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ILAR Journal V37(2) 1995
Laboratory Animal Care Policies and Regulations

Call for Comments

Future Challenges and Opportunities for Microbial Culture Collections

INTRODUCTION

The U.S. National Committee for the International Union of Microbiological Societies (USNC/IUMS) held a one-day fact-finding meeting on March 27, 1995 to gather information about the current and potential uses of microbial collections and the challenges and opportunities they face in the future. The meeting was organized to answer questions posed by the National Research Council (NRC) Commission on Life Sciences in response to the Committee's proposal for a full-scale NRC study on microbial collections.

The Committee proposed the study after hearing reports of continuing financial difficulties of several collections in the United States and abroad. Although long-term funding has always been a chronic issue for collections, the USNC/ IUMS is concerned that substantial resources and expertise are falling into jeopardy at a time when biomedical, basic research, agricultural, biotechnological, and environmental uses of microbes are expanding.

In requesting additional information about the dimensions and magnitude of the collections situation, the Commission on Life Sciences (CLS) asked the following questions: Are funding levels for collections declining relative to past support levels? What are the indicators that under-investment may lead to negative long-term consequences'? Are there new demands being placed on collections that have increased costs or services? Is the maintenance of collections changing in fundamental ways? Have the needs of academic scientists for access to collections changed over the last decade? What is the significance of "commercial" collections to both academic and industrial scientists?

To answer these questions, the meeting brought the USNC/IUMS together with representatives from funding agencies, academic and commercial microbiologists, and curators and database developers from several different collections including the ATCC, the Drosophila collection at Indiana University, the former anaerobe collection at Virginia Polytechnic Institute, the DOE subsurface microbe collection at Florida State University, the U.S. Department of Agriculture (USDA) entomopathologic fungi collection at Cornell, and the Merck Company collection.

The one-day workshop could not completely answer all of the questions asked by the CLS in part because there are no recent systematic data on collections. Nevertheless, the meeting did elucidate major trends influencing the use of collections and the changes underway that have, on the one hand increased funding, space, and personnel problems, and on the other hand, led to the expanded substantive involvement of collections in answering research and commercial questions.

This report is a summary of the major issues presented and discussed at the meeting. It will be used as a basis for the USNC/1UMS response to the Commission on Life Sciences.

CHANGING ROLE OF CULTURE COLLECTIONS

Traditionally viewed as part of the scientific infrastructure, culture collections have long been considered a useful but peripheral tool in support of research. Over the last decade, however, collections have begun to play an increasingly diverse role within companies, research institutions, and in the scientific community at large. In their evolving roles, the expertise and information housed in collections are more closely tied to research, development, and production activities.

What is responsible for encouraging these developing roles? Throughout the workshop, several factors were reiterated as major forces changing the environment in which culture collections operate. These include the economic importance of microorganisms, the emergence of new areas of research, the effect of regulations on activities related to microorganisms, and the expansion of electronic information capabilities.

Economic Importance of Microorganisms

Microorganisms are an expanding part of the world economy and of increasing interest to the pharmaceutical industry. Antibiotic production alone provides annual worldwide revenues of over 16 billion dollars. In the last 10 years, advances in molecular biology have allowed the machinery of microorganisms to be manipulated and exploited, fostering the advent of the biotechnology industry. This 10 billion dollar industry has relied on microbes and their metabolites as the source of many of its products.

Natural products screening is a flourishing enterprise. Microbial collections, the contents of which have been carefully characterized and identified, are a source of value-added material/'or bio-prospectors--those seeking an organism or metabolic product for the burgeoning new industries of bio-sensors, bioremediation, energy conservation, environmentally friendly products, and biocontrol.

Polymerase chain reaction (PCR), a method used to amplify small quantities of nucleic acids, was developed using the DNA polymerase of Thermus aquaticus, a thermophilic organism deposited in the American Type Culture Collection in the 1960s. PCR is now widely used in basic and applied research, from diagnostics to forensics. The utilization of this unique characteristic of Thermus aquaticus has generated interest in exploring other "extremophiles" maintained at the ATCC.

