TARGETED RESEARCH ON ORAL MICROBIAL BIOFILMS

Release Date:  March 6, 1998

RFA:  DE-98-006

P.T.

National Institute of Dental Research
National Institute of Allergy and Infectious Diseases

Letter of Intent Receipt Date: June 23, 1998
Application Receipt Date:  July 21, 1998

PURPOSE

The National Institute of Dental Research (NIDR) and National Institute of
Allergy and Infectious Diseases (NIAID), invite research grant applications to
investigate basic research on microbial oral biofilms, specifically in the areas
of antimicrobial resistance, gene transfer, and host defense.  Such research
projects in these targeted areas will improve strategies to diagnose, prevent and
treat biofilm-associated  infectious diseases in the oral cavity (e.g.,
periodontitis, dental caries and candidiasis).  Collaborative projects that bring
together investigators in diverse scientific disciplines studying biofilms,
including microbiology, immunology (including mucosal immunology), biochemistry,
pathology, engineering, imaging technology, and mathematical modeling are
encouraged.  Applications and collaborations are also encouraged with foreign
scientists conducting unique research on biofilms.

HEALTHY PEOPLE 2000

The Public Health Service (PHS) is committed to achieving the health promotion
and disease prevention objectives of "Healthy People 2000," a PHS-led national
activity for setting priority areas.  This Request for Applications (RFA),
Targeted Research on Microbial Biofilms, is related to the priority area of oral
health.  Potential applicants may obtain a copy of "Healthy People 2000" (Full
Report: Stock No. 017-001-00474-0 or Summary Report:  Stock No. 017-001-00473-1)
through the Superintendent of Documents, Government Printing Office, Washington,
DC 20402-9325 (Telephone: 202-512-1800).  The documents are also available on the
Internet at http://odphp.osophs.dhhs.gov/pubs/hp2000/default.htm.

ELIGIBILITY REQUIREMENTS

Applications may be submitted by domestic and foreign, for-profit and non-profit
organizations, public and private, including universities, colleges, hospitals,
laboratories, units of State and local governments and eligible agencies of the
Federal government.  The NIH encourages applications that include investigators
who are racial/ethnic minority individuals, women and persons with disabilities. 
Although an application must be submitted from a single institution,
collaborative arrangements with other institutions are encouraged.  Also,
applications are not restricted to traditional dental, oral and craniofacial
research settings.

MECHANISM OF SUPPORT

The mechanism of support utilized will be the individual research project grant
(R01).  Responsibility for the planning, direction, and execution of the proposed
project will be solely that of the applicant.  The total project period for an
application submitted in response to this RFA may not exceed five years.  This
RFA is a one-time solicitation for new and competing renewal awards.  Future
competitive renewal applications will compete with all investigator-initiated
applications and will be reviewed according to customary referral and review
procedures.  New investigators without prior R29 or R01 support are strongly
encouraged to apply.  They should identify their status in a cover letter as well
as in the application and provide evidence of institutional support for the
research.

FUNDS AVAILABLE

The estimated total costs (direct plus indirect) available for the first year of
support for NIDR awards will be $3 million.  The NIDR plans to support or
contribute to the support of a total of approximately ten grants in response to
this RFA in fiscal year 1999.  Although NIAID has not set aside funds for this
initiative, they will consider support for highly meritorious applications that
meet their missions.  Grant awards will be contingent on the receipt of high
quality applications and the availability of funds. Applications may not request
more than three percent annual increases for inflation over subsequent years. 
Usual PHS policies governing grants administration and management will apply. 
Funding beyond the first and subsequent years of the grant will be contingent
upon satisfactory progress during the preceding years and the availability of
funds.

RESEARCH OBJECTIVES

Background

A biofilm is an accumulation of  bacterial, fungi, or protozoa on solid surfaces,
such as tissue cells, teeth and artificial implants.  Biofilms are medically
important since few diseases are caused by microbes that are planktonic, that is,
non-adherent and free-floating.  Over 80% of microbial infections in the body are
caused by bacteria growing as a biofilm.  Examples include the layer of
Pseudomonas aeruginosa that forms in the trachea of cystic fibrosis patients, the
E. coli bacterial biofilm that forms in urinary tract and intestinal infections,
Staphylococcus sp. infections from biofilms on indwelling catheters, and eye
infections from biofilms that form on contact lenses. In the oral cavity,
microbial biofilms, commonly referred to as dental plaque, are involved in the
pathogenesis of caries, periodontitis, dental implant failures, denture
stomatitis and oral yeast infections such as candidiasis.  Biofilms that form in
the water lines of dental operatories are also an important public health concern
as a potential vector for infection, particularly to patients with depressed
immune systems.

