Conditions: Microbiota Anti-bacterial Agents
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 20
Healthy volunteers: yes

Brief summary:
The objective of this study is to evaluate the impact of antimicrobial (antibiotic) exposures on the microbiome in healthy adults, specifically during and after usual courses of the antimicrobials used to treat community acquired pneumonia (CAP). Pneumonia is a lung infection, and community-acquired pneumonia is pneumonia that develops outside of a healthcare facility (i.e., in the community). A microbiome is a the community of microorganisms living in a particular location, such as the gut or the mouth. Disruptions to a person's microbiome may reduce his/her "colonization resistance" (resistance to colonization with pathogenic microorganisms) and make him/her more susceptible to multidrug resistant organism (MDRO) colonization and infection.

To study changes in the microbiome, the investigators will recruit 20 healthy adult volunteers and obtain fecal, salivary, skin, and urine specimens at multiple time points before, during, and after administration of antimicrobials. Participants will be randomized to one of 4 antimicrobial regimens, all of which are FDA-approved for treatment of community-acquired pneumonia. Stool specimens will be analyzed via stool culture and genetic sequencing, and all remaining specimens will be frozen and used to create a biospecimen repository for future analysis. The rationale for using healthy volunteers (instead of patients already prescribed antibiotics by their physicians) is because the human microbiome is very complex and can be affected by a variety of medical conditions and other medications. In addition, the presence or absence of patient-specific factors means people with infections may not be prescribed the specific courses of antibiotics the investigators are trying to study. Studying the effect of antibiotics on healthy volunteers will provide baseline data that are more applicable to the population at large.

Detailed description:
Each year, antimicrobial resistance causes over two million infections and 23,000 deaths in the US alone, representing a critical global public health issue. Some of the most feared multidrug resistant organisms (MDROs) include Clostridium difficile, carbapenem-resistant Enterobacteriaceae (CRE), extended spectrum beta-lactamase producing Enterobacteriaceae (ESBL), MDRO Acinetobacter, and MDRO Pseudomonas aeruginosa; there are few antimicrobials effective against these MDROs, and available antimicrobials often have rate-limiting toxicities. The major risk factor for MDRO colonization and subsequent MDRO infections is exposure to antimicrobials. The use of antimicrobials has been associated with an altered and often less diverse composition of the fecal microbiome, and expansion of the resistome. A "healthy" microbiome provides "colonization resistance" against potentially pathogenic bacteria; antimicrobials disrupt this protective community, providing selective pressure that favors MDRO colonization, persistence, and transmission to others.

Methods to proactively prevent MDRO colonization, rather than reliance on reactive approaches to this problem, are urgently needed. Antimicrobial stewardship is a key component of MDRO prevention efforts; however, there is no method to determine which antimicrobials cause the greatest degree of microbiome disruption. A better understanding of exactly how antimicrobials alter the microbiome is necessary to optimally guide future MDRO prevention efforts and antimicrobial stewardship. The development of microbiome disruption indices (MDIs) would help characterize the risk associated with specific antimicrobials, and can be used during antimicrobial development, patient monitoring while on antimicrobials, and to facilitate infection prevention efforts to contain MDRO spread. Additionally, MDIs can be used as an alert when microbiome disruptions reach a critical level and MDRO colonization is imminent. At that point, interventions to restore the microbiome could be implemented.

Community-acquired pneumonia (CAP) is one of the leading causes of death in the United States, with an estimated \>900,000 cases each year in adults age 65 and older. Large amounts of antimicrobials are used in treating patients with CAP because the disease is relatively common. A better understanding of the effect of CAP antimicrobial treatment on the microbiome could result in improved treatment options for patients with CAP and protect CAP patients from colonization or infection with MDROs.

