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Bloom and bust: intestinal microbiota dynamics in response to hospital exposures and Clostridium difficile… Vincent, Caroline; Miller, Mark A; Edens, Thaddeus J; Mehrotra, Sudeep; Dewar, Ken; Manges, Amee R Mar 14, 2016

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RESEARCH Open AccessBloom and bust: intestinal microbiotadynamics in response to hospital exposuresand Clostridium difficile colonization orinfectionCaroline Vincent1,2, Mark A. Miller3, Thaddeus J. Edens4, Sudeep Mehrotra5, Ken Dewar2,6 and Amee R. Manges7*AbstractBackground: Clostridium difficile infection (CDI) is the leading infectious cause of nosocomial diarrhea. Hospitalizedpatients are at increased risk of developing CDI because they are exposed to C. difficile spores through contact withthe hospital environment and often receive antibiotics and other medications that can disrupt the integrity of theindigenous intestinal microbiota and impair colonization resistance. Using whole metagenome shotgun sequencing,we examined the diversity and composition of the fecal microbiota in a prospective cohort study of 98 hospitalizedpatients.Results: Four patients had asymptomatic C. difficile colonization, and four patients developed CDI. We observeddramatic shifts in the structure of the gut microbiota during hospitalization. In contrast to CDI cases, asymptomaticpatients exhibited elevated relative abundance of potentially protective bacterial taxa in their gut at the onset of C.difficile colonization. Use of laxatives was associated with significant reductions in the relative abundance ofClostridium and Eubacterium; species within these genera have previously been shown to enhance resistanceto CDI via the production of secondary bile acids. Cephalosporin and fluoroquinolone exposure decreased thefrequency of Clostridiales Family XI Incertae Sedis, a bacterial family that has been previously associated withdecreased CDI risk.Conclusions: This study underscores the detrimental impact of antibiotics as well as other medications, particularlylaxatives, on the intestinal microbiota and suggests that co-colonization with key bacterial taxa may prevent C. difficileovergrowth or the transition from asymptomatic C. difficile colonization to CDI.Keywords: Clostridium difficile infection, Whole metagenome shotgun sequencing, Intestinal microbiota, Antimicrobials,MedicationsBackgroundClostridium difficile infection (CDI) is the leading causeof infectious diarrhea in hospitalized patients. In theUSA alone, there are an estimated 453,000 cases and29,300 deaths from CDI each year [1]. CDI is associatedwith a wide range of syndromes, from asymptomaticcolonization to mild diarrhea or more severe pseudomem-branous colitis that may progress to toxic megacolon,intestinal perforation, sepsis, and death [2]. Despite ad-vances in infection control practices and the developmentof new treatment options, there has been a steady increasein the incidence and severity of CDI in the last decade andoutbreaks continue to occur in hospitals and health-careinstitutions worldwide [3, 4].Hospitalized patients are at increased risk of developingCDI because they are exposed to C. difficile sporesthrough contact with the hospital environment and oftenreceive broad-spectrum antimicrobials that disrupt the in-tegrity of the indigenous intestinal microbiota and impaircolonization resistance (i.e., the ability of the microbiota* Correspondence: amee.manges@ubc.ca7School of Population and Public Health, University of British Columbia,Vancouver, British Columbia, CanadaFull list of author information is available at the end of the article© 2016 Vincent et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Vincent et al. Microbiome  (2016) 4:12 DOI 10.1186/s40168-016-0156-3to prevent the establishment of enteropathogens like C.difficile in the gut). Nearly all classes of antibiotics havebeen associated with CDI, but clindamycin, penicillins,cephalosporins, and fluoroquinolones seem to pose thegreatest risk [5–7]. Additional risk factors for CDI includeadvanced age, underlying diseases, gastrointestinal sur-gery, nasogastric tube feeding, and use of proton pump in-hibitors (PPIs, a class of medications that inhibit theproduction of gastric acid in the stomach) [2, 8].Among patients who acquire C. difficile in their gut,some will remain asymptomatically colonized whileothers may go on to develop diarrhea or more severeforms of CDI. Differences in pathogen or host factorslike the immune status or the integrity of the intestinalmicrobiota may affect the clinical presentation of CDI.In hospitals and health-care facilities, asymptomatic car-riers often outnumber symptomatic patients and mayrepresent a considerable reservoir of C. difficile that con-tributes to environmental contamination and diseasetransmission among patients [9, 10]. It has been sug-gested that patients with asymptomatic C. difficilecolonization are at decreased risk of developing CDI, buta recent meta-analysis has suggested this may not be thecase [11, 12]. Previously, we showed that patients whohave higher levels of Clostridiales Family XI IncertaeSedis were at a decreased risk of developing CDI [13],and others have demonstrated that the presence of sec-ondary bile acid-producing bacteria such as Clostridiumscindens was associated with resistance to CDI [14].Despite the strong relationship between the intestinalmicrobiota