Three recent conferences on microbial biofilms, the International Conference on
Oral Biology meeting on oral biofilms (March, 1996), the American Society for
Microbiology conference on Microbial Biofilms (September, 1996), and the Seattle
conference on antimicrobial resistance in oral bacteria (September, 1996),
highlighted the importance of biofilms in infectious diseases and progress being
made in this scientific area by both domestic and foreign scientists.  A recent
NIDR Infectious Diseases Planning Workshop held November 10-12, 1997 recommended
increased support for research on the topic of oral biofilms.  The need for
increased research on biofilms is based on many factors:

Biofilms are remarkably difficult to treat with antimicrobials.  The reasons for
this are not clear.  Antimicrobials may be readily inactivated or fail to
penetrate into the biofilm.  In addition, bacteria within biofilms have an
inherent increased (up to 1000-fold higher) resistance to antimicrobial
compounds, even though these same bacteria are sensitive to these agents if grown
under planktonic conditions. Techniques such as ultrasound or small electrical
pulses may enhance the penetration and effectiveness of antimicrobials in
biofilms.

Biofilms increase the opportunity for gene transfer between/among bacteria.  This
is important since bacteria resistant to antimicrobials or chemical biocides can
transfer the genes for resistance to neighboring susceptible bacteria.  Gene
transfer can convert a previous avirulent commensal organism into a highly
virulent pathogen.

Certain species of bacteria communicate with each other within the biofilm.  As
their density increases, the organisms secrete low molecular weight molecules
that signal when the population has reached a critical threshold.  This process,
called quorum sensing, is responsible for the expression of virulence factors by,
for example, Pseudomonas aeruginosa, which produces destructive proteinases when
the number of these bacteria reach a high enough density in the airway biofilms
of cystic fibrosis patients.

Bacteria express new, and sometimes more virulent phenotypes when growing within
a biofilm.  Such phenotypes may not have been detected in the past because the
organisms were grown on rich nutrient media under planktonic conditions.  The
growth conditions are quite different particularly in the depths of biofilms,
where nutrients and oxygen are usually limited, and waste products from neighbors
can be toxic.  In short, bacteria found at the bottom of the biofilm look and act
different than species located at the surface.

Bacteria embedded within biofilms are protected from many of the natural host
defenses and are notoriously difficult for the host to remove.  Contact with a
solid surface triggers the expression of a panel of bacterial enzymes which
catalyze the formation of sticky polysaccharides that promote colonization and
protection.  The structure of biofilms is such that immune responses may be
directed only at those antigens found on the outer surface of the biofilm, and
antibodies and other serum or salivary proteins often fail to penetrate into the
biofilm.  Studies have shown that the host may synthesize antibodies that are
ineffective in killing a bacterium in a biofilm, yet highly effective in killing
the planktonic form of the organism. In addition, phagocytes are unable to
effectively engulf a bacterium growing within a complex polysaccharide matrix
attached to a solid surface.  This causes the phagocyte to release large amounts
of pro-inflammatory enzymes and cytokines, leading to inflammation and
destruction of nearby tissues.

The field of biofilm research has traditionally been hindered by an inability to
study the biofilm in non-destructive, three dimensional ways.  In this regard,
most plaque samples are scraped from the tooth surface and smeared on a glass
slide or agar plate.  In addition, it has been difficult or impossible to assess
gene expression and metabolism of the microbe at the single cell level within a
biofilm.  However, as a result of  advances in laser technology, digital imaging,
scanning electron microscopy, and new fluorescent probes, researchers can now
build a three dimensional model of biofilms and identify the location in the
biofilm where specific genes are being expressed.

