YJINF3550_proof ■ 8 June 2015 ■ 1/10 Journal of Infection (2015) xx, 1e10

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www.elsevierhealth.com/journals/jinf

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Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference Deborah Horwitz a,b,c,f, Tyler McCue d,f, Abigail C. Mapes a,b, Nadim J. Ajami d, Joseph F. Petrosino d, Robert F. Ramig e, Barbara W. Trautner a,b,c,* a

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Section of Infectious Diseases, Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA b Section of Infectious Diseases, Department of Surgery, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA c Center for Innovations in Quality, Effectiveness, and Safety, Michael E. DeBakey Veterans Affairs Medical Center, 2002 Holcombe Blvd., Houston, TX, 77030, USA d Alkek Center for Metagenomics and Microbiome Research, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA e Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA Accepted 26 May 2015 Available online - - -

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KEYWORDS Urinary tract infection; Probiotics; Microbiota; Biodiversity; Microbiome

Summary Background: Patients with long-term indwelling catheters are at high risk of catheter-associated urinary tract infection (CAUTI). We hypothesized that colonizing the bladder with a benign Escherichia coli strain (E. coli HU2117, a derivative of E. coli 83972 would prevent CAUTI in older, catheterized adults. Materials and methods: Adults with chronic, indwelling urinary catheters received study catheters that had been pre-coated with E. coli HU2117. We monitored the cultivatable organisms in the bladder for 28 days or until loss of E. coli HU2117. Urine from 4 subjects was collected longitudinally for 16S rRNA gene profiling. Results: Eight of the ten subjects (average age 70.9 years) became colonized with E. coli HU2117, with a mean duration of 57.7 days (median: 28.5, range 0e266). All subjects also remained colonized by uropathogens. Five subjects suffered invasive UTI, 3 febrile UTI and 2 urosepsis/bacteremia, all associated with overgrowth of a urinary pathogen. Colonization with E.

* Corresponding author. One Baylor Plaza, BCM 504, NA 422, Houston, TX, 77030, USA. Tel.: þ1 713 798 2320; fax: þ1 713 748 7359. E-mail address: [email protected] (B.W. Trautner). f These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.jinf.2015.05.014 0163-4453/Published by Elsevier Ltd on behalf of The British Infection Association. Please cite this article in press as: Horwitz D, et al., Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.014

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D. Horwitz et al. coli HU2117 did not impact bacterial bladder diversity, but subjects who developed infections had less diverse bladder microbiota. Conclusions: Colonization with E. coli HU2117 did not prevent bladder colonization or subsequent invasive disease by uropathogens. Microbial diversity may play a protective role against invasive infection of the catheterized bladder. Trial Registration: ClinicalTrials.gov, NCT00554996 http://clinicaltrials.gov/ct2/show/ NCT00554996. Published by Elsevier Ltd on behalf of The British Infection Association.

Introduction

Materials and methods

Catheter-associated urinary tract infection (CAUTI) is the most common infection in nursing homes and one of the top four healthcare-associated infections in acute care.1,2 Patients with long-term indwelling catheters are chronically bacteriuric and are thus at high risk for CAUTI.3 No current approach is effective at preventing bladder colonization and subsequent CAUTI in persons who require long-term urinary catheters.4 Bacterial interference using potential bladder probiotic Escherichia coli 83972 has promise as a novel, antimicrobial sparing method of preventing CAUTI.5 In Sweden, this organism has been studied in several clinical trials.6e8 The version of E. coli 83972 used in clinical trials in the United States, HU2117, has a deletion in the P-fimbriae adhesion molecule (papG) as a safety measure.9 Trials of direct bladder inoculation with this organism in persons with neurogenic bladders suggested protection from symptomatic UTI, as did two studies of E. coli HU2117-coated catheters in persons with spinal cord injury (SCI).5,10,11 No prior trial was performed specifically in older residents of long-term care facilities, a population with high prevalence of long-term catheterization and symptomatic UTI. CAUTI is often a polymicrobial infection, particularly in persons with indwelling catheters. This observation first arose through traditional microbiological methods and has been reinforced by more recent studies using 16S ribosomal RNA sequencing.12e15 However, the relationship between bladder bacterial diversity and predisposition to infection has not yet been explored. We hypothesized that such a relationship might exist, based on the established relationship between loss of gastrointestinal microbiota diversity and Clostridium difficile proliferation,16 and the fact that catheterized bladders normally have polymicrobial colonization.3 We performed a pilot clinical trial of urinary catheters coated with E. coli HU2117 in older adults. This trial allowed us to study clinical outcomes in parallel with both traditional microbiological culture and 16s rRNA sequencing of urine specimens. We hypothesized that urinary catheters coated with benign E. coli HU2117 would be a safe and effective means to colonize bladders of older patients requiring long-term indwelling urinary catheters and that rates of symptomatic UTI would decrease during bladder colonization with HU2117. We expected HU2117 to competitively exclude bladder uropathogens and induce favorable clinical outcomes.