The catalogue of information built by collections can provide a roadmap to researchers trying to find organisms with special properties. For example, Fujisawa was granted the patent on the compound Immunosuppressant FK506, sending its competitors scrambling to find comparable substances. Within months it was discovered that Immunosuppressant FK506 was similar to an antifungal material isolated 20 years ago from an organism that was freely available. Dr. Keith Bostian, CEO of Microcide Pharmaceuticals, estimated that at least 3 years of effort was saved by the information in collections' databases.

New Areas of Research

In addition to research related to medical and industrial biotechnology, microorganisms are also being reexamined in the context of their diversity and ecology. Investigations into the function and relationships of microorganisms to one another and to all other biota in their ecosystems will be aided by the information already recorded in collection databases. Indeed, the taxonomic expertise of curators will be challenged by the expected inundation of biodiversity samples for identification. Given the projection of enormous numbers of undiscovered organisms in the environment, collections may have difficulty meeting the needs of storing and maintaining samples. For example, special handling is needed for microorganisms found in the depths of ocean vents.

Culture collections will also play an important role in research on antibiotic resistance, the emergence of new and old diseases, and accordingly, the causes of virulence. Archival collections, sometimes regarded as microbial "museums'' are stockpiles of information waiting to be unlocked with the right scientific tools and questions. An example is the mycobacterium collection, which was rescued by the American Type Culture Collection. As tuberculosis re-emerges as a problem in the United States, these cultures are essential resources.

Regulations

Microorganisms used in industry and academia are subject to a number of national and international regulations on their use and handling. Collections are often asked to take responsibility in meeting these requirements. For example, the Food and Drug Administration (FDA) requires biotechnology firms to be able to verify the DNA sequence of any organism producing a product in a fermentation system. The collections department will often develop a standard assay to comply with this regulation.

The number of patents granted on microorganisms, from wild-type to mutants to genetically modified is growing. Any patented organism must be maintained by the patentee for 30 years (in addition to deposit in an International Depository Authority such as the ATCC or Northern Regional Research Laboratory). Thus, viability and plasmid retention checks are increasingly important work for culture collections.

Collections that ship microorganisms nationally and internationally are affected by overlapping and sometimes conflicting shipping regulations of various U.S. agencies. Collections are also sometimes required to have export licenses for international shipments.

In the case of biocontrol, both Animal and Plant Health Inspection Service of the USDA, and the Environmental Protection Agency regulate the movement and transfer of pathogens and nonindigenous organisms. Although the concerns are primarily related to industrial use and release of microorganisms, collections are caught up in the regulations even though the cultures are contained and simply sent from one qualified lab to another.

Accessibility of Information

Advances in information technology bring the possibility of greater accessibility to information as well as the ability to compare information held in different locations. Many collections have put information about their holdings on electronic databases, but going online is a more recent development that opens up the collection to a broad audience.

Examples of networks being established are the Microbial Strain Data Network, for which fees are associated; the Microbial Germplasm Database in the United States, which focuses mainly on plant pathogens; the World Data Center at RIKEN, funded by the Japanese government; the United Nations Environment Programme and the World Federation for Culture Collections; and the Brazilian Tropical Database, funded by the Brazilian government. Microbial Information Network Europe, funded initially by the CEC, now must stand on its own through user fees. The Microbial Information Network of China is a newly established entity.

Networks provide users with the means to find out about an organism and its properties, and to make comparisons with other organisms. Computer databases are most effective if the information is presented in a consistent way; there will be many challenges to accomplishing this goal in a manner that ensures the integrity of the data. Questions of who owns the data and how electronic access should be paid for must also be addressed in the future.