and CDI susceptibility, the impact of non-antimicrobial medications on the microbiota has notbeen examined in detail.In this study, we prospectively examined the intestinalmicrobiota of hospitalized patients at-risk for CDI. Usingwhole metagenome shotgun (WMGS) DNA sequencing,we specifically assessed (i) the changes in the relativeabundance of microbial taxa in patients who were iden-tified as colonized or infected with C. difficile and (ii)the impact of antibiotics and other medications on thediversity and composition of the intestinal microbiotaamong patients who were neither colonized nor infectedwith C. difficile. We postulated that protective micro-biota members which are thought to mediate competi-tive inhibition against C. difficile (such as ClostridialesFamily XI Incertae Sedis and non-toxigenic C. difficile)[13, 15] or limit the germination and growth of C. diffi-cile via the production of secondary bile acids (Clostrid-ium and Eubacterium genera) [14, 16] are present incolonized but not in infected patients. We also hypothe-sized that not only antibiotics but also other medicationssuch as PPIs will decrease the overall diversity of the in-testinal microbiota and increase the relative abundanceof opportunistic microorganisms such as enterococciand yeasts [17–20]. We report that the relative abun-dance of Clostridiales Family XI Incertae Sedis, Clostrid-ium, and Eubacterium is higher in asymptomaticallycolonized patients than in CDI cases. Moreover, antibi-otics and other medications such as laxatives have sub-stantial effects on the intestinal microbiota of hospitalizedpatients and reduce the relative abundance of these poten-tially protective bacterial taxa. Even though the term “in-fected” is sometimes used to designate people who areasymptomatically colonized with C. difficile, in this report,we will use the term “infected” (or CDI) to describe pa-tients who are symptomatic and “colonized” to describepatients who are asymptomatically colonized with C.difficile.ResultsSubject characteristicsA total of 104 patients were enrolled in the study. Fourpatients did not provide any stool sample, and six stoolsamples were excluded because of poor library quality orsequencing results; this left a total of 98 patients and229 fecal samples in the study. We analyzed a median oftwo fecal samples per patient (range, 1–15). Patientcharacteristics are shown in Table 1. The majority of pa-tients (63 %) suffered from osteoarthritis and were ad-mitted for orthopedic surgery; most of them (89 %)received cefazolin (a first-generation cephalosporin) asperioperative antimicrobial prophylaxis. One patient(1.0 %) had asymptomatic C. difficile colonization on ad-mission, three patients (3.1 %) became asymptomaticallycolonized during hospitalization, and four patients(4.1 %) developed hospital-acquired CDI. All of the CDIcases had diarrhea during their initial episode but noneof them developed recurrent CDI.WMGS sequencingFecal samples were evaluated by WMGS sequencing toassess the diversity and composition of the intestinalmicrobiota. We obtained a median of 6.1 million high-quality reads per sample (range, 0.6–41.9 million). Theproportion of human DNA reads was highly variable(range, 0.1–94.9 %) but half of the samples contained<20 % of human reads. After removing the host DNA,we computed the frequency of 16S ribosomal RNA(rRNA) genes in each sample as a measure of bacterialDNA content. The mean frequency of 16S rRNAgenes per megabase (Mb) of sequence data was 0.39(standard deviation 0.18). Across all samples, we ob-tained a median of 2.1 % of reads (range, 0.1–4.8 %)with a hit to MetaPhlAn2’s taxonomic marker data-base, with at least 2700 hits per sample. Bacteroides(99.0 %), Prevotella (98.0 %), and Subdoligranulum(98.0 %) were the most common genera, and Por-phyromonadaceae (100 %), Bacteroidaceae (99.0 %),Vincent et al. Microbiome  (2016) 4:12 Page 2 of 11Enterobacteriaceae (98.0 %), Prevotellaceae (98.0 %),and Ruminococcaceae (98.0 %) were the most com-mon families detected across patients (percentage ofpatients in which the corresponding taxa wasdetected).C. difficile detectionResults from the detection of C. difficile or its toxins ineight patients who developed C. difficile colonization(n = 4) or infection (n = 4) are provided in Table 2.Out of these eight patients, six had a positive C.Table 1 Characteristics of the study patientsVariable Neither C. difficile infectionnor colonization (n = 90)C. difficile infection(n = 4)C. difficile colonization(n = 4)Age, mean years (range) 74 (61–91) 71 (66–80) 74 (71–78)Male sex 44 (49) 2 (50) 2 (50)Horn’s indexa, median (range) 1 (1–2) 1 (1–1) 1 (1–2)Duration of hospitalizationb, median days (range) 5 (1–69) 21 (2–61) 5 (1–15)Hospitalization in past 12 monthsc 11 (12) 0 0Fecal specimens analyzed, median (range) 2 (1–12) 4 (1–15) 2 (1–4)Reason for hospital admissionOsteoarthritis/Rheumatoid arthritis 62 (69) 0 1 (25)Pneumonia 8 (9) 1 (25) 1 (25)Cellulitis 4 (4) 1 (25) 1 (25)Fever 2 (2) 0 0Chronic obstructive pulmonary disease 3 (3) 0 0Othersd 11 (12) 2 (50) 1 (25)Medication usebNon-steroidal anti-inflammatory drugs 33 (37) 4 (100) 2 (50)Proton pump inhibitors 26 (29) 2 (50) 2 (50)Glucocorticoids 12 (13) 2 (50) 1 (25)Opioids 25 (28) 3 (75) 1 (25)Laxatives 16 (18) 3 (75) 0Propulsive agents 3 (3) 0 0Antipropulsive agents 1 (1) 1 (25) 0Chemotherapeutic agents 0 0 1 (25)Any antibiotic 80 (89) 4 (100) 4 (100)Cephalosporins 