During the pre-transplant period, the average Shannon diversity index ranged approximately 3–4, similar to values observed for healthy volunteers in both our prior observations (not shown) and previous reports [12]. However, over the course of allo-HSCT, the mean diversity index decreased to approximately 2 and remained low until the end of our observation period. Similar decreases were observed with other measures of microbial diversity (Supplementary Figure 1). By subgroup, patients with leukemia and those receiving either metronidazole or beta-lactam antibiotics experienced comparatively greater decreases in Shannon diversity index (Supplementary Figure 2).

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Figure 1.

Changes in microbial diversity within the intestinal tract during allogeneic hematopoietic stem cell transplantation. Microbial diversity, quantified by the Shannon diversity index, was calculated for each fecal specimen of each patient and plotted over day of transplant (circles). A diversity trend (solid black line; 95% confidence intervals shown in gray) was constructed using moving average filtering.

Development of Intestinal Domination

The microbial composition for 12 selected patients over time, with concurrent episodes of bacteremia and administration of antibiotics, is shown in Figure 2A. In some patients, the microbiota remained relatively diverse, experiencing only mild fluctuations in composition. In these patients, the genus Blautia was frequently an abundant inhabitant and appeared to be associated with diminished antibiotic administration.

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Figure 2.

Characterization of the intestinal microbiota during allogeneic hematopoietic stem cell transplantation. A, Genus-level taxonomy of 12 selected patients, plotted over day of transplant. Each stacked bar represents the microbial composition of a single fecal specimen, based on taxonomic classification. Shading between specimens are provided for interpolation and visualization of changes in relative abundances. Concurrent antibiotic administration (vancomycin, fluoroquinolones, metronidazole, and beta-lactams) and detected bacteremias are shown for each patient. Patients in the first row generally maintained diversity with few changes in composition; patients in the second row developed intestinal domination by Enterococcus; patients in the third row developed intestinal domination by Streptococcus; and patients in the fourth row developed domination with Proteobacteria. Note that patient 58 in the top row maintained diversity but developed Enterococcus domination shortly after metronidazole administration. (Plots of all 94 patients can be found in Supplementary Figure 3.) B, Kaplan-Meier plot of intestinal domination as a survival event. Crosshairs represent censored observations. Intestinal domination by Enterococcus, Streptococcus, and Proteobacteria was common and occurred at various times throughout the observation period.

However, other patients demonstrated marked changes in composition, with transitions from a relatively diverse microbiota to a simpler one with fewer members. In many instances, the microbial composition became dominated by a single bacterial taxon. Enterococcus was the most frequent dominating genus, occurring in 38 (40.4%) patients. Of these patients, most (92%) had detectable expression of the vanA gene, suggesting the presence of VRE. Streptococcus was the next most frequent dominating genus, observed in 35 (37.2%) patients. Domination by phylum Proteobacteria, which includes Enterobacteriaceae and other aerobic gram-negative bacteria, was observed in 12 (12.8%) patients. Domination by these bacteria occurred at various times relative to transplant (Figure 2B). The composition of all 94 patients is provided (Supplementary Figure 3).

Hierarchical clustering of the phylogenetic composition of all 439 specimens demonstrated that specimens can be categorized into specific states of either biodiversity or domination by specific taxa (Figure 3A), with biodiverse specimens being more common during the pre-transplant period. Circos plots demonstrated a large number of state-to-state transitions (Figure 3B–E). Although most pre-transplant specimens remained biodiverse, transitions to states of domination were most prevalent between the pre-transplant specimen and the first post-transplant specimen and intermediate between sequential post-transplant specimens.

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Figure 3.

Microbiota states and their transitions. A, Hierarchical clustering of all fecal specimens according to microbiota state based on phylogenetic classification. There was wide variation in phylogenetic composition; some specimens showed a relatively biodiverse membership, whereas others were largely dominated by a single bacterial taxon. B, Circos plots representing microbiota state transitions between consecutive specimens within the observation period. Subplots of transitions during specific transplant periods are shown in C–E. C, Pre-transplant transitions revealing that patients in the “biodiverse microbiota” state tend to remain in that state; resilience of the “biodiverse” state, measured as the percentage of consecutive specimens that remain in that state, is 77%. D, Resilience of “biodiverse” state decreasing to 35% during the intra-transplant period (consecutive specimens straddling the day of stem cell infusion). E, Post-transplant transitions with the “biodiverse” state resilience staying low at 48%.