NEW ROLES, NEW FUNCTIONS, NEW CHALLENGES

As research on microorganisms intensifies, it is not surprising that collections and their managers have had to adapt to new developments in the field. As a result, the specific functions of collections have diversified.

Dr. Jenny Hunter Cevera of the Lawrence Berkeley Lab, and the former curator of the Cetus collection, described how the obvious function of a collection to maintain the growth of organisms overlays an expanding list of other activities, including, for example, the identification of isolates and contaminants, providing taxonomic descriptions of organisms, characterizing plasmids and hosts, comparing 16S and 23S rRNA sequences, conducting photomicroscopy, providing quality assurance and quality control, validating organisms in production and their products, determining the DNA sequence of organisms used in fermentation, fine-tuning fermentation, conducting patent deposits, shipping organisms, and constructing and maintaining computer databases, and conducting research on the organisms themselves.

Compared to the traditional notion of a microbial "warehouse," a successful modern culture collection requires an expanded and diverse source of expertise and adequate resources. In fact, according to Dr. Kathleen Matthews, curator of Indiana University's Drosophila Center, a 1993 NSF-Genetics Society Workshop found that the most successful collections shared three aspects:

  1. expert and committed management, including constant evaluation and quality control, and a knowledge of users of the collection and their research;
  2. integration with users in the community; that is, act as a center for active dialogue on microbe related issues, such as maintenance of a genetic map, nomenclature issues, databases, or newsletter; and
  3. adequate and stable funding.

OPERATIONS OF REPRESENTATIVE CULTURE COLLECTIONS

In order to hear first-hand how different kinds of collections are attempting to successfully meet the needs of the research community, the USNC/IUMS listened to five curators discuss the specific challenges and opportunities facing their collections, and the ways those issues are being addressed.

A Federal Collection

Dr. Richard Humber is the Director of the Agricultural Research Service collection of entomopathogenic fungi (ARSEF). Originally set up as a source of germplasm for biocontrol of invertebrate pests such as insects, mites, and nematodes, it holds approximately 5,000 strains. It is a potentially valuable source of compounds for pharmaceutical and biorational agricultural use, and in fact, through the Boyce Thompson Institute, ARSEF has organized cooperative research and development agreements (CRADAs) with private firms to conduct screening of the collection.

Challenges facing the ARSEF collection include funding, information resources, regulations, staffing, and decisions about accessions and deaccessions. In contrast to the plant germplasm collections of the USDA, ARSEF is maintained through research funding which does not take into consideration the burden of servicing the collection and user requests. ARSEF has a blanket importation permit and is used widely by the USDA as a quarantine facility for the importation of microbes. Meeting the screening requests of industry can also stretch the staffing and other resources of the collection.

ARSEF publishes a catalog and maintains an elaborate database, which is not yet on the Internet. The collection needs programming expertise to put the database online. Dr. Humber also noted that getting accurate geographical and host (insect) data for these organisms is a major challenge for the collection. Completion of these missing data can increase the utility of the collection.

Taxonomic skills in the area of entomopathogenic fungi is specialized and scarce. Few funds have been available to train the next generation of systematists, leaving the future care of the collection in jeopardy.

Like all collections, space at ARSEF is limited, and a critical concern is how to absorb endangered collections such as the Australian fungi collection, while trying to make more room for new isolates resulting from biodiversity research.

The American Type Culture Collection

The ATCC is often thought of as a national service collection. It is a patent repository, and one of the largest collections in the United States. It is respected and trusted by the academic and private research communities. It is the primary source of "standards" or "reference strains" for quality control, sterility testing, susceptibility testing, evaluation of drug candidates, toxicity testing, and diagnostic reagents. It is a major source of microbial, plant, and tissue cultures. The ATCC distributed 139,000 cultures in 1994.

According to Dr. Robert Gherna, head of bacteriology for the ATCC, 80 percent of the collection is not distributed. Nevertheless, in addition to maintaining these archival cultures, the ATCC continues to absorb parts of endangered or dismantled collections, such as the mycobacterium collection. How to save endangered collections is a primary concern of the ATCC, professional societies, and others.