A review of currently funded NIDR grants indicates the scope of research on
biofilms is primarily limited to projects which involve: (i) identification of
bacterial species in plaque; (ii) mechanisms of plaque formation and survival
(e.g., adherence, polysaccharide formation, acidurance, and pellicle deposition);
or (iii) epidemiology studies that sample dental plaque.  Although these studies
are important and provide the framework for contemporary research on biofilms,
they do not address the fact that bacteria change their phenotype and sometimes
genotype when grown in biofilms, and become resistant to antimicrobial agents and
host defenses.  For example, although several grants have supported research on
the resistance of oral bacteria to antimicrobials, these studies were conducted
after the bacteria were isolated from dental plaque and grown under planktonic
conditions.  Such conditions are artificial and do not reflect the real in
vivo/in situ environmental conditions in which bacteria grow as biofilms.  Thus,
data from these ex vivo or in vitro studies have questionable relevance to
disease pathogenesis, treatment and prevention.

The areas of focus for this initiative, namely antimicrobial resistance, gene
transfer, and host defenses in biofilm research, are currently understudied and
will address the critical problems associated with prevention and treatment of
bacterial oral biofilm diseases.  Moreover, they capitalize on contemporary areas
of immunology, microbiology, bioengineering and computer technology that dovetail
with our current portfolio in dental plaque research.

Research Objectives and Scope

The goal of this RFA is to promote a fuller understanding of selected biological
aspects of oral biofilms; namely the development of increased resistance to
antimicrobials and antimicrobial agents, transfer of genetic information
among/between organisms in the biofilm, and effective host responses that prevent
or limit the formation of biofilms on implanted materials or natural substrates. 
Studies concerned with the development of biofilms,  or with the adherence of
bacterial species to the substrate or to other microorganisms would, for example,
not be an appropriate response to this RFA.  Similarly, studies on the microbial
and biochemical composition of biofilms are not within the scope of this
initiative.  Examples of relevant research topics are listed below; however the
list should not be construed as complete or restrictive.

Antimicrobial resistance:

development of simple and accurate procedures to determine the minimum inhibitory
concentration of antimicrobials in oral biofilm

determination of the physical and chemical factors or conditions that cause
antimicrobial resistance by bacteria growing in oral biofilms

examination of transport limitations as a mechanism of resistance to
antimicrobials in oral biofilms

characterization of the extracellular and intracellular signals involved in up-
regulation of antimicrobial resistance genes in microorganisms growing in oral
biofilms

identification of novel and potentially clinically useful ways to enhance
antimicrobial sensitivity in microorganisms growing in oral biofilms

control of biofilms with biocides generated under selective environmental
conditions

development of computer models that can be used to design optimal antimicrobial
levels for biofilms on an individual subject basis

Genetic transfer:

development of advanced imaging and detection techniques for biofilm-associated
microbial gene expression

plasmid expression and transfer in oral biofilms

characterization of the mechanisms used by bacteria to transfer virulence genes
to neighboring bacteria in oral biofilms

identification of the genes most likely to be transferred as a result of growth
in an oral biofilm

characterization of the physiological status of donor and recipient cells

Host response:

characterization and effectiveness of the immunoglobulin responses to specific
microorganisms in oral biofilms

development of ways to enhance the effectiveness of antibody-microorganism
interactions in oral biofilms through the use of, for example, novel adjuvants

evaluation and enhancement of the role of the innate immune system (for example,
complement, defensins, lysozyme) in prevention or elimination of oral biofilms

evaluation of the biochemical aspects responsible for the enhanced
proinflammatory nature of oral biofilms

role of the host immune response in oral biofilm formation and prevention

INCLUSION OF WOMEN AND MINORITIES IN RESEARCH INVOLVING HUMAN SUBJECTS

It is the policy of the NIH that women and members of minority groups and their
subpopulations must be included in all NIH supported biomedical and behavioral
research projects involving human subjects, unless a clear and compelling
rationale and justification is provided that inclusion is inappropriate with
respect to the health of the subjects or the purpose of the research.  This
policy results from the NIH Revitalization Act of 1993 (Section 492B of Public
Law 103-43).

All investigators proposing research involving human subjects should read the
"NIH Guidelines for Inclusion of Women and Minorities as Subjects in Clinical
Research," which have been published in the Federal Register of March 28, 1994
(FR 59 14508-14513) and in the NIH Guide for Grants and Contracts, Vol. 23, No.
11, March 18, 1994.

LETTER OF INTENT

Prospective applicants are asked to submit, by June 23, 1998, a letter of intent
that includes a descriptive title of the proposed research, the name, mailing
address, FAX number, email address and telephone number of the Principal
Investigator and the identities of other key personnel and participating
institutions and departments; and the number and title of this RFA.

