Saccharomyces cerevisiae risk assessment (1997)

Saccharomyces cerevisiae Final Risk Assessment

ATTACHMENT I--FINAL RISK ASSESSMENT OF
SACCHAROMYCES CEREVISIAE
(February 1997)
I. INTRODUCTION
Saccharomyces cerevisiae has an extensive history of use in the area of food processing. Also known as Baker's Yeast or Brewer's Yeast, this organism has been used for centuries as leavening for bread and as a fermenter of alcoholic beverages. With a prolonged history of industrial applications, this yeast has been either the subject of or model for various studies in the principles of microbiology. Jacob Henle based his theories of disease transmission on studies of strains of Brewer's Yeast. Currently, S. cerevisiae is the subject of a major international effort to characterize a eucaryotic genome (Anderson, 1992).
History of Commercial Use and Products Subject to TSCA Jurisdiction
Saccharomyces cerevisiae, in addition to its use in food processing, is widely used for the production of macromolecular cellular components such as lipids, proteins including enzymes, and vitamins (Bigelis, 1985; Stewart and Russell, 1985).
The Food and Drug Administration rates Brewer's Yeast extract as Generally Recognized as Safe (FDA, 1986). Furthermore, the National Institutes of Health in its Guidelines for Research Involving Recombinant DNA Molecules (DHHS, 1986) considers S. cerevisiae a safe organism. Most experiments involving S. cerevisiae have been exempted from the NIH Guidelines based on an analysis of safety (see Appendix C-II of the NIH Guidelines). While alcoholic beverages, vitamins, and bread leavening are covered under the Federal Food, Drug and Cosmetic Act, the production of enzymes and other macromolecules may be subject to TSCA regulation. The abundance of information on S. cerevisiae, derived from its role in industry, has positioned it as a primary model for genetic studies and, by extension, as a strong candidate for genetic manipulation for TSCA applications (Dynamac, 1990).
II. IDENTIFICATION AND CLASSIFICATION
A. Taxonomy and Characterization
Saccharomyces cerevisiae is a yeast. The organism can exist either as a singlecelled organism or as pseudomycelia. The cells reproduce by multilateral budding. It produces from one to four ellipsoidal, smoothwalled ascospores. S. cerevisiae can be differentiated from other yeasts based on growth characteristics and physiological traits: principally the ability to ferment individual sugars. Clinical identification of yeast is conducted using commercially available diagnostic kits which classify the organism through analysis of the ability of the yeast to utilize distinct carbohydrates as sole sources of carbon (Buesching et al., 1979; Rosini et al., 1982). More recently, developments in systematics have led to the design of sophisticated techniques for classification, including gasliquid chromatography of lysed whole cells (Brondz and Olsen, 1979).
As a result of the application of newer techniques arising from innovative approaches, the taxonomy of Saccharomyces is subject to greater scrutiny. The initial classification was based principally on morphological characteristics with specific physiological and biochemical traits used to differentiate between isolates with similar morphological traits. Using these criteria, there are as many as 18 species listed in the literature. In addition, what had been classified as one large heterogeneous species, S. cerevisiae, may, in the future, be divided into four distinct species based on DNA homology studies. The four species are S. cerevisiae, S. bayanus (also known as S. uvarum), S. pasteurianus (also known as S. carlsbergensis), and S. paradoxus. All four represent industrially important species. None of these organisms or other closely related species has been associated with pathogenicity toward humans or has been shown to have adverse effects on the environment.
Any assessment of Saccharomyces must take into consideration the malleability of the current classification. For this assessment of S. cerevisiae the reviews of the organism are based on the classification proposed by Van der Walt (1971).
B. Related Species of Concern
None of the above strains or other closely related species has been associated with pathogenicity toward humans or has been shown to have adverse effects on the environment.
III. HAZARD ASSESSMENT
A. Human Health Hazards
1. Colonization and Pathogenicity
S. cerevisiae is a commonly used industrial microorganism and is ubiquitous in nature, being present on fruits and vegetables. Industrial workers and the general public come into contact with S. cerevisiae on a daily basis through bothinhalation and ingestion (see section IV). Saccharomyces spp. are frequently recovered from the stools and throats of normally healthy individuals. This indicates that humans are in constant contact with these yeasts.