60 (67) 1 (25) 1 (25)Fluoroquinolones 13 (14) 2 (50) 0Penicillin with β-lactamase inhibitors 12 (13) 3 (75) 1 (25)Vancomycin (intravenous) 7 (8) 2 (50) 0Carbapenems 6 (7) 2 (50) 2 (50)Penicillins 5 (6) 0 0Azithromycin 4 (4) 1 (25) 0Metronidazole 3 (3) 1 (25) 0Cotrimoxazole 2 (2) 0 0Otherse 2 (2) 3 (75) 0Data are number (%) of subjects unless otherwise specifiedaEvaluated at study enrollmentbFrom admission until diagnosis of C. difficile infection or colonization (for infected and colonized patients, respectively) or until discharge (for patients withneither infection nor colonization)cInformation about prior hospitalization was unknown for one of the 90 patients with neither C. difficile infection nor colonizationdOther reasons include bladder/kidney/urinary tract infection, closed fracture, urosepsis, cholecystitis, chronic stasis dermatitis, perinephric infection, diverticulitis,abdominal pain, abdominal hernia, ureteral stone, gangrene, and hip paineOther antimicrobial agents include oral vancomycin, clindamycin, daptomycin, gentamicin, Tigecycline, antivirals, and antifungalsVincent et al. Microbiome  (2016) 4:12 Page 3 of 11difficile culture result (two of the four CDI cases didnot have C. difficile detected by toxigenic culture butrather with an enzyme immunoassay). All patientswith a positive C. difficile culture result had a singleisolate recovered and characterized. Two of the fourasymptomatic carriers were colonized with a toxigenicstrain. C. difficile was also detected by WMGS se-quencing in two of the four asymptomatically colo-nized patients as well as in two CDI patients with apositive C. difficile culture result. No other patientshad C. difficile detected by culture or sequencing.Intestinal microbiota dynamics in patients with C. difficilecolonization or infectionFigure 1 shows variations in the relative abundance ofmicrobial taxa, overall microbial diversity, as well as bac-terial and human DNA proportions in relation to hos-pital exposures and length of stay for patients whobecame colonized (Fig. 1a–d) or infected (Fig. 1e–h)with C. difficile. The human DNA content and the com-position and diversity of the intestinal microbiota werehighly variable during patient hospitalization. In con-trast, the proportion of bacterial DNA (measured afterthe exclusion of human DNA) was more stable.We specifically looked at the relative abundance of po-tentially protective bacterial taxa, including ClostridialesFamily XI Incertae Sedis, Clostridium, and Eubacterium. Afew days prior to or at the time of C. difficile colonization,three out of four asymptomatic patients exhibited highrelative abundance of these taxa in their intestinal micro-biota: the relative abundance of Eubacterium was 24.3 % inpatient 30 on the day C. difficile colonization was detected,the relative abundance of Anaerococcus (a ClostridialesFamily XI Incertae Sedis member) was 31.3 % in patient 87on the day C. difficile colonization was detected, and therelative abundance of Clostridium spp. other than C. diffi-cile (mainly Clostridium bolteae) was 22.2 % in patient 994 days prior to C. difficile colonization. We also observedtransient increases in the relative abundance of Clostrid-ium spp. other than C. difficile (31.0 % [mainly C. bolteaeand Clostridium hathewayi] 13 days prior to CDI diagno-sis, patient 55) or Clostridiales Family XI Incertae Sedis(17.4 % 37 days prior to CDI diagnosis, patient 98; data notshown) in two of the four patients who developed CDI.However, these increases occurred several days prior toCDI onset and the corresponding bacterial taxa had a rela-tive abundance of less than 5 % upon CDI diagnosis. In thefecal samples (n = 196) from the 90 patients who were nei-ther colonized nor infected with C. difficile, the medianabundance (range) of Clostridiales Family XI IncertaeSedis, Clostridium, and Eubacterium was 3.1 (0.0–62.3 %),0.4 (0.0–67.6 %), and 0.2 % (0.0–37.0 %), respectively.We observed transient but large increases of Micro-virus (reaching a relative abundance of ≥50 % in patients30, 98, and 99), Candida (reaching a relative abundanceof >30 % in patients 98 and 99), and Leuconostoc (reach-ing a relative abundance of >15 % in patients 98 and 99).In patients 98 and 99, the increase in the relative abun-dance of Microvirus was concomitant with an increaseof human read proportions and a decrease of microbialdiversity. In patient 98, striking blooms of Enterococcusoccurred on days 16 (98.0 %), 31 (60.8 %), and 47–62(64.8–97.5 %) after admission and appeared to followthe administration of intravenous vancomycin. In thefecal samples (n = 196) from the 90 patients who wereneither colonized nor infected with C. difficile, theTable 2 Detection of C. difficile and its toxins in four asymptomatically colonized and four CDI patientsPatient ID Sample type Toxigenic culturea EnzymeimmunoassaycWMGS sequencingCulture Toxinb No. of reads Relative abundanceof C. difficile (%)Asymptomatically colonized30 Rectal swab Positive Negative ND 8,487,324 0.00063 Rectal swab Positive Positive ND 10,366,360 0.00087 Rectal swab Positive Negative ND 4,489,956 0.36399 Stool Positive Positive ND 14,952,432 0.064CDI35 Stoold Negative ND Positive 5,926,081 0.00036 Stoold Positive Positive ND 7,068,998 0.01755 Stool Positive Positive Positive 9,950,687 0.00398 Stool Negative ND Positive 4,337,018 0.000WMGS whole-metagenome shotgun, ND not doneaPerformed by Dale N. Gerding’s laboratorybDetected