Participants Participants were adults over age 50 residing in a long-term care facility, with a requirement for urinary catheter use, at least one prior symptomatic UTI, and pre-existing bladder colonization. Participants with bladder anatomic abnormalities were excluded, as well as people with active malignancies, immunocompromised states such as AIDS/ HIV, or uncontrolled diabetes.

Study design Subjects received 7e10 days of antibiotics targeted to Gram-negative urinary pathogens and enterococci present in their urine. The existing urinary catheter was replaced with a standard urinary catheter during the course of antibiotics. Antibiotic therapy was followed by a 2e4 day washout period prior to placement of a study catheter that had been pre-coated with E. coli HU2117 (see catheter preparation below). Subjects had UTI symptoms assessment and serial urine cultures in a clinical microbiology laboratory on study days 0, 1, 3, 7, 14, 21, and 28. On study day 0, the day of catheter placement, urine was collected for culture pre and post catheter insertion if possible. The study catheter was removed and processed (see below) on day 28. Monthly urine samples were collected after study catheter removal until HU2117 did not grow from 2 consecutive cultures. Symptomatic CAUTI was defined by the Infectious Diseases Society of America criteria,17 using our diagnostic algorithm.18 This protocol was approved by the FDA (IND 14007), the Baylor College of Medicine IRB, and the Houston VA Research & Development committee (NCT00554996).

Preparation of the study catheter Following a standardized protocol,10,11 a Foley catheter (Bardex; Lubricath) was placed into a sterile plastic bottle containing Luria Bertani (LB) broth (Lennox, Fisher Scientific). The broth was inoculated with one colony of E. coli HU2117 growing on LB agar and incubated for 48 h at 37  C. At 24 and 48 h, a sample of the broth was streaked on various selective media. Bottles that contained contaminants were discarded. Two study catheters were prepared in parallel for each patient: one was inserted on study day 0 and the other was processed for quality control.

Please cite this article in press as: Horwitz D, et al., Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.014

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Table 1

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Clinical characteristics and clinical outcomes of subjects.

Subject number

Age

Gender

Reason for catheter dependence

# Days colonized with HU2117

Clinical outcome

1

80

M

114

Colonization

2

88

M

Bed-bound Healing sacral wound Bed-bound Healing sacral wound

3 4 5

60 58 64

F M M

Hemiparesis Urinary retention Healing sacral wound

300 14 43

6

66

M

Bed-bound Incontinence

11

7

87

M

14

9

70

M

Obstruction secondary to prostatic enlargement Neurogenic bladder

10 11

67 74

M F

Urinary retention Areflexic bladder

58

57

0 0

Processing of study catheter The balloon tip was cut off and discarded. The proximal 7e10 cm of the catheter was further processed. This catheter section was washed three times for 30 s in 45 mL 1 phosphate buffered saline (PBS), then flushed with 30 mL 1 PBS using a needle and syringe. Three 1 cm pieces were cut from that section, placed into 1.5 mL tubes with 1 mL PBS þ 0.1% SDS, and sonicated for 10 min. The sonicated sample was serially diluted and plated onto selective agar. Bacterial colonies were quantified after overnight incubation at 37  C.