Arms:
arm 1: 1 750mg tab of levofloxacin by mouth for 5 days arm 2: 1 500mg tab by mouth on day 1, then 1 250 mg tab per day by mouth for 4 days (total 5 days) arm 3: 200mg tab by mouth twice per day for 5 days arm 4: Azithromycin: 1 500mg tab by mouth on day 1, then 1 250 mg tab per day by mouth for 4 days (total 5 days)

Cefpodoxime: 200mg tab by mouth twice per day for 5 days

Interventions: intervention 1: Levofloxacin intervention 2: Azithromycin intervention 3: Cefpodoxime
Intervention descriptions: intervention 1: 5 days of levofloxacin administration intervention 2: 5 days of azithromycin administration intervention 3: 5 days of cefpodoxime administration

Protocol document:
Prot_SAP_000.pdf: 
1 
 
Prospective Study Characterizing Fecal Microbiome Disruptions  
During and After Receipt of Antimicrobials 
 
Clinical Protocol 
August 30, 2022 
Version #3 
ID: NCT03098485 
Principal Investigator:  
Jennie H. Kwon, DO, MSCI 
Washington University School of Medicine 
4523 Clayton Ave., Box 8051 
Saint Louis, MO 63110 
 
 
Co-investigators:  
Erik R. Dubberke, MD, MSPH  
Carey-Ann D. Burnham, PhD  
Gautam Dantas, PhD 
Washington University School of Medicine 
Saint Louis, MO 63110 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
This document is the property of Washington University. No part of it may be transmitted, reproduced, published, or 
used by other persons without prior written authorization from the study sponsor 


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Introduction________________________________________________________________________________________ 
Study Purpose:  
The purpose of this study is to evaluate the impact of antimicrobial exposures on the microbiome in healthy adults, 
specifically during and after usual courses of the antimicrobials used to treat community acquired pneumonia (CAP). 
Background: 
In the United States, approximately 2 million people develop infections due to multidrug-resistant organisms (MDRO), 
and approximately 23,000 die as a result.1 The major risk factor to acquire MDRO colonization and develop subsequent 
MDRO infections is exposure to antimicrobials.2,3 A healthy fecal microbiome provides “colonization resistance” against 
MDROs and antimicrobial-exposure induced disruptions facilitate MDRO selection and colonization.4,5 The use of 
antimicrobials has also been associated with an altered and often less diverse composition of the fecal microbiome, and 
expansion of the resistome (i.e. the compendium of resistance genes present in the microbiome).6-8  
Controlling MDRO threats is a multifaceted task, requiring the prevention of healthcare-associated infections (HAI), 
preventing the spread of MDROs within and between healthcare facilities, and by improving antimicrobial stewardship.9 
With regards to antimicrobial stewardship, what is not definitively known is whether certain antimicrobials lead to 
greater or lesser degrees and/or durations of microbiome alterations.10,11 A better understanding of exactly how 
antimicrobials impact the microbiome is necessary to optimally guide MDRO prevention efforts and antimicrobial 
stewardship.11  
To address this knowledge gap, we propose a prospective study of the impact of antimicrobial exposures on the 
microbiome in healthy adults. The goal of this study is to characterize microbiome disruptions observed during and after 
usual courses of antimicrobials used to treat community acquired pneumonia (CAP). Healthy volunteers will be recruited 
and then be randomized to receive one of four standard antimicrobial courses for CAP. Fecal, salivary, skin, and urine 
specimens will be collected before, during, and after receipt of antimicrobials and analyzed to determine the impact of 
the antimicrobials on the microbiome. These data will allows us to develop microbiome disruption indices (MDI), which 
can be used to characterize the MDRO risk associated with specific antimicrobials.  
A better understanding of how courses of antimicrobials alter the microbiome can be used to improve antimicrobial 
prescribing practices, allowing for selection of antimicrobials that minimize MDRO colonization risk. Through this study, 
we will develop standardized protocols for subject recruitment, antibiotic administration, specimen collection, culture, 
and metagenomic specimen analysis. These standardized protocols can then be used to compare the impact a variety of 
antimicrobial treatment regimens on the microbiome. These data will lead the development of MDIs, which have a 
multitude of potential uses in the fields of antimicrobial development, antimicrobial stewardship, infection control, and 
identification of patients who require microbiome remediation.12 With the tremendous burden of MDRO infections and 
a paucity of safe and effective therapies against MDROs, data to develop MDIs are necessary to improve our 
antimicrobial stewardship and infection control measures.  
Specific aims and hypotheses: 
Specific aim 1: Determine the effects of antimicrobials used to treat community acquired pneumonia (CAP) on the fecal 
microbiota architecture and resistome (i.e. compendium of antibiotic resistance genes within the microbiota) in healthy 
adults.  
Hypothesis 1: We hypothesize that, compared to the pre-antimicrobial state, antimicrobials will cause a decrease in 
microbial diversity, increase the concentration of antimicrobial resistance genes and MDROs in the fecal microbiome, 
and the microbiome alterations will vary between antimicrobials.  
 
