SUMMARY STATEMENT

PROGRAM CONTACT: ( Privileged Communication ) Release Date: 02/27/2017

Revised Date:

Principal Investigator 

GORDON, VERNITA

Application Number: 1 R01 AI121500-01A1

Applicant Organization: UNIVERSITY OF TEXAS, AUSTIN

Review Group: BMBI

Biomaterials and Biointerfaces Study Section

Meeting Date: 02/15/2017

Council: MAY 2017

RFA/PA: PAR16-242

PCC: M36

Requested Start: 05/01/2017

Project Title: Assessing the roles of biofilm structure and mechanics in pathogenic, persistent 

infections

SRG Action:

Next Steps: Visithttp://grants.nih.gov/grants/next_steps.htm

Human Subjects: 10-No human subjects involved

Animal Subjects: 30-Vertebrate animals involved - no SRG concerns noted

Project

Year

1 

2 

3 

4 

TOTAL

Direct Costs

Requested

Estimated

Total Cost 

ADMINISTRATIVE BUDGET NOTE: The budget shown is the requested budget and has not been adjusted 

to reflect any recommendations made by reviewers. If an award is planned, the costs will be calculated by 

Institute grants management staff based on the recommendations outlined below in the COMMITTEE 

BUDGET RECOMMENDATIONS section.

NEW INVESTIGATOR

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1 R01 AI121500-01A1 2 BMBI

GORDON, V 

1R01AI121500-01A1 Gordon, Vernita

NEW INVESTIGATOR

RESUME AND SUMMARY OF DISCUSSION: This application proposes to determine the mechanics 

and structure of biofilm infections of the opportunistic pathogen Pseudomonas aeruginosa in chronic 

wounds and how these physical properties impact disease course. The impact of these studies, if 

successful, will address the major physical factors controlling virulence, antibiotic resistance, and

immune evasion in biofilm infections and is expected to give rise to new types of treatments and 

diagnostics for chronic biofilm infections that specifically target structure and mechanics. The strengths 

of the application are the dramatically improved focus; the supportive preliminary data; and the novel 

mechanism to treat biofilms. Concerns areb that it will require a large amount of coordination between 

groups. Overall, the application receives much enthusiasm in the area of biofilm infections.

DESCRIPTION (provided by applicant): What spatial structure and mechanics develops in biofilm 

infections, and how such spatial structure and mechanics impact the persistence and virulence of 

biofilm infections, is not known. The long-term goal is to find diagnostic and treatment approaches that 

address the structure and mechanics of multicellular, three- dimensional biofilm infections within the 

host. The objective of this application is to determine the mechanics and structure of biofilm infections 

of the opportunistic pathogen Pseudomonas aeruginosa in chronic wounds, and how these physical

properties impact disease course. The central hypothesis is that spatial structure and mechanics are 

the major physical factors controlling virulence, antibiotic resistance, and immune evasion in biofilm 

infections. The rationale underlying this application is that completion will identify key physical targets 

for preventing, disrupting, or ameliorating biofilm infections for an important biofilm-forming pathogen. 

The proposed work will also develop a widely-applicable platform for assessing the state and impact of 

biofilm structure and mechanics for other infecting organisms. The central hypothesis will be tested by 

pursuing three specific aims: 1) Determine the spatial structure and mechanics of in vivo biofilm 

infections; 2) Determine how spatial arrangements differentiate into distinct microenvironments; 3) 

Determine the role of spatial structure and mechanics in biofilm-neutrophil interactions. We will pursue

these aims using an innovative combination of analytical and manipulative techniques from both 

biological and physical sciences. These include both recently-developed techniques specific to biofilm 

studies, and more-established techniques that have been applied very little to the study of biofilm 

materials. The proposed research is significant, because it will determine which structural and 

mechanical characteristics should be therapeutic targets. It is also significant because it will develop a 

platform that can be extended to study other pathogens (or commensals) and synergies to open new 

avenues for biofilm therapies. This work will develop foundational resources that will be used by other 

researchers, for P. aeruginosa and other organisms. The proximate expected outcome of this work an 

understanding of which biofilm structural and mechanical characteristics contribute to clinical impact. 

The results will have an important positive impact immediately because they will establish better 

understanding of biofilm infection, virulence, and resistance to antibiotics and the immune system for an 

important pathogen, and long-term because they lay the groundwork to develop a suite of techniques 

for better treatment of biofilm infections.

PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because 

understanding what biofilm structures and mechanics exist and the degree to which they influence the 

medical outcomes of biofilm infections is expected to give rise to new types of treatments and 

diagnostics for chronic biofilm infections that specifically target structure and mechanics. Thus, this 

application is relevant to the part of NIH’s mission that pertains to fostering fundamental creative 

discoveries and innovative research strategies as a basis for ultimately protecting health.

CRITIQUES: The written critiques of individual reviewers are provided in essentially unedited form 

below. These critiques were prepared prior to the meeting and may not have been revised afterwards. 
1 R01 AI121500-01A1 3 BMBI

GORDON, V 

CRITIQUE 1:

Significance: 2

Investigator(s): 1

Innovation: 1

Approach: 2

Environment: 1

Overall Impact: This application is to investigate the structural and physical properties of biofilms

using a wide range of novel techniques developed by the team and how these properties affect 

infections, antibiotic resistance, resistance to immune invasion and virulence. The proposed study is 

very novel in several aspects including novel techniques used, different properties to be studied, new 

insights into biofilm development, etc. This application has been improved substantially from the 

previous applications and the team has made several progresses with published records to support the 

current study. A few concerns still exist including coordination of the research activities, unclear 

description of budgets, inconsistence in description of research activities and justification of animal to 

be used. 

1. Significance:

Strengths 

 It is novel to investigate the structural and physical properties of biofilms.

 Better understanding of such properties will likely lead to develop novel control strategies.

Weaknesses

2. Investigator(s):

Strengths

 All investigators have published extensively in the areas of proposed research.

Weaknesses

 It is not well defined specifics tasks for each investigator.

3. Innovation:

Strengths

 Novel techniques will be developed to study biofilms.

 The application focus on the structural and mechanical properties of biofilms.

 The application will also study interaction of immune cells with biofilms. 

Weaknesses

4. Approach:

Strengths

 The application will use both in vivo and in vitro models for characterization of biofilm.
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GORDON, V 

 Several cutting-age techniques have been proposed for the study.

 End-point measurement are clearly defined. 

Weaknesses

 In aim 1, how to image biofilms in specific sites of wounded area over time is not clearly 

described.

 In aim 2, in vitro antibiotic resistance test will be performed. Why not use in vivo model?

 In aim 3, in vitro model will be used; however, mouse is needed in the animal protocol section? 

5. Environment:

Strengths

 Excellent environment.

Weaknesses

Protections for Human Subjects: Not Applicable (No Human Subjects)

Vertebrate Animals: NO, animal welfare concerns or incomplete

 Mouse is still required for the aim 3 but not described in the approach section

Biohazards: Acceptable

Resubmission:

 It is a much-improved submission comparing to the previous one with a more focused mission. 

However, there are several inconsistences in the application including animal use, the model to 

be used for aim 1 studies. 

Resource Sharing Plans: Unacceptable

 The proposed meetings and data exchange is not sufficient for such complex projects

Authentication of Key Biological and/or Chemical Resources: Not Applicable (No Relevant 

Resources)

Budget and Period of Support:

Recommended budget modifications or possible overlap identified:

 Certain portions of budget justification are missing. Support for several graduate 

students/postdocs is requested without clearly defined tasks on the project. 

CRITIQUE 2:

Significance: 1
1 R01 AI121500-01A1 5 BMBI

GORDON, V 

Investigator(s): 1

Innovation: 2

Approach: 1

Environment: 1

Overall Impact: The central hypothesis to be tested in this resubmission of an R01 application is that 

spatial structure and mechanics are the major physical factors controlling the development of 

pathogenicity, antibiotic resistance, and immune evasion in biofilm infections. To test this hypothesis, 

they will use Pseudomonas aeruginosa as a model microorganisms and determine the spatial structure 

and mechanics of biofilm infections in wounds; determine how spatial arrangements impact bacterial 

growth, biofilm microenvironments, antibiotic resistance, and virulence; and determine the role of 

spatial structure and mechanics in biofilm leucocyte interactions. This is a much-reduced set of 

objectives compared to the original application, which was to examine two microorganisms in three

environments. Other significant changes have been made in response to SS’s critique, including a 

reorganization of the research team. The overall effect is a tighter application with high probability of 

success.

1. Significance:

Strengths

 The scientific premise of this application is that biofilm microarchitecture and mechanics are key 

to the development, antibiotic resistance and immune evasion in biofilm infections. 

Understanding this may prove to be key in preventing and treating biofilms.

 The data resulting from this study will be important to the field and for the development of drugs 

that target novel sites, leading to prevention, disruption or amelioration of biofilms.

