Board of Governors, American Academy of Microbiology

Eugene W. Nester, Ph.D. (Chair) University of Washington

Kenneth I. Berns, M.D., Ph.D. Mount Sinai Medical Center, New York

James E. Dahlberg, Ph.D. University of Wisconsin, Madison

Arnold L. Demain, Ph.D. Drew University

E. Peter Greenberg, Ph.D. University of Iowa

J. Michael Miller, Ph.D. Centers for Disease Control and Prevention

Stephen A. Morse, Ph.D. Centers for Disease Control and Prevention

Harriet L. Robinson, Ph.D. Emory University

Abraham L. Sonenshein, Ph.D. Tufts University Medical Center

David A. Stahl, Ph.D. University of Washington

Judy A. Wall, Ph.D. University of Missouri

Colloquium Steering Committee

J. Willliam Costerton, Ph.D. (Co-Chair) Montana State University

E. Peter Greenberg, Ph.D. (Co-Chair) University of Iowa

Anne K. Camper, Ph.D. Montana State University

Clay Fuqua, Ph.D. Indiana University

Paul E. Kolenbrander, Ph.D. National Institutes of Health

Roberto G. Kolter, Ph.D. Harvard Medical School

James A. Shapiro, Ph.D. University of Chicago

Carol A. Colgan American Academy of Microbiology

Colloquium Participants

Douglas H. Bartlett, Ph.D. Scripps Institute of Oceanography

Anne K. Camper, Ph.D. Montana State University

J. Willliam Costerton, Ph.D. Montana State University

Rodney Donlan, Ph.D. Centers for Disease Control and Prevention

Gary M. Dunny, Ph.D. University of Minnesota

Clay Fuqua, Ph.D. Indiana University

E. Peter Greenberg, Ph.D. University of Iowa

Jo Handelsman, Ph.D. University of Wisconsin, Madison

Caroline S. Harwood, Ph.D. University of Iowa

Barbara H. Iglewski, Ph.D. University of Rochester Medical School

Howard F. Jenkinson, Ph.D. University of Bristol Dental School, UK

Heidi B. Kaplan, Ph.D. University of Texas Medical School

Paul E. Kolenbrander, Ph.D. National Institutes of Health

Roberto G. Kolter, Ph.D. Harvard Medical School

Howard K. Kuramitsu, Ph.D. State University of New York, Buffalo

Jared R. Leadbetter, Ph.D. California Institute of Technology

Dennis Mangan, Ph.D. NIDCR, National Institutes of Health

Soeren A. Molin, Ph.D. Technical University of Denmark

Leland S. Pierson, Ph.D. University of Arizona

James A. Shapiro, Ph.D. University of Chicago

Joan Strassmann, Ph.D. Rice University

Graham C. Walker, Ph.D. Massachusetts Institute of Technology

Stephen C. Winans, Ph.D. Cornell University

Fitting Microbial Ecology Into Macroecology Paradigms

A communication gap has developed between microbiologists and ecologists, occasionally growing so wide that some in ecology have claimed that microbial ecology falls short of “true” ecology. However, microbial communities have been found to adhere to the same basic rules of ecology that macro-communities follow, including the maintenance of different levels of organization, temporal progression, and the existence climax communities. However, there are characteristics inherent to microbial communities that are due to the small distances and microenvironments relevant to microbes.

Another issue is the concern that microbes may engage in horizontal genetic exchange within communities. These attributes set microbial communities apart from communities of metazoan organisms and require that general ecological principles be adapted to the particular idioms of microbial ecology.

Resilient and Stable

One of the more fascinating attributes of microbial communities is the paired properties of resiliency and stability they exhibit. Microbial communities are capable of recovering from and adapting to radical habitat alterations by altering community physiology and composition. In this way, they are able to maintain great stability in structure and function over time. Environmental alterations bring about community change by impacting individual components of the community, but the results are manifested at the level of the whole community. It is tempting for microbiologists to attribute community stability to genetic diversity and functional redundancy, but experimental work has not yet proven this principle to hold true in every situation.