by restriction endonuclease analysis or cytotoxicity assaycPerformed on a stool sample at the Jewish General HospitaldA rectal swab was used for WMGS sequencingVincent et al. Microbiome  (2016) 4:12 Page 4 of 11median abundance (range) of Microvirus, Candida,Leuconostoc, and Enterococcus was 0.0 (0.0–95.6 %), 0.0(0.0–3.5 %), 0.0 (0.0–51.0 %), and 0.1 % (0.0–91.7 %),respectively.Medication use and intestinal microbiota diversity andcompositionWe examined the effect of antibiotics as well as othermedications on the diversity and composition of the gutmicrobiota. To avoid confounding the results, we ex-cluded patients who were colonized or infected with C.difficile (n = 8) from these analyses; therefore, the Clos-tridium genera does not include C. difficile. Utilizationof fluoroquinolone was moderately correlated with theuse of metronidazole (ρ = 0.45), as were use of penicillinwith use of gentamicin (ρ = 0.57), and use of opioidswith use of laxatives (ρ = 0.41).Among patients who were neither infected nor colo-nized with C. difficile, we obtained a mean Shannon di-versity index of 2.0 (standard deviation 0.6). Exposure tofluoroquinolones and intravenous vancomycin was associ-ated with a significant decrease in intestinal microbiotadiversity, while use of opioids was associated with an in-crease in diversity (Table 3).A large number of microbial taxa were affected bycommon hospital exposures; out of a total of 125 generaand 59 families identified in at least 5 % of the patients,88 genera (70.4 %) and 42 families (71.2 %) were associ-ated with a statistically significant increase or decreasein relative abundance in univariable analyses (Additionalfile 1: Table S1 and Additional file 2: Table S2). With theexception of non-steroidal anti-inflammatory drugs(NSAIDs) and opioids, most medications tended todecrease, rather than increase, the relative abundance ofaffected microbial taxa. Carbapenems (33 genera and 13families) and intravenous vancomycin (34 genera and 15families) influenced the largest number of microbial taxa.In multivariable analyses, we specifically looked at theimpact of medications on the relative abundance of mi-crobial taxa that are thought to be protective againstCDI (Clostridiales Family XI Incertae Sedis, Clostridium,and Eubacterium) as well as opportunistic microorgan-isms that can overgrow as a result of antimicrobial use(Enterococcus and Candida) (Table 4). Use of laxativesA)E) F) G) H)C) D)B)Fig. 1 Intestinal microbiota dynamics in patients with C. difficile colonization or infection. The figure shows changes in intestinal microbiotacomposition (area charts or bar charts for patients with a single measurement), Shannon diversity (dash-dotted line), bacterial DNA content (dotted line),and human DNA content (solid line) during hospitalization for patients who developed C. difficile colonization (a–d) or infection (e–h). Only thosemicrobial taxa with a relative abundance of ≥10 % in at least one sample are depicted. The y-axis on the left shows the relative abundance ofmicrobial taxa or human DNA proportions, while the y-axis on the right shows the Shannon diversity index or bacterial DNA content(expressed as number of 16S rRNA genes per Mb). The x-axis shows the number of days after hospital admission (day 0). The figures onlydisplay information for the period from hospital admission until the last stool collection. The bars underneath the graphs indicate hospitalexposures: Azithro azithromycin, Ceph cephalosporin, Chemo chemotherapeutic agent, Metro metronidazole, NSAID nonsteroidal anti-inflammatorydrug, Penicillin combination penicillin with β-lactamase inhibitor, PPI proton pump inhibitor, Steroid, glucocorticoid, Vanco IV intravenous vancomycin,Vanco PO oral vancomycin. The day on which a patient had a positive C. difficile culture (Culture+), the presence of C. difficile infection symptoms, but anegative C. difficile enzyme immunoassay (CDI symptoms) or a diagnosis of C. difficile infection (CDI diagnosis) is also indicatedVincent et al. Microbiome  (2016) 4:12 Page 5 of 11was associated with significant reductions in the relativeabundance of Clostridium and Eubacterium. Adminis-tration of cephalosporins, fluoroquinolones, or penicillinwith β-lactamase inhibitor was associated with signifi-cant reductions in the frequency of Clostridiales FamilyXI Incertae Sedis. Exposure to fluoroquinolones signifi-cantly increased the relative abundance of Enterococcus,while intravenous vancomycin was of borderline signifi-cance. None of the medications were associated with sig-nificant variations in the relative abundance of Candidain multivariable analyses.DiscussionWe previously demonstrated that a reduction in Clostri-diales Family XI Incertae Sedis is significantly and inde-pendently associated with the risk of CDI in a distinctpatient population [13]. Certain species within the Eubac-terium and Clostridium genera, notably C. scindens, havethe ability to convert primary bile acids into secondary bileacids [16] and have been shown to strongly inhibit C. diffi-cile in the intestinal microbiota of antibiotic-treated miceand humans [14]. In this study, asymptomatically colo-nized patients, but not CDI cases, exhibited elevatedTable 3 Associations between medication use and intestinal microbiota diversityMedicationa No. of patientsexposedNo. of samplesexposedEstimateb Standard error P valuecAntibioticsCephalosporins 57 81 −0.06 0.07 