Specific aim 2: Determine the time to restoration of the fecal microbiome after exposure to antimicrobials used to treat 
CAP.  
Hypothesis 2: We hypothesize that the degree and time to stabilization of the fecal microbiome will vary between 
antimicrobials.  
 
Specific aim 3: Create microbiome disruption indices (MDIs) based on 1) the degree of fecal microbiota disruption, 2) 


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duration of microbiota disruption, and 3) resistome expansion.  
Hypothesis 3: We hypothesize that the impact of antimicrobials will vary in regards to the degree of microbiome 
disruption, duration of microbiome disruption, and potential for resistome expansion. 
 
Methods___________________________________________________________________________________________ 
Study design:  Prospective cohort study. 
Study population:  20 healthy adults. Subjects will be randomized to receive a five day course of azithromycin, 
levofloxacin, cefpodoxime, or cefpodoxime with azithromycin, which are recommended antimicrobials for the treatment 
of CAP.13 
Inclusion criteria: Healthy adults age 21-60 who provide written, informed consent. 
Exclusion criteria: 
History of allergic reaction to study antimicrobial(s)  
Contraindication(s) to study antimicrobial(s)  
Inability to provide regular stool samples  
Any non-topical antimicrobial exposure in previous 6 months  
Tube feeds as primary source of nutrition in previous 6 months  
Pregnant or risk of becoming pregnant during study period  
Breastfeeding during study period  
Gastroenteritis in last 3 months  
Any non-elective hospitalization in the previous 12 months  
Incontinent of stool  
Known colonization with an MDRO  
Anticipated change in diet or medications during study period  
Elective surgery during study period  
History of an intestinal disorder  
Inability to provide written, informed consent 
 
Recruitment: The following recruitment strategies will be used: 
1. Word of mouth 
2. Volunteer for Health website posting 
3. Volunteer for Health Facebook post with study flyer 
4. Post flyers around the WUSM campus 
5. Email blast to WUSM employees. 
Because the number of subjects needed is small, study investigators may not need to use all recruitment strategies. 
Study Procedures: 
Institutional Board Review (IRB) approval through the WUSM Human Research Protection Office (HPRO) will be obtained 
prior to study implementation. Subjects interested in participation will contact study personnel via phone. Study 
personnel will describe the study and screen potential subjects by asking whether they have any allergies to antibiotics 
and whether they are able to provide regular stool samples. If the subject is still interested in participation, a clinic visit 
(WUSM Infectious Diseases Clinic) will be scheduled. At this visit, the study investigator will review the informed consent 
document and answer any questions the subject may have. If the subject agrees to participate, he/she will then provide 
written, informed consent. Upon enrollment, subjects will undergo a clinical evaluation and interview. Women of child-
bearing age will take a pregnancy test. Demographic data, dietary preferences, medical/surgical history will be obtained. 
Each subject will be provided with a specimen collection kit with appropriate materials and instructions for stool, saliva, 


4 
 
skin, and urine collection. The subject will collect specimens at the designated time points (Table 1) and complete a 
questionnaire on bowel movement consistency, frequency, diet, medications, and any changes in medical history at 
each time point. A total of 15 specimen sets will be collected per subject, and the subject will be provided all materials 
for specimen collection. Prior to each specimen collection date, study personnel will contact the subject via phone and 
remind him/her to collect specimens and fill out a food log, stool log, and medical update form.  
 