Microbial communities share the ability to maintain biological stability, known as homeostasis, with other biological and ecological entities. Microbes within communities are often buffered from changing environmental conditions, but when exposed to conditions that exceed this adaptive tolerance, a microbial community will destabilize. These conditions, which can include nutrient starvation, viral infection, and other environmental insults, can be viewed as catastrophes for the community. Human activities, in particular, can be responsible for destabilization of communities. For example, supplying a diet of grain to cattle has been shown to lead to a lower rumen pH, which alters the gut community and fosters the growth of pathogenic strains like Escherichia coli 0157:H7. Oftentimes, a radically different microbial community may take the place of the former under the new conditions.

Development

Mechanisms for Positioning Cells

Within many kinds of microbial communities, secondary structure can buffer sensitive members from environmental fluxes for sensitive members, maximize the ability of two or more species to carry out coupled functions, and create micro-environments conducive to exotic, localized forms of metabolism. For some communities, this structural organization is apparently governed on a community-wide scale, via mechanisms like quorum sensing, in which a signal from every member of the community contributes to the architecture of the community. In other communities, complex configurations can come about in seemingly uncontrolled ways that are actually closely guided by pair-wise interactions. For example, the communities harbored in termite guts that allow the insects to digest cellulose are highly structured, but it is thought that this organization results from small-scale interactions, rather than from an overall architectural plan.

In order to achieve the appropriate secondary structure for a community, cells must position themselves, moving within the community to the appropriate locations. It is likely that many different mechanisms control this cellular positioning, but most communities apparently employ chemical gradients to dictate the appropriate movements. An example of gradients in action is the migration of cells within microbial mat communities. Microbial mats are characterized by steep gradients of sulfide and oxygen. Sulfide-oxidizing bacteria, like Beggiatoa, position themselves along this gradient to optimize their ability to make energy from transferring electrons from sulfide to oxygen. At night, when oxygen is limiting in the mat, Beggiatoa glide close to the surface, where atmospheric oxygen is available. During the day, when oxygen is produced by photosynthetic members, oxygen is present deeper in the mat, and Beggiatoa glide downwards, where oxygen and sulfide concentrations are optimal.

Scales of distance

The distances between cells in a microbial community can be critical to community functions, and distances ranging from the length of the entire community down to the length of an individual cell can play a role. Many communities require that very short distances be maintained between cells. Myxococcus, for example, requires “head-to-head” contact between cells in order to initiate fruiting body development. Likewise, the collaborative interspecies hydrogen transfer between methanogens and ethanol fermenters requires extremely close contact, as do all known communities that carry out interspecies hydrogen transfer.

In Situ Techniques for Examining Microbial Communities

Two distinct types of in situ techniques are available: nondestructive real-time monitoring and destructive techniques that kill the bacteria while retaining the original structure of the community. To study microscopic biofilm communities, both types of methods are heavily dependent on confocal scanning laser microscopy imaging techniques. In destructive techniques, cells from any microscopic microbial community can be fixed and embedded in resin prior to staining and imaging. In non-destructive techniques, the community must be grown in a flow cell that allows microscopy of the living community from early colonization through to the achievement of a climax community. Among nondestructive in situ techniques for studying microbial communities, optical fiber imaging holds particular promise for the field. Advanced fiber optical imaging systems are being developed that can provide a confocal image, allowing a three-dimensional view of the community. Advances are also being made in the resolution and depth that can be achieved by the technique.

Both nondestructive real-time monitoring and destructive techniques are critically important to the study of microbial communities. However, there is a tendency for these approaches to be descriptive or qualitative and poorly controlled. Efforts are being made to develop more rigorous techniques that allow quantitative measurements and appropriate controls while retaining the structure and viability of the community under study. It must also be kept in mind that in situ techniques like these are a complement to invasive, reductionist approaches, not a replacement for them. Destructive techniques that dissect the community are of equal importance and need to be incorporated into the experimental design.

Molecular Techniques

The use of molecular techniques has radically changed the practice of microbiology research, allowing fine-scale analyses of microbial communities that were never before possible. Within the past 20-30 years, microbiologists have implemented these powerful genetic and biochemical techniques, shedding light on the development of microbial communities, the relationships within communities, and the contributions of individual populations. Some specific issues and cautions associated with the use of individual molecular techniques are discussed below.