0.3764Fluoroquinolones 8 15 −0.20 0.10 0.0367Penicillin with β-lactamase inhibitors 7 12 −0.17 0.19 0.3805Carbapenems 5 11 −0.30 0.26 0.2417Intravenous vancomycin 6 12 −0.41 0.15 0.0080Other medicationsGlucocorticoids 7 13 −0.02 0.17 0.8977Laxatives 13 24 −0.05 0.09 0.5976Non-steroidal anti-inflammatory drugs 30 57 0.08 0.08 0.3264Opioids 23 38 0.13 0.06 0.0202Proton pump inhibitors 21 54 −0.11 0.10 0.2738Among patients who were neither colonized nor infected with C. difficileaReceived within 3 days prior to stool collectionbA positive value indicates that intestinal microbiota diversity was higher among patients exposed to the medication, while a negative value indicates thatintestinal microbiota diversity was lowercP values were determined by using the GEE-derived robust z scoresTable 4 Multivariable analysis of medications associated with selected microbial taxaTaxa (outcome) Medicationa Estimateb Standard error P valuecClostridium Glucocorticoids 3.72 1.65 0.0239Laxatives −2.46 0.99 0.0126Non-steroidal anti-inflammatory drugs −2.11 0.90 0.0197Eubacterium Fluoroquinolones −0.99 0.44 0.0237Laxatives −1.21 0.48 0.0115Clostridiales Family XI Incertae Sedis Cephalosporins −5.78 2.08 0.0055Fluoroquinolones −5.95 2.21 0.0071Non-steroidal anti-inflammatory drugs 3.76 1.99 0.0580Penicillin with β-lactamase inhibitors −5.70 2.54 0.0248Enterococcus Fluoroquinolones 215.43 102.34 0.0353Intravenous vancomycin 181.32 95.02 0.0564Among patients who were neither colonized nor infected with C. difficile. All of the multivariable models were adjusted for age, sex, and durationof hospitalizationaReceived within 3 days prior to stool collectionbA positive value indicates that the relative abundance of the corresponding taxa was higher among patients exposed to the medication, while a negative valueindicates that the relative abundance was lowercP values were determined by using the GEE-derived robust z scoresVincent et al. Microbiome  (2016) 4:12 Page 6 of 11relative abundance of Clostridiales Family XI IncertaeSedis, Clostridium, or Eubacterium in their gut shortlyprior to or when C. difficile colonization was detected.These observations are in agreement with our initial hy-pothesis and suggest that co-colonization with any ofthese potentially protective bacterial taxa might prevent C.difficile overgrowth in colonized subjects or the transitionfrom asymptomatic colonization to a full-blown infection.Previous studies have shown that in the absence ofgross perturbation, the intestinal microbiota of healthysubjects is relatively stable over time [21, 22]. In con-trast, we observed dramatic shifts in the compositionand diversity of the intestinal microbiota in patients whodeveloped C. difficile colonization or infection, as well asin other hospitalized patients (data not shown). Theseobservations suggest that single measurements of the in-testinal microbiota may be problematic when studyinghospitalized patients, in particular, as their microbiotacomposition is strongly influenced by their illness andmedical interventions. This offers one explanation whysome studies examining the role of the intestinal micro-biota on health outcomes may yield conflicting results.In a previous study, we showed that fecal excretion ofhuman DNA was significantly increased in patients withCDI or having other gastrointestinal problems and ap-peared to be an outcome of intestinal inflammation [23].In this study, the proportion of human DNA was highlyvariable over time but was observed to be higher priorto CDI development in two of the four cases. In some ofour patients, increases in the proportion of human DNAwere concomitant with a reduction of microbial diversityand increases in the relative abundance of Microvirus.Microviruses are single-stranded DNA bacteriophagesthat infect enterobacteria and are commonly found inhuman gut samples [24]. High levels of human DNA ex-cretion have been associated with a reduction of intes-tinal microbiota diversity [23].One of the patients who developed CDI (patient 98)exhibited striking blooms of Enterococcus (with frequen-cies reaching 98 %) on multiple occasions during hishospitalization. These blooms appeared to follow theadministration of intravenous vancomycin and mayrepresent an overgrowth of vancomycin-resistant Entero-coccus (VRE). Studies have shown that the use of intra-venous vancomycin is significantly associated with VREcolonization or infection [25]. Although we do not knowwhether this patient actually carried VRE or developedsepsis, Ubeda et al. have previously shown that intestinaldomination by VRE typically precedes bloodstream in-fection in hospitalized patients [26]. In the cohort of pa-tients who were neither colonized nor infected with C.difficile, we confirm that the use of intravenous vanco-mycin is associated with increases in the relative abun-dance of Enterococcus.Receipt of opioids was associated with significant in-creases in microbial diversity. This medication typicallydelays gastrointestinal transit time and may facilitate mi-crobial growth in the colon, thereby increasing diversity.Significant reductions in intestinal microbiota diversitywere only associated with the use of fluoroquinolonesand intravenous vancomycin. Surprisingly, exposure toother antibiotics and PPIs did not reduce overall micro-biota diversity in our cohort but did influence the rela-tive abundance of numerous microbial taxa. Theassociation between PPIs and CDI development