Although a letter of intent is not required, is not binding, and does not enter
into the review of a subsequent application, the information that it contains is
helpful in estimating the potential review workload and avoiding conflict of
interest in the review.

The letter of intent is to be sent to Dr. Dennis Mangan at the address listed
under INQUIRIES.

APPLICATION PROCEDURES

The research grant application form PHS 398 (rev. 5/95) is to be used in applying
for these grants.  These forms are available at most institutional offices of
sponsored research and may be obtained from the Division of Extramural Outreach
and Information Resources, National Institutes of Health, 6701 Rockledge Drive,
MSC 7910,Bethesda, MD 20892-7910, telephone 301/435-0714, email:
ASKNIH@od.nih.gov.  The forms are also available on the NIH World Wide Web Home
Page at http://grants.nih.gov/grants/funding/phs398/phs398.html.

The RFA label available in the PHS 398 application form kit must be affixed to
the bottom of the face page of the original and the original must be placed on
top of the entire package.  Failure to use this label could result in delayed
processing of the application such that it may not reach the review committee in
time for review.  In addition, in order to identify the application as a response
to this RFA, the RFA title (Targeted Research on Microbial Biofilms) and number
DE-98-006 must be typed in item 2 of the face page of the application form and
the YES box must be checked.  The instructions accompanying Form PHS 398 must be
followed.

Applicants from institutions that have a General Clinical Research Centers (GCRC)
funded by the NIH National Center for Research Resources may wish to identify the
Center as a resource for conducting the proposed research.  If so, a letter of
agreement from the GCRC Program Director must be included in the application
material.

Submit a signed, typewritten original of the application, including a cover
letter, the checklist and three signed photocopies in one package to:

CENTER FOR SCIENTIFIC REVIEW
NATIONAL INSTITUTES OF HEALTH
6701 ROCKLEDGE DRIVE, ROOM 1040 - MSC 7710
BETHESDA, MD  20892-7710
BETHESDA, MD  20817 (for express/courier service)

At the time of submission, two additional copies of the application must also be
sent to:

Dr. H. George Hausch
Division of Extramural Research
National Institute of Dental Research
45 Center Drive, Room 4AN-38D - MSC 6402
Bethesda, MD  20892-6402

Applications must be received by July 21, 1998.  If an application is received
after that date, it will be returned to the applicant without review.

REVIEW CONSIDERATIONS

Review Procedures

Upon receipt, applications will be reviewed for completeness by the Center for
Scientific Review (CSR) and responsiveness by the NIDR.  Incomplete and/or non-
responsive applications will be returned to the applicant without further
consideration.  Applications that are complete and responsive to the RFA will be
evaluated for scientific and technical merit by a special emphasis panel convened
by the Scientific Review Section, NIDR.  As part of the initial merit review, all
applications will receive a written critique and undergo a process in which only
those applications deemed to have the highest scientific merit, generally the top
half of applications under review, will be discussed, assigned a priority score,
and receive a second level review by the appropriate national advisory council
or board.

The NIAID has an interest in the role of biofilms in the pathogenesis of
infectious diseases in humans.  Therefore, applications that address oral
biofilms are likely to be given a secondary assignment to NIAID.

Review Criteria

The goals of NIH-supported research are to advance our understanding of
biological systems, improve the control of disease, and enhance health.  The
reviewers will comment on the following aspects of the application in their
written critiques in order to judge the likelihood that the proposed research
will have a substantial impact on the pursuit of these goals.  Each of these
criteria will be addressed and considered by the reviewers in assigning the
overall score and weighting them as appropriate for each application.  Note that
the application does not need to be strong in all categories to be judged likely
to have a major scientific impact and thus deserve a high priority score.  For
example, an investigator may propose to carry out important work that by its
nature is not innovative but is essential to move a field forward.

Significance:  Does this study address an important problem?  If the aims of the
application are achieved, how will scientific knowledge be advanced?  What will
be the effect of these studies on the concepts or methods that drive this field?

Approach:  Are the conceptual framework, design, methods, and analyses adequately
developed, well-integrated, and appropriate to the aims of the project?  Does the
applicant acknowledge potential problem areas and consider alternative tactics?