Technologies Needed to Advance the Field

There are many areas of microbial communities research that require development of new analytical methods. For example, new staining and imaging approaches, coupled with improved confocal microscopes that allow more rapid data collection, would provide clearer views of the physical structures of microbial communities. The lack of methods for analysis of cell surface structures, such as matrix material within biofilms, is severely limiting for the field and presents an area that should be developed. Moreover, tools for rapid diversity analyses, like microarrays that allow assessments of phylogenetic composition within communities, are needed.

Improvements of existing analytical methods are also necessary. For example, enhancement of noninvasive, microscale sampling and physical measurements should be explored. In research of microbial biofilms, the shortage of available technologies often limits the rate of advancement in the field, and more focus on methods and technology development for this area in particular is warranted.

TABLE 2.

WIDER APPLICATION OF TECHNOLOGIES TO BENEFIT MICROBIAL COMMUNITIES RESEARCH.

What are the Contributions of Not Yet Cultivated Microorganisms?

The issue of uncultivated organisms rings loudly in the ears of all microbial ecologists, particularly those concerned with the interactions of cells in communities. It has been estimated that as many as 99% of all microbial species have not yet been grown in culture, and many may, in fact, be resistant to cultivation by available techniques. As a result, there are limited opportunities for studying the genetic, biochemical, and metabolic capacities of the vast majority of single-celled organisms. This discrepancy—that most of the microbes in the world cannot be studied in fine detail in the lab—is at the root of what may be an immense gap in our understanding of the microbial world.

Oral biofilms offer a good demonstration of this gap in the current knowledge. When viewed with a microscope, it is clear that half of the organisms in dental plaque are spirochetes, with distinct spiral-shaped morphology and corkscrew motility, but most of the identified cell types have not yet been grown in the lab. Many such uncultivated organisms are apparently active in the community, and they likely play a role in oral health. In many communities, including the oral flora, it is not known whether active but uncultivated microbes contribute a large extent or a small percentage to community activity, and it remains a source of active debate and investigation.

A number of explanations have been proposed for the apparent unculturability of many microbial species. First, it is highly likely that many microbes are, in fact, culturable through the use of current technology, but they resist cultivation because the precise growth conditions have not been made available. Some recent successes bear this theory out. In the case of Campylobacter, it was only lately understood that micro-aerophilic conditions are needed for its growth. A recent study evaluated the percentage of clinical vaginal swabs from which Staphylococcus aureus can be grown, and showed that 90% of swabs were reported back as negative for this species. However, when growth plates were incubated for a longer period than that originally dictated by the clinical protocol, the organism was found to have developed from many previously “negative” samples; this almost universal presence of this potential pathogen was confirmed in a separate study by the use of FISH probes and PCR. In this case, the culture conditions were defined by clinicians’ need to get fast results, but they limited the validity of the final result.

Another cause for poor culturability may be that critical elements present within the microbial community environment are lacking in traditional cultivation techniques. Current cultivation techniques will need improvement if investigators hope to capture the organisms that require the nutritional, chemical, or physical support of the surrounding microbial community.

It is important to distinguish between metabolically active microbes that cannot be cultivated due to our limited knowledge of their physical and nutritional requirements and those organisms that are quiescent or dormant and cannot be revived in culture. Clearly, microbes in the first category are expected to have enormous impacts on the activity of their respective microbial communities. Microbes that fall in the second category may also impact the community, as they may contribute to the structure of biofilms, for example, but do not deplete resources. Dead cells may, in fact, release nutrients into the community that support the growth of other members.

More research is needed to investigate the contribution of non-cultivatable cells within microbial communities. Fortunately, we now have some tools that allow for this type of research. The application of meta-genomics approaches, in which DNA is extracted from a total sample without separating out the individual organisms and the resulting library of genetic sequences is analyzed, is proving fruitful, and driving new questions for research. For example, research has shown that a meta-genomic library of soil microorganisms that is probed for 16S ribosomal RNA (rRNA) sequences may contain up to 25-30% Acidobacter sequences. Only three members of this genus have been cultured, leaving investigators with many questions (and few answers) regarding the metabolic role of these organisms in soil. An important caveat when interpreting non-cultivation based discoveries like this is the all too frequent disconnect between genetic fingerprints and metabolic capabilities. The genetic sequences of marker loci like the 16S rRNA gene are only hints about the phenotypic characteristics of a given organism. Nevertheless, meta-genomics and other molecular techniques will allow investigators to make major inroads into the world of viable, but not yet cultivated, microbes.