is still acontroversial issue [27]. The notion of reduced micro-biota diversity may suggest that the number and relativeabundance of microbial taxa are both declining. How-ever, our study suggests that microbiota dynamics aremuch more complex; many taxa are being depletedwhile others are blooming. Therefore, measurement ofthe overall diversity is masking important distortions inthe composition of the microbial community.In multivariable analyses, we showed that the use oflaxatives is associated with significant reductions in therelative abundance of Clostridium and Eubacterium; cer-tain species among these genera may confer protectionagainst CDI via the production of secondary bile acids.However, in a post hoc analysis, the Kyoto Encyclopediaof Genes and Genomes (KEGG) pathway correspondingto secondary bile acid biosynthesis (ko00121) was notdetected in any our metagenomic samples. Laxatives ac-celerate gastrointestinal transit time and may have detri-mental effects on the intestinal microbiota. van derWulp et al. previously showed that treatment with poly-ethylene glycol (an osmotic laxative) changes the com-position of the intestinal microbiota and decreases theproduction of secondary bile acids in rats [28]. We alsodemonstrated that the use of cephalosporins, fluoroqui-nolones, and penicillin with β-lactamase inhibitors is sig-nificantly and independently associated with reductionsin the relative abundance of Clostridiales Family XIIncertae Sedis representatives. Since cephalosporin andfluoroquinolone exposure, as well as depletions of Clos-tridiales Family XI Incertae Sedis, have been associatedwith CDI risk [13], this provides a potential explanationfor why these antibiotics increase the susceptibility toCDI in hospitalized patients.We observed discrepancies in the detection of C. diffi-cile or its toxins by culture, WMGS sequencing, and en-zyme immunoassay. In two of the four asymptomaticallycolonized patients, C. difficile was detected by culturebut not by WMGS sequencing. In two of the four CDIcases, C. difficile could not be detected, either by cultureor sequencing (their CDI diagnosis was based on a posi-tive enzyme immunoassay performed at the hospital).This may be due to a low organism load, the lack of bio-mass collected with the rectal swabs, or insufficientVincent et al. Microbiome  (2016) 4:12 Page 7 of 11sequencing depth. One patient (patient 98) receivedvancomycin and metronidazole treatment for presump-tive CDI prior to the actual diagnosis by enzyme im-munoassay, which may have reduced the quantity of C.difficile organisms available for detection.The number of patients who developed C. difficilecolonization or infection was limited in our study; there-fore, we could not perform statistical analyses to assessthe role of hospital exposures and microbial taxa for pa-tients with these outcomes. In all of the four patientswho developed asymptomatic colonization, C. difficilewas detected by culture in the last stool collected priorto patient discharge. Therefore, we could not determinewhether C. difficile colonization was transient (presentin only one occasion) or not in these patients. In the fullcohort, insufficient numbers of patients exposed to cer-tain medications (e.g., metronidazole and cotrimoxazole)or colonized by specific microbial taxa (e.g., Aerococcusand Microbacteriaceae) precluded statistical analyses.Diet and other factors such as underlying disease statecan affect the intestinal microbiota and could still con-found the associations identified in our study.ConclusionsThe integrity of the intestinal microbiota is intricatelyrelated to the health of the host. In this study, we showthat the diversity and composition of the intestinalmicrobiota in hospitalized patients is highly dynamic.Use of antibiotics, as well as other non-antimicrobialmedications, particularly laxatives, has a profound andrapid effect on the structure of the intestinal microbiotaand significantly decreased the relative abundance of keybacterial taxa that may be involved in CDI protection.Our results also support the hypothesis that co-colonization with key bacterial taxa such as ClostridialesFamily XI Incertae Sedis, Clostridium, or Eubacteriummay prevent C. difficile overgrowth or the transitionfrom asymptomatic C. difficile colonization to CDI. Abetter understanding of CDI pathogenesis, including themedical exposures that undermine the effectiveness ofcolonization resistance and the specific microbiota alter-ations that allow C. difficile to infect the gut, will con-tribute to our ability to develop novel clinical strategiesto prevent or treat this life-threatening infection.MethodsPatient recruitment and follow-upResearch subjects were recruited as part of a prospectivecohort study conducted at the Jewish General Hospital(JGH) in Montréal between October 2009 and April2011. Patients ≥60 years of age, who were expected tostay more than 2 days in the hospital at the time of studyenrolment, and who had received antimicrobials in theprevious 48 h or were expected to receive some in thenext 24 h were eligible to participate in the study. Pa-tients admitted to the hospital for CDI were excluded.Subjects were enrolled in the study within 5 days afteradmission to selected medical wards or surgical units.Patients were followed for 60 days after study enrolmentor 30 days after hospital discharge (whichever came first)to ascertain the development of CDI. CDI