As a non-profit organization, the ATCC survives through federal and state contracts and grants, through revenues on charges for cultures and patent deposits, and teaching courses. In 1994, the ATCC received 17 percent of its revenues from federal sources, a decline from 24 percent in 1991. The level of federal support can be contrasted to the Deutsche Sammlung von Mikroorganismen (DSM) in Germany, which gets 82 percent of funding from government, the RIKEN in Japan which is fully funded by the government, and the decentralized national collections of the United Kingdom, which receive 35 percent of their funding from government sources.

ATCC's funding concerns are based on the increased cost of personnel (due to competition from the biotechnology industry and others), its need for expansion and renovated space, new requests for storage of DNA material, the preparation of biological materials under strict quality control procedures, and the recent need to purchase cultures in demand (when once it received them gratis).

In addition to funding concerns and accession concerns, the ATCC faces new issues in its role as a patent repository. For example, the ATCC is receiving cultures for storage with restrictions on their distribution. It anticipates being involved in litigation regarding the misappropriation of a patented strain by a third party who received it from the ATCC. The ATCC is even being sued by Gulf War veterans for its distribution of cultures to Iraq, even though the Department of Commerce had issued an export license for the material.

Academic Research Collection

Dr. Edward Moore, former curator of the Virginia Technical Institute anaerobe collection which was recently dismantled, explained its history and background. The collection consisted of 60,000 cultures from clinical infections, periodontal disease, and colon cancer including 6,000 strains not available elsewhere and some isolated before World War I when antibiotics were not in wide use. Dr. Moore estimated that it took 300 person-years of work to build the collection, which was used to develop positive identification techniques for anaerobes.

Dr. Moore was able to distribute most of the collection to the Centers for Disease Control, the ATCC, a dental lab in Boston, and a company called Microbial I.D. Inc. Some of the strains, however, were discarded.

The situation of the Virginia Tech anaerobe collection is representative of many academic collections. Dr. Moore was about to retire when it became clear that the University did not have plans to continue support for the collection. Yet the collection itself was built on publicly funded grants.

Biodiversity Collection

The Department of Energy's subsurface microbial collection housed at Florida State University (FSU) contains microbes isolated from deep aquifer coastal plain sediments. At the time of its inception, it was thought that the unusual locations might reveal novel organisms with interesting properties.

According to Dr. David Balkwill, the collection's curator, the collection has received substantial inquiries from private industry anxious to look for new products. The biggest challenge facing the collection is that it is too young, that is, most of its cultures are uncharacterized microorganisms. Lack of data diminishes the value of the strains to industry, which would have to pay $25-40 per culture not knowing what it was getting or even if many of the cultures are duplicates. Therefore, a critical issue for the collection is developing fast identification techniques. Much of the work being done at FSU is the design of molecular probes to help determine the phylogeny of the cultures.

A second important issue is that most of the funding for the collection is from the Department of Energy, a situation that is very unstable. Like the VPI collection, there is no guarantee that the University will continue to "host" the collection. The need to diversify funding is therefore critical.

An Industrial Collection

The Merck Co. culture collection for antibiotics was started in 1954 to conduct natural products screening and to have a taxonomic capability for patent purposes. It is one of two major collections at Merck, the other being clinical microbiology.

The antibiotics screening collection holds 17,665 pro-karyotic and eukaryotic strains, mostly in lyophilized and frozen forms. It serves as an archival collection, as a depository for Merck's patented strains, as a disaster protection backup for Merck's production line, and as a research center. The collection maintains and characterizes cultures, checks purity and viability, and is the hub for distribution of microorganism inside and outside the company. The collection maintains historical information, growth requirements, utility and other strain data on an internal database. Dr. George Garrity, curator of the collection, estimates that the value of the services it provides to the company is directly around 3.15 million dollars (based on a comparison with ATCC prices). The collection is the basis for many company products.