Clinical Factors Associated With Development of Intestinal Domination

Clinical predictors of intestinal domination with Enterococcus, Streptococcus, and Proteobacteria are shown in Table 2. Age, sex, conditioning regimen intensity, and stem cell source were not associated with intestinal domination by the 3 organisms evaluated. An underlying diagnosis of leukemia was associated with a higher risk of domination by Enterococcus.

Table 2.

Clinical Predictors of Intestinal Domination

	Enterococcus Domination		Streptococcus Domination		Proteobacteria Domination
Predictor	HR (95% CI)	P	HR (95% CI)	P	HR (95% CI)	P
Age, years	1.00 (.98–1.04)	.790	0.99 (.97–1.03)	.681	1.00 (.95–1.05)	.978
Female sex	0.84 (.42–1.64)	.611	1.07 (.50–2.27)	.852	1.12 (.33–3.78)	.854
Underlying diagnosis (leukemia vs other)	3.22 (1.60–6.94)	.001	0.71 (.32–1.51)	.375	0.66 (.18–2.19)	.498
Prior antibiotics (14 days)a	1.49 (.77–2.94)	.237	1.03 (.48–2.17)	.945	1.31 (.39–4.44)	.651
Conditioning regimen (myeloablative or reduced intensity vs non-myeloablative)	1.01 (.44–2.84)	.977	0.61 (.25–1.75)	.329	0.98 (.22–9.25)	.983
T-cell depleted graft	0.81 (.40–1.61)	.551	0.91 (.39–2.00)	.812	1.07 (.29–3.62)	.910
Stem cell source (cord vs other)	1.22 (.55–2.52)	.607	0.54 (.19–1.34)	.196	1.36 (.36–4.69)	.633
Feverb	1.68 (.78–3.74)	.182	0.90 (.36–2.39)	.826	1.28 (.30–6.34)	.747
Antibioticsb
Vancomycin	2.12 (.67–10.21)	.222	0.95 (.33–3.77)	.938	5.17 (.52–707.15)	.192
Metronidazole	3.38 (1.65–6.73)	.001	1.94 (.81–4.30)	.131	1.73 (.41–6.03)	.426
Fluoroquinolonesc	1.09 (.49–2.24)	.832	1.19 (.51–2.60)	.677	0.09 (.00–.75)	.020
Beta-lactamd	1.64 (.74–3.99)	.232	1.69 (.62–5.64)	.319	1.23 (.27–7.50)	.800

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Abbreviations: CI, confidence interval; HR, hazard ratio.

^a Defined as administration of any antibacterial drug within 14 days prior to observation period.

^b Analyzed as a time-varying predictor.

^c Fluoroquinolones consist of ciprofloxacin and levofloxacin.

^d Beta-lactams include cephalosporins, beta-lactam–beta-lactamase combinations, and carbapenems.

Antibiotics, given at various time points for various indications (eg, prophylaxis or empiric/directed treatment) were associated with subsequent enterococcal domination. Metronidazole was associated with a 3-fold increase in risk of enterococcal domination. Metronidazole was given for various reasons, including treatment for Clostridium difficile infection (14 of 30 metronidazole recipients). Vancomycin, fluoroquinolone, and beta-lactam administration did not increase the risk of domination. Fluoroquinolone administration was associated with a 10-fold decreased risk of intestinal domination by Proteobacteria.

DISCUSSION

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, 27], 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 [28–30]. 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, 32].

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 [4–8]. Prior to 2002, viridans streptococci were an important cause of bacteremia at our center, with significant morbidity [4, 6]. 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, 8, 33], 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, 36] and supports the notion that anaerobic bacteria contribute to colonization resistance [30, 31]. 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, 39] 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, 37]. 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 [40–43]. 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.

Supplementary Data

Supplementary materials are available at Clinical Infectious Diseases online (http://cid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

References