For many years, microbiologists relied solely on traditional culture-based approaches, in which only those microorganisms that could be grown in a lab were used to extrapolate to the whole of microbial diversity and microbial communities. Over-reliance on these techniques has likely introduced a number of misunderstandings with regard to the in situ activity of microorganisms. The use of cultivation-based techniques in modern-day research should be examined carefully, and extraneous experimentation that would lead only to conclusions relevant to the bench-top behavior of pure-culture microorganisms should be limited. The focus of current and future research should be on the wider world of microbial diversity, and should not be limited to those few non-representative organisms that are easily maintained in the laboratory environment.

Enhanced Resistance to Antimicrobials in Microbial Communities

Biofilm microbial communities often exhibit heightened resistance to antimicrobial treatment as compared with their free-living counterparts. Antimicrobial resistance in biofilms and other microbial assemblages is a critically important issue and has extensive practical implications for medicine, industry, and the environment. It is widely agreed that the field merits a great deal more experimental work.

Who is a member of the community and who isn't?

One point that is so fundamental to the topic of microbial communities that is often overlooked is the question of how to define the limits of a microbial community. In the environment, functionally defined microbial communities exist in continuity with one another, and the distinctions between them blur. It is difficult to delimit these communities in almost any habitat, separating the cells and species that are members from those that are not. Resolving this issue should be a key theme for discussions among investigators, and an attempt should be made to come to some agreement on what defines a member of a given microbial community.

What are the right model systems for community interactions?

A major missing component in microbial community research is the development of one or a few good model systems to determine the fundamental mechanisms at work in communities. An ideal model system would be comprised of well-characterized individual members that together accomplish functions that are beyond the capabilities of the individual species. While the study of this model system should not preclude investigating the diversity of microbial communities, some significant effort should be directed towards developing one or more systems. Initially, work should focus on characterizing communities with only two member species. Chlorochromatium is an excellent candidate for such a model. Once inroads have been made into understanding two-member communities, further research can branch into investigating multi-species communities. Multispecies candidates include dental communities, microbial mats, termite or gypsy moth gut communities, and the communities that inhabit the rumens of cows.

The Role of Multidisciplinary Collaboration

Microbial communities are complex and phenomena related to their development, function, dynamics, and impacts are not limited to the domain of any single traditional scientific discipline. Hence, research into microbial communities requires the intellectual and technical skills of professionals from many different fields, including microbiology, ecology, medicine, population biology, environmental chemistry, molecular biology, biochemistry, soil science, plant science, hydrology, geology, engineering, and others. Because of the breadth of skills required in this field, the development of multidisciplinary collaborations is strongly recommended.

Table 3 identifies pairs of traditional disciplines that are particularly well suited to collaborative efforts and areas of research that are best suited to these types of investigations. Interest in collaboration depends to a large extent on attracting diverse researchers to this arena and may be accomplished through seminar or symposium sponsorship at large scientific meetings, such as the meetings of the American Society for Microbiology (ASM). Recruitment may also be accomplished by individual scientists, who, already engaged in research on microbial communities, recognize persons outside the field who may contribute and propose collaborations. However, successful collaborations can only be established if financial resources are available to support them. Scientists need to encourage funding agencies and corporations to both recognize the need for multi-disciplinary work and to encourage joint projects by funding specific grants and research.

TABLE 3.

COLLABORATIVE OPPORTUNITIES IN MICROBIAL COMMUNITIES RESEARCH.

Education and Training

In addition to multidisciplinary research efforts, the complexity of microbial communities necessitates multidisciplinary education and training. In the future, more students at the undergraduate and graduate levels should be exposed to the “communities view” of microbial biology. Moreover, students should receive integrated training in order to develop experience in multiple relevant disciplines.

Small meetings, such as ASM and Gordon conferences, have advanced the field of microbial communities research and education and their continued support is encouraged. Furthermore, short courses for graduate students and early faculty that incorporate the study of microbial communities have proven useful.

International Collaboration

International collaboration between scientists active in the field of microbial communities research is highly desirable. Scientific research should have no borders, and international collaborations should be fostered, particularly in the study of microbial communities, which holds great significance the vitality of the planet and all the world's citizens.