Nearly one-third of the cultures have come from outside the company, for example, 2000 strains from the ATCC, 600 from the NRRL, others from the U.S. Army, and from universities.

Dr. Garrity noted that the most important challenges to the Merck collection in the future will be finding trained individuals, meeting user needs, keeping up with fixed costs, and keeping management aware of what the collection does and its importance in maintaining the rigor of science, product development, and quality control.

IS A NATIONAL RESEARCH COUNCIL STUDY WARRANTED?

In a final roundtable session of the meeting, participants were asked what would be accomplished by an NRC study. Aside from the specific challenges faced by individual collections, the participants focused on the contribution of an NRC study to the examination of broader policy issues such as the effects of underinvestment in collections, funding and priority issues, and on steps to capitalize on what collections have to offer.

Underinvestment

Although there was disagreement about which parties should carry the burden of collections expenses, there was a strong sentiment among many of the workshop participants that the United States, as a whole, was undervaluing its microbial germplasm collections.

Loss of germplasm. Indicators of this underinvestment included the loss of important germplasm such as that in the Virginia Tech collection, a scenario being repeated frequently across the country and the world. Meeting participants were unable to quantify the loss, however, as a national registry of collections is lacking. It was noted that past efforts to obtain modest funding to establish such a registry were unsuccessful.

Another area of concern is the purchase of entire academic collections by private industry, both domestic and foreign, often inexpensively. That industry might benefit from collections is not the issue. Rather, the fear is that industry might not have the expertise to handle the materials, that access to the cultures will be curtailed, and that resources built with public funds have been transferred to the private sector with too little return.

The latter point leads to the question of who owns a collection (university, individual researchers, federal government) and to the undervalued status of microbial collections at most universities. For example, in one anecdote related at the meeting, Rutgers University was unaware that one of its collections had been dismantled and distributed several years prior by its retiring curator when it established a Center of Biodiversity Research and began frantically looking for the materials.

Lack of trained microbiologists. Another indicator of underinvestment is the lack of adequately trained next generation curators. As one participant noted, taxonomists are a dying breed. The fact that industry must often "contract out" to consultants for microbiological advice signifies a lack of professionals trained not only in classical molecular biology techniques, but with a system of characterization that includes biochemistry, taxonomy, and molecular biology. It was noted at the meeting that a "Microbiology 101" course was no longer offered in many academic institutions. Students may never be taught even the elementary technique of how to transfer a culture. The United States has fallen behind other countries in bringing its students into the microbiology field. A related issue is that manuals on how to preserve germplasm are virtually nonexistent.

Nonstandard materials in circulation. An additional indication of underinvestment in collections is the exchange of nonstandard culture materials by researchers. This practice, which can affect the integrity of data, is possibly due to the increased cost of obtaining standard cultures. The ATCC's decision to increase the prices of cultures for both academic and industrial purchasers 10% over the last 4 years and 5% this year is a result of increasing costs and declining revenues. The ATCC's prices must also compete with commercial germplasm distributors who buy the most popular strains and grow and resell them, without the burden of maintaining large numbers of infrequently distributed cultures.

Poor public image. The level of public knowledge about microorganisms and the significance of their storage in collections continues to be a concern. Although the public hears about thc emergence of new diseases such as the Hanta viruses and HIV, the reemergence of disease such as the plague and tuberculosis, of the growing problem of antibiotic resistance, and of deaths from contaminated food products, workshop participants felt that it was unlikely that the public understands and appreciates the role of microbiology and of collections in addressing these problems.

Some participants argued that this importance was not understood even by the federal agencies that support collections. Collections remain labeled as infrastructure, and as infrastructure, are easy to underfund.