Weaknesses

 None

2. Investigator(s):

Strengths

 This is an outstanding team of investigators, each bringing critical expertise to the problem.

 The team is already interactive.

Weaknesses

 None

3. Innovation:

Strengths

 The approach that will be used for this study is innovative and will result in new tools for 

studying biofilms. The team will develop a new technique for measuring the mechanism of 

biofilm infections.

 Sophisticated imaging will be used to characterize biofilm structure and manipulative techniques 

we be employed to recreate these structures to better understand their role.

 A fresh approach will be used to examine the interplay between neutrophils and bacteria.

Weaknesses
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GORDON, V 

 None

4. Approach:

Strengths

 Use of in vivo biofilms rather than in vitro biofilms.

 Selection of a panel of mutants to examine the role of alginate, Psl, and Pel.

 Multiple assays for disease outcomes: systemic spread of bacteria, quorum sensing, antibiotic 

effectiveness, inflammation (neutrophil invasion), wound closure and healing.

 Physical structures of biofilm will be determined by CLSM and quantitative image analysis.

 Use of quantum dots to assess diffusion characteristics.

 AFM and microrheology will be used to assess biomechanics.

 In vivo data will be compared to biofilms grown in vitro.

 Aim 2 will take advantage of novel methods for building biofilms that allows monitoring while 

testing different microarchitectures on biofilm development, etc. This will enable them to

examine different properties noted in the mouse model.

 Aim 3 will add neutrophils to the equation. The three aims build on each other nicely.

Weaknesses

 The study relies on close coordination among laboratories.

5. Environment:

Strengths

 The environments at the two partnering institutions are mutually synergistic.

Weaknesses

 None

Protections for Human Subjects: Not Applicable (No Human Subject)

Vertebrate Animals: YES, all four points addressed

 Care has been taken to ensure the least number of animals will be used for the studies.

Biohazards: Acceptable

 Pseudomonas will be handled in appropriate biosafety hoods.

Resubmission:

 The application has responded well to SS critique. Where appropriate, they have explained

their rationale for the choices they have made. 

 The application is much more focused than originally.
1 R01 AI121500-01A1 7 BMBI

GORDON, V 

 Preliminary data are now published.

Resource Sharing Plans: Acceptable

Authentication of Key Biological and/or Chemical Resources: Acceptable

Budget and Period of Support: Recommend as Requested

CRITIQUE 3:

Significance: 2

Investigator(s): 2

Innovation: 1

Approach: 2

Environment: 1

Overall Impact: This application seeks to test the hypothesis that virulence, antibiotic resistance, and 

immune evasion in biofilm infections of the pathogen Pseudomonas aeruginosa within chronic wounds 

are influenced by the biofilm’s physical properties. Three aims are proposed to test this hypothesis. In 

Aim 1, P. aeruginosa applied to surgical excisions (in mice) will form biofilms; systemic bacterial load 

will be evaluated along with quorum sensing, antibiotic efficacy, and inflammation. Biofilms will be 

evaluated for a comprehensive set of outcomes including mass transport, heterogeneity, and 

mechanical properties. Aim 2 seeks to determine how spatial structures within the film develop 

microenvironments that influence biofilm disease, affect antibiotic resistance, and virulence. These two 

aims are well founded in the literature that was reviewed within the Research Strategy, and both aims 

were also well supported by a robust body of preliminary data. Aim 2 is particularly compelling as the 

application and team seek to recreate what are effectively biomimetic biofilms (based on observations 

made in Aim 1) and then evaluate these ‘synthetic’ biofilms for controlled studies of the influence of 

biofilm structure. Finally, in Aim 3, how properties including elasticity and yielding relate to 

immunological clearance are evaluated in a systematic manner. Overall this is a strong team with a 

strong application that has clearly benefitted from focus added as a result of the initial review. This work 

has high potential translational potential as a better understanding of factors that contribute to 

virulence, persistence, and function of biofilms may lead to identification of new therapeutic targets.

1. Significance:

Strengths 

 Chronic infections affect millions of Americans and may result in death and sometimes result in

life-limiting comorbidities including amputation.

 The influence of biofilm physical properties and structures are considered important to the 

survival and persistence of the biofilms, yet comprehensive characterization of these factors has 

not been performed and so the influence of these factors on virulence, antibiotic resistance, and 

immune evasion remain unknown. Yet elucidating these factors will enable identification of 

potential therapeutic targets.