 
Once the specimens are ready, the subject will contact a pre-identified courier (JS Logistics, St. Louis, MO) for specimen 
pick up, and then the specimens will then be promptly delivered to the WUSM lab. After the pre-antimicrobial stools are 
collected, subjects will be provided with a 5 day course of azithromycin, levofloxacin, cefpodoxime, or cefpodoxime with 
azithromycin. Explicit directions on how to take the antimicrobial will be provided, as well as memory aids and 
instructions to send the pill bottle back to confirm all doses were taken. 
The specimen on days -14, -10, and -7 days before antimicrobials can be submitted +/- 48 hours from the scheduled 
date. The -1 day specimens may be submitted -48 hours from the scheduled date but not after the subject has begun 
antimicrobial therapy. The day 3 during antimicrobials specimen may be submitted +/- 48 hours from the scheduled 
date.  The specimens on day +1, +3, +7, and +10 post-antimicrobials may be submitted +/- 48 hours from the scheduled 
date. The specimens on day +14 may be submitted +/- 72 hours from the scheduled date. The specimens on days +30-
+180 may be submitted +/- 1 week from the scheduled date. 
Study activities will be complete after the subject submits the 180-day specimens. 
Subject Remuneration 
Subjects will be provided with $30 per specimen set for the first 14 specimen sets and $80 for the final (day 180) 
specimen set. Total remuneration for subjects who submit all specimens will be $500. No compensation will be provided 
if a subject fails to submit a specimen set. Subjects who drop out of the study early will receive compensation only for 
the specimens already submitted. 
 
Data management___________________________________________________________________________________ 
Specimen storage and transport: 
Specimens will be shipped to WUSM by study subjects via courier. All specimens will be de-identified and labeled with a 
study ID number and will be stored without PHI. All salivary, urine, skin and all remaining stool specimens will be frozen 
at -80°C for future analyses. Prior to freezing, specimens will be divided into aliquots and labeled in formats and 
containers appropriate for future metabolomic, metagenomics, and culture-based analyses. Specimens will be stored in 
locked laboratories. 
 
Electronic data: 


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All electronic records will be stored on password-protected computers on secure network servers. Portable data 
storage, such as laptops, jump drives, etc., will not be used. Data on biological specimens will be stored without PHI and 
separately from the primary enrollment database. 
 
Paper records: 
All paper/hard copy records, such as signed consent forms, interview and exam findings, stool and food logs, and 
medical history updates, will be stored in locked files in locked offices. When being transported via courier, they will be 
in sealed packages without any visible identifying information. Only key study personnel will have access to identified 
data. 
 
Data analysis_______________________________________________________________________________________ 
Specimen processing: 
A subset of 7 stool specimens per subject will be analyzed by culture and shotgun sequencing (Table 1). All saliva, skin, 
urine, and remaining stool specimens will be stored at -80°C for future analyses.  All study investigators will remain 
blinded to study group assignment until after the culture and metagenomic data are analyzed. 
 
Culture methods: 
Semi-quantitative cultures will be performed to assess the relative abundance of aerobic and anaerobic bacteria in the 
fecal specimens. A series of 10, 10-fold dilutions will be made of the fecal specimen then plated and incubated at 35°C. A 
“sweep” of the growth on each blood agar plate will be frozen in TSB with glycerol for additional analysis. Overall burden 
of MDRO in the fecal specimens will also be assessed by culture. 
 
Shotgun sequencing: 
Metagenomic DNA will be extracted from the fecal material using a standardized phenol chloroform method as we and 
others have previously published.14-18 Each metagenomic sample will be tagged with a unique 7 bp nucleotide barcode. 
We will use the Illumina NextSeq platform to generate at least 400 million reads per lane. Total reads will be 
demultiplexed by barcode into individual sample sequence bins. The adaptors and barcodes will be removed by 
Trimmomatic and simultaneously filtered for quality. The Dantas lab has extensive experience in the Illumina library 
preparation pipeline for metagenomic sequencing.19-21 Further details of metagenomic methods are delineated in SOW 
section, task 6: shotgun sequencing.   
  