cases werefollowed for 60 days after successfully completing CDItreatment to monitor the development of recurrent CDI.Ethics, consent, and permissionsAll study participants provided informed written con-sent. The Research Ethics Boards of the JGH (08-118A)and the McGill University Health Centre (11-205 GEN)approved the research protocol.Sample and epidemiologic data collectionFecal specimens were obtained from each patient atstudy enrollment, every 3 days during hospitalization, atthe onset of diarrhea (if applicable), and at discharge.The samples were collected as bulk stool or with the useof a rectal swab if the patient was not producing stool.Rectal swabs are a convenient means of sampling thehuman gut and give highly reproducible microbiota pro-files that can closely resemble that of fecal samples [29].Epidemiologic information was extracted from the pa-tients’ medical charts and included patient demographics,previous hospitalizations, reason of admission, disease se-verity index, dates of admission and discharge, CDI diag-nosis, and the use of antimicrobials and other medicationsduring hospitalization (with corresponding start and stopdates).Toxigenic C. difficile cultureToxigenic culture test results were provided as acourtesy of Dr. Dale N. Gerding (Hines VA Hospitaland Loyola University Chicago Stritch School ofMedicine, IL). Briefly, stool or rectal swab specimenswere streaked onto selective pre-reduced cefoxitin-cycloserine-fructose agar supplemented with taurocholate(TCCFA) and incubated anaerobically for 48 h. Presump-tive identification of C. difficile was based on typical col-ony morphology. If colony morphologies suggested that amixture of C. difficile strains were present, multiple col-onies were picked and saved individually. All rectal swabswere also inoculated onto non-selective blood agar to de-tect the growth of intestinal microbiota and confirmproper sampling technique, thereby excluding the possi-bility of false-negative results on the TCCFA. Purified iso-lates were characterized by restriction endonucleaseanalysis (REA) to determine their toxin classification [30].The Bartels Cytotoxicity Assay (Trinity Biotech) was per-formed to determine the presence of toxin B in C. difficileisolates with an undetermined REA group.Vincent et al. Microbiome  (2016) 4:12 Page 8 of 11Study definitionsCDI was defined as follows: (i) the presence of symp-toms (diarrhea, fever, abdominal pain, or ileus) and apositive C. difficile enzyme immunoassay (ImmunoCardToxins A&B, Meridian Bioscience, Inc.) or toxigenic cul-ture, (ii) an endoscopic diagnosis of pseudomembranes,or (iii) a pathological/histological diagnosis of CDI. Diar-rhea was defined as the passage of ≥3 new unformedstools within 24 h. Recurrent CDI was defined as a CDIdiagnosis that occurs within 60 days after successfullycompleting the treatment for the initial episode of CDI.Asymptomatic C. difficile colonization was defined as apositive stool culture for C. difficile and the absence of aCDI diagnosis during follow-up. Colonization or infec-tion was considered hospital-acquired if the diagnosiswas made ≥48 h after hospital admission and during thestudy follow-up period.WMGS sequencing and data analysisTotal DNA was extracted from rectal swabs with useof the DNA IQ System (Promega) and from bulkstool aliquots with the use of the QIAamp DNA StoolMini Kit (Qiagen). Multiplexed DNA libraries wereprepared according to a previously described protocolthat allows the generation of libraries from lowamounts of input DNA [31]. WMGS sequencing wasperformed with 150-nucleotide read lengths on theIllumina HiSeq 2000 and 2500 platforms at theMcGill University and Génome Québec InnovationCentre. Up to 14 samples were multiplexed in each se-quencing lane. Reads derived from the human genomewere identified and removed with BMTagger [32]. Add-itional quality filtering steps included the trimming of se-quencing adapters and low-quality bases from the 3′ endof reads as well as the removal of short (<60 bases) andduplicate reads [33, 34]. We retrieved all reads containingthe V1–V3 reverse primer sequence (which targets a seg-ment of the 16S rRNA gene) [35] and their 3′ sequencesin order to obtain 55-mers originating with the primer se-quence. The frequency of 16S rRNA genes (55-mers) perMb of sequence data was used as a measure of bacterialDNA content in each sample. High-quality reads were alsoanalyzed by MetaPhlAn2 [36] in order to determine thefamily, genus, and species level relative abundances. Diver-sity was calculated at the genus level with the Shannonindex. HUMAnN version 0.99 [37] was used in conjunc-tion with RAPSearch2 [38] and the KEGG database [39]to evaluate the presence of the pathway for secondary bileacid biosynthesis (ko00121) in each metagenomic sample.Statistical analysesWe provide a descriptive analysis of microbiota com-position for patients who were either colonized or in-fected by C. difficile; we specifically examined changesin the relative abundance of potential CDI-protecting mi-croorganisms (i.e., Clostridiales Family XI Incertae Sedis,Clostridium, and Eubacterium). In order to assess the rela-tionship between hospital exposures (e.g., antibiotics andother medications) and either overall microbiota diversityor relative abundance of microbiota members, we usedgeneralized estimating equation (GEE) analyses with aGaussian link. GEE takes into account the multiple sam-ples per subject