Biofilms are matrix-enclosed microbial assemblies adhering to non-biological and biological surfaces. They undergo dynamic environment-dependent changes. Many biofilms constitute complex microbial communities rather than assemblies composed of one or a few species. Species uncultivable under most in vitro growth conditions may contribute to these biofilms. The most frequently occurring biofilm-associated infection world- wide is the urinary tract infection (UTI). Nosocomial indwelling catheter-associated urinary tract infections (CA- UTI) are contracted by more than 1 million patients per year in the U.S. alone. Bacteria colonizing the catheters are highly adapted to the production of biofilms. Reasons as to why bacterial colonization of long term-inserted urethral catheters results in CA-UTI versus asymptomatic bacteriuria (CA-ASB) are essentially not known, and causative factors pertaining to the host environment and complexity of microbial biofilms may be implicated. Recent 16S rRNA gene surveys have indicated that microbial complexity of CA biofilms and urinary precipitates is higher than previously thought. The overall objective of this proposal is to characterize the dynamic formation and dispersal of these biofilms, as well as the triggers controlling relative human host inertia versus inflammatory responses. We hypothesize thata dynamic balance is established among pathogenic and lesser-characterized generally harmless bacteria, influencing the extent of the human host's immune response. A systems biology approach allows integration of diverse molecular datasets to elucidate signaling among microbial species responsible for cooperative as well as competitive behaviors and with the urothelial host defense system. The first Specific Aim is to profile the metagenome, metaproteome and metabolome of CA biofilms and dispersed bacterial aggregates from clinical cases in a longitudinal study design. The second Specific Aim is to develop and evaluate in vitro model systems inoculated with CA biofilm isolates and perform 'omics analysis on in vitro developing biofilms. The in vitro model systems will be based on cultivation of CA biofilm isolates with controlled level of oxygenation and synthetic urine as growth media. The third Specific Aim is to integrate and analyze metagenomic, metaproteomic and metabolomic datasets using advanced bioinformatics and multivariate statistics methods. We intend to identify biosignatures at five different levels: species, bacterial and host proteins and metabolites that characterize patterns of microbial communication with each other in the time domain and allow assessments of host tolerance towards and host defense against polybacterial colonization. We predict that the results will not only have profound implications as regards biofilm dynamics, but will also reveal biosignatures relevant in the context of diseases not limited to one infectious agent other than CA-UTI. 2.

Public Health Relevance

Urinary catheter-associated microbial biofilms are a highly important health problem since more than 1 million patients in the U.S. alone, particularly in hospital environments, are affected. Discerning asymptomatic bacteriuria which does not require antibiotic treatment from urinary tract infections which do require antibiotic treatment remains clinically difficult. A systematic study of such biofilms with modern technologies (genomics, proteomics, metabolomics) will allow us to understand for the first time how these biofilms form, how they disperse and how human defenses (fail to) interfere with biofilm establishment on catheters, which in turn may provide solutions to the problem of urinary tract infection diagnosis. Revised Scope of Work (Details) The Specific Aims are changed in two components. First, we will reduce the scope of work in Specific Aim 1 to ~75% of the previously proposed scope of work. This is reflected in the reduced sample numbers for each of the four omics technologies (see Table below). This moderately reduced scope of work will not negatively impact the anticipated results because we will still be able to integrate metagenomic, metaproteomic and metabolomic data from in vivo biofilm isolates using bioinformatics and multivariate statistics methods. Sample type Patients Sampling time points Analysis method No. of analyses Catheter- associated biofilm (CAB) 8 6 (catheter removed every 1-2 weeks);these 6 samples represent a longitudinal sampling approach X A 16S rRNA metagenomics 48 A Metagenomic sequencing 10 B LC-MS/MS metaproteomics 32 B 2D-LC-MS/MS metaproteomics 8 B, C LC-MS metabolomics 96 Urinary precipitate (UP) 8 6 (precipitate collected every 1-2 weeks);these 6 samples a represent longitudinal sampling approach and pertain to biofilm dispersal X A 16S rRNA metagenomics 48 A Metagenomic sequencing 10 B LC-MS/MS metaproteomics 32 B 2D-LC-MS/MS metaproteomics 8 B, C LC-MS metabolomics 96 X Foley catheters of patients in Dr. Wolcott's clinic are replaced every 1-2 weeks;the objective is to obtain at least six samples from each patient for each sample type;AAll samples will be subjected to bacterial species diversity surveys (16S rRNA), with samples from two patients to be subjected to metagenomic sequencing as well;BMetaproteomics and metabolomics experiments will be performed on 2/3rd of the samples;CMetabolomics experiments will be performed using three separate extraction/ionization modes. Second, we will reduce the scope of work in Specific Aim 2 leaving out the co-cultivation of catheter- associated bacterial isolates. We will still be able to culture individual bacterial isolates derived from the patient samples. We will still be able to carry out the experiments pertaining to the in vitro multi-species biofilm models which includes setting up six different reactors, three each (aerobic, micraerophilic, anaerobic) for two different kinds of biofilms. Both of these biofilms will be analyzed using the 2D-LC-MS/MS metaproteomic and LC-MS metabolomic analyses also described in Specific Aim 1 in three developmental stages.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM103598-02
Application #
8636490
Study Section
Special Emphasis Panel (ZGM1-GDB-2 (MC))
Program Officer
Sledjeski, Darren D
Project Start
2013-03-15
Project End
2017-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
2
Fiscal Year
2014
Total Cost
$311,802
Indirect Cost
$111,802
Name
J. Craig Venter Institute, Inc.
Department
Type
DUNS #
076364392
City
Rockville
State
MD
Country
United States
Zip Code
20850
Yu, Yanbao; Suh, Moo-Jin; Sikorski, Patricia et al. (2014) Urine sample preparation in 96-well filter plates for quantitative clinical proteomics. Anal Chem 86:5470-7