There is a need for more direct mechanisms of exchange among international laboratories, such as exchange programs for graduate students and postdoctoral fellows and increased support for visiting international professorships. European efforts of this type, which include money for travel and meetings among collaborating scientists, could serve as examples. Institutions that might sponsor such international collaborations include the Fogarty Center at the National Institutes of Health, the World Health Organization (WHO), and the Gates Foundation. Biofilm workshops throughout the world sponsored by the National Science Foundation have been successful in advancing international research on the topic, and more ventures like them should be encouraged.

Communicating the Importance and Practical Benefits of Research on Microbial Communities to the Public

Scientists need to communicate to the public the crucial role of microbial communities in everyday life. From the major biogeochemical cycles to medical implications to the possibilities for new commercial applications, microbial communities impact our lives in innumerable ways, and a greater awareness of this fact among the lay public would foster a greater ability to enhance the well-being of humans and the environment.

Importantly, these functions and potential applications are accomplished by microbes about which we know little, that have never been cultivated, and that operate in nature not as single species or individuals of a species, but in aggregates or communities. With greater recognition of the importance of microbial communities will come greater support in the public sector for the research that fills these knowledge gaps and improves the human condition.

In certain areas, improved understanding of microbial communities can directly benefit the public. For example, in the medical profession, disease is now recognized as a perturbation of the natural state, and microbial communities frequently play a role in maintaining that state. A grasp of this role can guide patients and consumers in preventing harmful perturbations in their own bodies, directed by the insight that maintaining a healthy flora is more attractive than medicines to amend perturbations.

In addition to receiving information about benefits to health and the environment, the public should be made aware that microbial communities comprise a vast, untapped commercial resource. The potential applications of microbial community functions are numerous and are limited only by the imaginations of scientists and engineers. Possible uses of microbial communities include large-scale applications for pollution reduction and water and sludge treatment. One can envision a next technological revolution after the electronic software revolution—a “bioware” revolution with microbial community products effectively reducing biofouling and corrosion, drug discovery, and application of mixed communities in industrial fermentation processes. Microbial processes can be exploited to their full potential if scientists can identify the basic mechanisms of community establishment, function, and maintenance.

Improved public communication of current understanding of microbial communities can be carried out by non-academic science advisory groups, academic researchers, and government agencies. Preparing and disseminating documents designed for public information purposes, such as this colloquium report, are crucial to this effort. The Internet is a particularly powerful tool for disseminating information. Individual researchers could make information available to an extremely large audience by posting websites on their own work or on the general topic of communities. These websites should be written in lay language, with visual aids and interactive elements.

Educational materials, like television programs and articles in popular magazines, can also be used to educate the public. When targeting young audiences, microbiology education needs to incorporate up-to-date subject matter regarding microbial communities so that young students will be familiar with these ideas as they are trained. In school, science laboratory exercises should include investigations of microbial communities.

Scientific societies are perfectly positioned to support education and communication activities, and they should be encouraged to continue to hold meetings on microbial communities. Both symposia at national scientific meetings, which communicate to fellow scientists, and meetings targeted to the public, which can attract journalists from the popular media, are helpful in educating a wide audience.

Recommendations for Research

• Determining the contributions of organisms that cannot be cultured in the lab to the development, structure, and function of microbial communities should be an overriding theme for future research in this field.
• Understanding the reasons for enhanced antibiotic resistance of microbial communities is pivotal to managing persistent microbial community infections.
• Development of suitable model systems for the study of microbial communities would prove profitable to the field by enabling a thorough understanding of the underlying order and processes at work in these complex and dynamic systems.

Recommendations for Education and Collaboration

• The phenomena relevant to microbial communities research are not the exclusive realm of any single scientific discipline. Multidisciplinary collaboration among scientists from many fields is most conducive to making important contributions in the areas where the current knowledge is weakest.
• International collaborations are critical if the strengths of research programs in the various nations involved in the field are to be used to their full potential.
• The public is largely unaware of the innumerable impacts that microbial communities have on daily life. Improving the education of the public through the use of publications, television, and the Internet is encouraged so that the public can come to recognize the importance of continued research in the field.