and the likelihood that the microbiotaprofiles are correlated over time within patients. Microbialdiversity and relative abundances were modeled using alog transformation. In these analyses, we excluded patientswho were colonized or infected with C. difficile and con-sidered only those microbial taxa and medications thatwere present in or administered to at least 5 % of thepatients (i.e., cephalosporins, fluoroquinolones, penicillinwith β-lactamase inhibitors, carbapenems, intravenousvancomycin, glucocorticoids, laxatives, NSAIDs, opioids,and PPIs). Use of medication was treated as a binaryvariable with “1” indicating exposure within 1 to 3 daysprior to stool collection and “0” indicating otherwise. Weincluded exposure to intravenous vancomycin in ouranalyses, as evidence suggests that substantial amountsof the drug can be excreted in the bowel and maytherefore affect the intestinal microbiota [40]. Multivar-iable analyses were used in order to take into accountthe correlation in the use of certain medications. Inaddition to the medications mentioned above, all multi-variable models included adjustment for age, sex, andduration of hospitalization. Final multivariable modelsretained significant exposure variables at a p value of<0.05. A positive estimate value indicates that the cor-responding exposure is associated with an increase ineither overall microbiota diversity or relative abundanceof the given microbial taxa. All statistical analyses wereperformed in R [41]; GEE was performed using the gee-pack package. P values were determined by using theGEE-derived robust z scores.Availability of supporting dataThe sequence data supporting the results of this articleare available in the National Center for BiotechnologyInformation Sequence Read Archive under accessionnumber SRP064400 (http://www.ncbi.nlm.nih.gov/sra/SRP064400).Additional filesAdditional file 1: Table S1. Significant associations betweenmedication use and intestinal microbiota genera (univariable analysis).(XLSX 433 KB)Additional file 2: Table S2. Significant associations betweenmedication use and intestinal microbiota families (univariable analysis).(XLSX 385 KB)Vincent et al. Microbiome  (2016) 4:12 Page 9 of 11AbbreviationsCDI: Clostridium difficile infection; PPI: proton pump inhibitors;WMGS: whole metagenome shotgun; TCCFA: cefoxitin-cycloserine-fructoseagar supplemented with taurocholate; REA: restriction endonuclease analysis;GEE: generalized estimating equations; rRNA: ribosomal RNA;VRE: vancomycin-resistant Enterococcus; JGH: Jewish General Hospital;NSAID: non-steroidal anti-inflammatory drug.Competing interestsMAM has been an employee of bioMerieux since October 2012. The otherauthors declare that they have no competing interests.Authors’ contributionsCV prepared the sequencing libraries, performed the bioinformatic analyses,participated in data analysis and interpretation, and drafted the manuscript.MAM provided patient epidemiologic information and clinical samples andparticipated in data analysis and interpretation. TJE performed the statisticalanalyses. SM assisted with bioinformatic analyses. KD participated in dataanalysis and interpretation. ARM conceived and designed the study,participated in data analysis and interpretation, and helped to draft themanuscript. All authors read and approved the final manuscript.AcknowledgementsThe authors would like to thank Dr. Dale N. Gerding for the toxigenic C.difficile culture, REA typing, and cytotoxicity assays done as part of a parallelstudy on the same patient cohort. They also thank the clinical research staffat the JGH, Jennifer Eastmond, Romina Gheorghe, and Sophie Florencio, fortheir valuable help with the collection of clinical samples and the chartreviews. This work was supported by a Pfizer ASPIRE Award in AntibacterialResearch [WS1954106 to A.R.M.], a Studentship from the Research Institute ofthe McGill University Health Centre [C.V.], as well as a Frederick Banting andCharles Best Canada Graduate Scholarship (Doctoral Award) from theCanadian Institutes of Health Research [GSD-113375 to C.V.]. The fundingbodies had no role in the study design, in the collection, analysis, andinterpretation of data, in the writing of the manuscript, and in the decisionto submit the manuscript for publication.Author details1Department of Microbiology and Immunology, McGill University, Montréal,Québec, Canada. 2Génome Québec Innovation Centre, McGill University,Montréal, Québec, Canada. 3Jewish General Hospital, Montréal, Québec,Canada. 4Devil’s Staircase Consulting, North Vancouver, British Columbia,Canada. 5New York Genome Center, New York, NY, USA. 6Department ofHuman Genetics, McGill University, Montréal, Québec, Canada. 7School ofPopulation and Public Health, University of British Columbia, Vancouver,British Columbia, Canada.Received: 4 September 2015 Accepted: 14 February 2016References1. Lessa FC, Mu Y, Bamberg WM, Beldavs ZG, Dumyati GK, Dunn JR, et al.Burden of Clostridium difficile infection in the United States. N Engl J Med.2015;372(9):825–34. doi:10.1056/NEJMoa1408913.2. 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Antimicrob AgentsChemother. 2004;48(11):4427–9. doi:10.1128/AAC.48.11.4427-4429.2004.41. The R project for statistical computing. http://www.r-project.org/.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Vincent et al. Microbiome  (2016) 4:12 Page 11 of 11


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