Metagenomic data analysis: 
We will use methods previously published to conduct metagenomic analyses, including quantitative statistical 
techniques to compare microbial community composition and structure.19,22 Antibiotic resistance genes composition and 
abundance will be identified directly from shotgun data using ShortBRED.23 Once annotated, resistomes will be 
compared between samples by adapting a species-oriented metric, such as Bray-Curtis, to resistance gene class 
abundances.24,25 For aim 1, we will compare microbial diversity, antimicrobial resistance gene abundance, and MDRO 
concentration in the pre-antimicrobial specimen to the post-antimicrobial specimens within a person, between persons, 
and to previously published data from healthy controls. For aim 2, we will define the duration of antimicrobial disruption 
that occurs until return to the pre-antimicrobial state within a person (if this achieved), between persons, and to 
previously published data. Alternatively, we will assay for return to an alternative stable microbiota state post-
antimicrobials, within and between the aforementioned comparisons. For aim 3, we will characterize the degree and 
disruption that occurs with each antimicrobial, and work with the Centers for Disease Control and Prevention (CDC) to 
develop and test a quantitative MDI metric. 
 
Risk to Subjects and Protection Against Risks_____________________________________________________________ 
The primary risk to subjects is loss of confidentiality. To minimize this risk, the data management practices described 
above will be utilized. Study personnel will maintain strict adherence to HIPAA guidelines throughout the conduct of the 
study. 


6 
 
There are also uncommon risks associated with the study antimicrobials. These will be detailed on the informed consent 
document and discussed with study subjects. They include: diarrhea, nausea, vomiting, abdominal pain or bloating, 
vaginal fungal infection, skin rash, and headache. 
There are rare risks associated with the study antimicrobials, including: allergic reaction, throat/tongue swelling, 
jaundice, abnormal heart rhythms/arrhythmias, death, liver or kidney failure or abnormal function, heart palpitations, C. 
difficile infection, chest pain, kidney infection, dizziness, sleepiness/fatigue, rash/itchy skin/hives, light sensitivity, 
indigestion/gas/belching, stroke, swelling, decrease appetite, insomnia, constipation, heart attack, tendon swelling or 
rupture, fungal infection, anemia, bleeding, loss of white blood cells, tremor, convulsions, nerve damage or changes in 
nerve sensation, anxiety/agitation/confusion, depression, hallucination, nightmares or other sleep disorder, muscle 
tightness or spasms, difficulty concentrating, or achiness. 
To minimize the risk of side effects from the study antimicrobials, study personnel will screen and exclude any 
participants with a history of allergic reaction to antimicrobials or any contraindications. Study investigators will perform 
a physical exam and interview subjects regarding their medical history; any subject with a history that might increase 
his/her risk of an adverse event will be excluded from participation. 
All participants will be provided with the contact information for the study team as well as the 24-hour number for the 
nurse line at the ID Clinic and instructed to call with any questions or concerns about adverse events. Dr. Kwon and/or 
Dr. Dubberke will supervise all enrollment and review data collection tools to identify adverse events or toxicities in 
enrolled subjects. They will document and report all adverse events in study participants. All adverse events will be 
reported to the WUSM Human Research Protection Office within 7 days of notification. 
Future Benefits_____________________________________________________________________________________ 
There are no direct benefits to subjects for participating in this study; however, there are future potential benefits to 
society. The prevalence, costs, morbidity, and mortality of MDRO infections continue to increase at a time when there 
are few antimicrobials being developed. Methods to proactively prevent MDRO colonization, rather than reliance on 
reactive approaches to this problem, are urgently needed. Antimicrobial stewardship is a key component of MDRO 
prevention efforts; however, there is no method to determine which antimicrobials cause the greatest degree of 
microbiome disruption. A better understanding of exactly how antimicrobials alter the microbiome is necessary to 
optimally guide future MDRO prevention efforts and antimicrobial stewardship. This study would elucidate the impact of 
antimicrobials on the fecal microbiome and the duration of this effect.  
 
The results of this study will improve our current body of knowledge by characterizing microbiome disruptions (from 
baseline) observed during and after usual courses of antimicrobials commonly used in medicine. This information can 
then be used to create microbial disruption indices (MDIs) for antimicrobial comparisons and future antimicrobial 
development. These indices, in turn, may be used to help physicians select treatments for their patients that will 
minimally disrupt their microbiomes.  
 
References_________________________________________________________________________________________ 
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