Bacterial culture, enumeration, and identification Bacteria were cultured aerobically on various agar media, including those selective for Gram-negative, Gram-positive, and fungal organisms. Two representative colonies with morphology consistent with E. coli HU2117 were picked and inoculated in LB with 10% glycerol and frozen at 80  C for subsequent molecular analysis. DNA was extracted using UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) following the manufacturer’s instructions. The identity of the HU2117-consistent colonies was verified by PCR with three HU2117identifying primer sets for regions unique to E. coli HU2117/83972 (fim, papG, and O-antigen) (Invitrogen).9

Colonization Catheter blockage day 6 Colonization Colonization Colonization Pseudomonas urosepsis and death day 53 Colonization Catheter blockage and febrile Proteus UTI day 11 Colonization E. coli urosepsis day 22 Colonization Febrile E. coli UTI day 76 No colonization No colonization Febrile E. coli UTI day 2

Was E. coli HU2117 present at time of UTI?

No

Yes

Yes (on catheter)

No

No

PCR was performed with Sapphire Amp Fast PCR Master Mix (Takara Bio, Otsu, Japan) using the manufacturer’s recommended reaction mixture and a GeneAmp System 9700 thermocycler (Applied Biosystems, Foster City, CA).

16S rRNA gene sequencing and profiling Sequential urine samples collected from the final four subjects in the trial before closure were frozen at 80  C. Bacterial DNA was extracted from thawed urine following methods adapted from the Human Microbiome Project.19 Briefly, the V4 region of the 16S rRNA gene was amplified through PCR and sequenced on the MiSeq platform (Illumina) using the 2  250 bp protocol.20 Sequencing read pairs were demultiplexed based on unique molecular barcodes, filtered for PhiX using Bowtie2 v2.2.1, and merged with SeqPrep. Sequences were demultiplexed using QIIME v1.8.019 and clustered using the UCLUST pipeline.20 Operational taxonomic unit (OTU) classification was achieved by mapping reads to the SILVA database,21 and abundances were recovered by mapping the demultiplexed reads to the UCLUST OTUs. The resulting OTU table was used in QIIME to calculate alphadiversity, beta-diversity, and taxonomic summaries.22,23

Please cite this article in press as: Horwitz D, et al., Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.014

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D. Horwitz et al. Table 2

Microbiologic outcomes using standard culture methods.

Subject Organisms at enrollment

Antibiotics pre-inoculation

Organisms at inoculation

Organisms during Colonization with E. coli HU2117

Organisms after loss of E. coli HU2117

1

Proteus Enterococcus Pseudomonas Mixed Gram positive

Cefepime

E. coli othera Enterococcus MRSA E. coli (HU2117)

Serratia Providencia S. epidermidis

2

Proteus

Amoxicillin

Proteus Mixed Gram positive Group B Strep E. coli (HU2117)

3

E. coli other

Sulfa methoxazoleTrimethoprim

Enterococcus E. coli (HU2117)

4

E. coli other Enterococcus Streptococcus, alpha hemolytic E. coli other Pseudomonas Klebsiella

Nitrofurantoin

E. coli other E. coli (HU2117)

Proteus E. coli other Enterococcus Providencia Mixed Gram positive HU2117 Proteus E. coli other, MRSA Mixed Gram positive Group B Strep E. coli (HU2117) Enterococcus Mixed Gram positive Group B Strep Aerococcus E. coli (HU2117) E. coli other Enterococcus E. coli (HU2117)

Ertapenem

E. coli other Pseudomonas E. coli (HU2117)

Sulfa methoxazoleTrimethoprim

Proteus Mixed Gram positive E. coli (HU2117) MRSA E. coli (HU2117)

5

6

7

9 10

11

Proteus Mixed Gram positive Providencia sp. Proteus

Rhizobacterium Acinetobacter sp. Enterococcus Pseudomonas Mixed Gram positive S. aureus Enterobacter E. coli other Enterococcus

Ertapenem

None (species

Decreased microbiota diversity associated with urinary tract infection in a trial of bacterial interference.

Patients with long-term indwelling catheters are at high risk of catheter-associated urinary tract infection (CAUTI). We hypothesized that colonizing ...
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