Awareness of the potential. In some cases, opportunities for collections to make a vital contribution are simply missed. For example, Dr. Milton Friend of the National Biological Service explained that the crane population in the United States has been affected by an epivirus believed to be an exotic organism responsible for crane deaths in Austria, Russia, and Japan. Because these organisms have never been cultured and maintained, there is no way to make a comparison.

Similarly, another major disease of water birds is avian botulinum. The Fish and Wildlife Service, in isolating Clostridium botulinum, also finds many botulism-inhibiting microbes. In addition to their potential role in finding the solution to the waterfowl condition, the organisms also have economic potential for the canning industry. Again, however, there is no systematic maintenance of these isolates.

Funding Issues

The rational basis for the sponsorship of collections by the federal government must be examined in relationship to benefits received and contributions made by other parties, including industry, universities, and researchers themselves.

Some determination of an appropriate level of investment in collections should be developed, whether that should be a national level in relation to other nations' spending, the contribution of thc resource to the economy, or relative to spending on research grants. The National Science Foundation, for example, supports 18 major collections at a cost of $3.5 million annually, or 3% of what is spent on research grants. Is this appropriate?

If there is indeed a role for federal sponsorship, the needs of those agencies in making coordinated, strategically sound funding decisions should also be addressed, including questions on the basis for initiating support for a collection and what a "phase-out" policy should be. Collections supported by public funds should be evaluated upon loss of funding or personnel.

The establishment of alternative sources of funding for collections should also be examined seriously, including the concept of an endowment, or a "royalty fee" fund from revenue on products developed using microorganisms. The potential for interaction with industry screening programs should also be examined.

In academe, the initiation of a collection implies a long-term commitment of which the curator and universities must be aware. An NRC study might help to formulate guidelines for planning a collection's "life-cycle." Although collections may be in existence an indeterminate number of years, plans on how the collection should be dismantled is an issue that should be examined from the start. Collections, and the universities that house them, must be accountable to their sponsors. How the collection will attempt to maximize its utilization by the broader community, and at what cost, should also be examined.

For example, neither users nor sponsors can expect that collections can house all the microbial diversity in the world. Although obviously an important part of understanding the working of ecosystems, the actual potential for holding all strains must be put into rational perspective. As one meeting participant put it, how can we seek funds for preserving biodiversity, when we can't afford to keep the biodiversity we already have preserved?

Scientists also need to develop consensus on the sharing of cultures mentioned in journal publications. The deposit of organisms with restrictions on their use (outside of patent rights) has serious ethical implications for research practice.

Steps to Maximize Use and Benefits of Collections

Throughout the workshop, participants identified many ways in which the contribution of collections could be enhanced to fully realize their potential. Many of these suggestions have applicability beyond collections, because they are tied to research and commercial interests as well. The are briefly listed here.

Need for centralized information. The absence of a comprehensive national or international registry of collections makes information gathering about the numbers of collections and their contents difficult. A registry would be a valuable tool, not only for the evaluation of microbial resources, but for the academic and industrial research communities, for sponsors of collections, and for collections themselves.

Collections related research. If collections are to be exploited for what they can provide, collections-related research should be expanded. The benefit of these kinds of research questions is that they have a much broader application than for collections. A study might consider the contributions of research on:

International/Regulatory Issues. Some attention should be given to issues that impede or have an impact on culture collections, such as:

CONCLUSION

The information presented at the meeting on the future challenges and opportunities for culture collections provided a glimpse of the potential impact of these institutions on academic research and commercial prospects. In an era of budgeting restraint, this potential may only be actualized if a national (and perhaps international) perspective on their effective use can be developed. For this reason, the U.S. National Committee for IUMS believes that an NRC study focusing on the issues related to collections may enhance the productive and dynamic capabilities of these important resources.

For more information contact Robin Schoen, Commission on Life Sciences, National Research Council, 2101 Constitution Avenue, N.W., Washington, DC 20418. Tel: 202-334-2233; Fax: 202-334-1687; E-mail: rschoen @ nas.edu.





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