 The scientific premise of this application is that the structure and mechanical properties and

behavior are critical to the persistence and antibiotic resistance of biofilms. Elucidating the role 
1 R01 AI121500-01A1 8 BMBI

GORDON, V 

of biofilm structure and mechanics may provide insight into novel approaches to treat and 

mitigating chronic biofilm infections.

Weaknesses

 None noted.

2. Investigator(s):

Strengths

 This investigative team is led by an exciting new investigator (Dr. Gordon), an Assistant 

Professor in the Department of Physics at UT Austin, with expertise in characterization of 

physical properties and biological manipulation of biofilms.

 The collaborative and interdisciplinary team members bring expertise in bacterial pathogenesis 

in wound and soft tissue infections (Runbaugh), advanced biomedical imaging a rheology of 

complex fluids (Christopher), and imaging and visualization of bacteria (Shear).

Weaknesses

 None noted. This is a strong team.

3. Innovation:

Strengths

 Relating the physical properties (i.e., spatial structure and mechanics) of Pseudomonas 

aeruginosa biofilms in chronic wounds to the virulence, antibiotic resistance, and immune 

evasion is novel and holds potential impact for improving patient outcomes and reducing

development of antibiotic resistant infections.

 Successful completion of the proposed work would enable development of novel therapeutic 

strategies with a specific focus on disrupting the biofilm structure and/or mechanics.

Weaknesses

 None noted.

4. Approach:

Strengths

 A key strength of this application is the assessment of only one pathogen Pseudomonas 

aeruginosa. This tight focus will enable the team to dig deeply into the questions posed in this 

application without losing focus as would likely happen with multiple strains of bacteria.

 Aim 1 – development of a new imaging technique using fluorescent beads and quantum dots to 

measure transport properties of in vivo infection. This technique will extend beyond studies of 

biofilms. Also, new technique in Aim 1 to evaluate heterogeneity of the matrix content. Both are 

strengths of this application.

 Aim 2 is particularly compelling as the application and team seek to recreate what are effectively 

2D biomimetic biofilms (based on observations made in Aim 1), e.g. using optical trapping to 

control heterogeneity, and then evaluate these ‘synthetic’ biofilms for controlled studies of the

influence of biofilm structure.

 Aim 3 makes a nice progression from in vitro to in vivo studies with a clearly defined and 

effective experimental design.
1 R01 AI121500-01A1 9 BMBI

GORDON, V 

Weaknesses

 Adhesion is likely to occur between the AFM tip and biofilms (e.g., see Activity 1.2); however, 

this is poorly accounted for in the research strategy. The citation #156 used in this context 

describes a soft film on a stiff substrate and does not review or provide reasonable solutions to 

deal with adhesive contact between a probe and a half space. 

 With regards to AFM testing, how will films be removed from the animal without disrupting the 

film structure or causing viscous dissipation (and thus affecting the measured properties)?

5. Environment:

Strengths

 The environments at UT Austin and in the lab of the application and her collaborators (at both 

UT Austin and at Texas Tech University) are excellent. The equipment and facilities needed to 

excel at the proposed research are available.

Weaknesses

 None noted.

Protections for Human Subjects: Not Applicable (No Human Subjects)

Vertebrate Animals: YES, all four points addressed

Biohazards: Acceptable

Resubmission:

 The application and her team appear to have responded to the previous reviewer's criticisms in 

a thorough and careful manner.

 Alterations from the original application should be but were not clearly denoted within the

Research Plan. 

Resource Sharing Plans: Acceptable

Authentication of Key Biological and/or Chemical Resources: Acceptable

Budget and Period of Support: Recommend as Requested

THE FOLLOWING SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO 

SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE, OR REVIEWERS’ 

WRITTEN CRITIQUES, ON THE FOLLOWING ISSUES:

VERTEBRATE ANIMAL: ACCEPTABLE

COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested.
1 R01 AI121500-01A1 10 BMBI

GORDON, V 

Footnotes for 1 R01 AI121500-01A1; PI Name: Gordon, Vernita 

NIH has modified its policy regarding the receipt of resubmissions (amended applications). 

See Guide Notice NOT-OD-14-074 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD14-074.html. The impact/priority score is calculated after discussion of an application by

averaging the overall scores (1-9) given by all voting reviewers on the committee and 

multiplying by 10. The criterion scores are submitted prior to the meeting by the individual 

reviewers assigned to an application, and are not discussed specifically at the review meeting 

or calculated into the overall impact score. Some applications also receive a percentile 

ranking. For details on the review process, see 

http://grants.nih.gov/grants/peer_review_process.htm#scoring.