Innovation:  Does the project employ novel concepts, approaches or methods?  Are
the aims original and innovative?  Does the project challenge existing paradigms
or develop new methodologies or technologies?

Investigator:  Is the investigator appropriately trained and well-suited to carry
out this work?  Is the work proposed appropriate to the experience level of the
principal investigator and other researchers (if any)?

Environment:  Does the scientific environment in which the work will be done
contribute to the probability of success?  Do the proposed experiments take
advantage of unique features of the scientific environment or employ useful
collaborative arrangements?  Is there evidence of institutional support?

The initial review will also examine: the appropriateness of proposed budget and
duration; the adequacy of plans to include both genders and minorities and their
subgroups as appropriate for the scientific goals of the research and plans for
the recruitment and retention of subjects; the provisions for the protection of
human and animal subjects; and the safety of the research environment.

AWARD CRITERIA

The earliest anticipated date of award is April 1, 1999.  Applicants should be
aware that, in addition to scientific merit, program priorities and program
balance, the total cost of the proposed project and the availability of funds
will be considered by NIH staff and the appropriate NIDR Advisory Council in
making funding recommendations.  In this regard, since the costs associated with
new equipment used to study biofilms, such as confocal scanning laser
microscopes, microbalances, microprobes, and infrared spectrometers can limit
support for research on oral biofilms, the establishment of consortial or
contractual arrangements with institutions that have these sophisticated
resources is welcomed.  In addition, the NIH values complementary funding from
other public and private sources including foundations and industrial concerns. 
In circumstances in which applications have similar scientific merit, but vary
in cost-competitiveness, the more cost-competitive application may be selected
for funding.

INQUIRIES

Written, email and telephone inquiries concerning this RFA are encouraged.  The
opportunity to clarify any issues or questions from potential applicants is
welcome.

Direct inquiries regarding programmatic issues to:

Dennis F. Mangan, Ph.D.
Division of Extramural Research,
National Institute of Dental Research
45 Center Drive, Room 4AN-32F, MSC 6402
Bethesda, MD  20892-6402
Telephone:  (301) 594-2421
FAX:  (301) 480-8318
Email:  Dennis.Mangan@nih.gov

Stephen P. Heyse, M.D., M.P.H.
Division of Microbiology and Infectious Diseases
National Institute of Allergy and Infectious Diseases
Solar Building, Room 3A32
Bethesda, MD  20892
Telephone:  (301) 496-7728
FAX:  (301) 402-2508
Email:  sh42i@nih.gov

Direct inquiries regarding grants management issues to:

Daniel Milstead
Division of Extramural Research
National Institute of Dental Research
45 Center Drive, Room 4AN-44A, MSC 6402
Bethesda, MD  20892-6402
Telephone:  (301) 594-4800
Email:  Daniel.Milstead@nih.gov

Robert Tarwater
Division of Extramural Activities
National Institute of Allergy and Infectious Diseases
6003 Executive Boulevard, Room 4A29
Bethesda, MD  20892-7610
Telephone:  (301) 496-7075
FAX:  (301) 480-3780
Email:  rt28o@nih.gov

Schedule

Letter of Intent Receipt Date:  June 23, 1998
Application Receipt Date:       July 21, 1998
Scientific Review Date:         October 1998
Advisory Council Date:          January 1999
Earliest Award Date:            April 1, 1999

AUTHORITY AND REGULATIONS

This program is described in the Catalog of Federal Domestic Assistance No.
93.121 -  Oral Diseases and Disorders Research Awards, and 93.856 - Microbiology
and Infectious Disease Research.  Awards are made under authorization of the
Public Health Service Act, Title IV, Part A (Public Law 78-410, as amended by
Public Law 99-158, 42 USC 241 and 285) and administered under PHS grants policies
and Federal Regulations 42 CFR 52 and 45 CFR Part 74.  This program is not
subject to the intergovernmental review requirements of Executive Order 12372 or
Health Systems Agency review.

The PHS strongly encourages all grant and contract recipients to provide a smoke-
free workplace and promote the non-use of all tobacco products.  In addition,
Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain
facilities (or in some cases, any portion of a facility) in which regular or
routine education, library, day care, health care or early childhood development
services are provided to children.  This is consistent with the PHS mission to
protect and advance the physical and mental health of the American people.



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