There are individuals who may ingest large quantities of S. cerevisiae every day, for example, people who take the yeast as part of a "health food" regimen. Therefore, studies were conducted to ascertain whether the ingestion of large numbers of these yeasts might result in either colonization, or colonization and secondary spread to other organs of the body. It was found that the installation of very large numbers of S. cerevisiae into the colons of animals would result in both colonization and passage of the yeasts to draining lymph nodes. It required up to 10¹⁰ S. cerevisiae in a single oral treatment to rats to achieve a detectable passage from the intestine to the lymph nodes (Wolochow et al., 1961). The concentrations of S. cerevisiae required were well beyond those that would be encountered through normal human daily exposure.
S. cerevisiae is not considered a pathogenic microorganism, but has been reported rarely as a cause of opportunistic infections. Eng et al. (1984) described five cases of such infections and reviewed the literature on eight other S. cerevisiae infections (also briefly reviewed by Walsh and Pizzo, 1988). All of the patients in the cases had underlying disease. Some of them had also received antibiotic therapy, thereby suppressing normal bacterial flora and allowing mycotic organisms to become established.
A low concern for the pathogenicity of S. cerevisiae is also illustrated by a series of surveys conducted at hospitals over the last several years. S. cerevisiae accounted for less than 1% of all yeast infections isolated at a cancer hospital and in most of the cases the organism was isolated from the respiratory system (Kiehn et al., 1980). At YaleNew Haven Hospital over the past five years, there have been 50 isolates of S. cerevisiae recovered from patients; however, most of the isolates were considered contaminants (Dynamac, 1991).
2. Toxin Production
There have been no reports of isolates of S. cerevisiae that produce toxins against either humans or animals. However, S. cerevisiae has been shown to produce toxins against other yeasts. These toxins, termed "killer toxins", are proteins or glycoproteins produced by a range of yeasts. The yeasts have been genetically modified to alter activity and are used in industrial settings as a means of controlling contamination of fermentation systems by other yeasts (Sid et al., 1988).
3. Measure of the Degree of Virulence
A number of individual virulence factors have been identified as being associated with the ability of yeasts to cause disease. The principal virulence factors associated with yeasts appear to be phospholipase A and lysophospholipase. It is believed that these enzymes enhance the ability of the yeast to adhere to the cellwall surface and result in colonization as a first step in the infectious process. Nonpathogenic yeast had considerably lower phospholipase activities. Of a wide range of fungi assayed for phospholipase production, S. cerevisiae was found to have the lowest level of activity (BarrettBee et al., 1985). Therefore, based on the phospholipase virulence factor S. cerevisiae is considered a nonpathogenic yeast.
A second factor associated with virulence in yeast is the ability of a fungus to impair the host's immune capabilities. The cell walls of most fungi have the capacity to impede the immune response of the host. In a study to determine the overall pathogenicity of a number of yeasts used in industrial processes, animals exposed to both high levels of S. cerevisiae and cortisone demonstrated a greater ability of the fungus to colonize compared with those animals treated with only the yeast. However, the animals suffered no illeffects from exposure to S. cerevisiae (Holzschu et al., 1979). Therefore, this study suggests that even with the addition of high levels of an immunosuppressant agent, S. cerevisiae appears to be nonpathogenic.
4. Ability to Transfer Virulence Factor Genes
S. cerevisiae does not carry virulence factors to humans or animals. However, the species does carry linear, doublestranded plasmids which can be transmitted to other Saccharomyces. These plasmids carry genes that encode the "killer toxins" discussed above can be transferred from one Saccharomyces to another. Therefore, gene constructs involving the incorporation of traits using these linear plasmids should be considered to be nonstable.
5. Summary
In conclusion, S. cerevisiae is a organism which has an extensive history of safe use. Despite considerable use of the organism in research and the presence of S. cerevisiae in food, there are limited reports in the literature of its pathogenicity to humans or animals, and only in those cases where the human had a debilitating condition. Factors associated with the virulence of yeasts (i.e., phospholipases) indicate that this organism is nonpathogenic. The organism has not been shown to produce toxins to humans.
B. Environmental Hazards
S. cerevisiae is ubiquitous in nature. It has been recovered from a variety of sites under varying ecological conditions. The organism is used in a variety of industrial scenarios. S. cerevisiae is commonly recovered from a variety of fresh fruits and vegetables, generally those fruits with high levels of fermentable sugars. However, it is not listed as the causative agent of food spoilage for fruits and vegetables (Phaff et al., 1966). The only adverse effect to the environment noted in the literature is the presence of the "killer toxins" which is active against other strains of Saccharomyces.
IV. EXPOSURE ASSESSMENT
A. Worker Exposure
S. cerevisiae is considered a Class 1 Containment Agent under the National Institute of Health (NIH) Guidelines for Recombinant DNA Molecules (U.S. Department of Health and Human Services, 1986).
No data were available for assessing the release and survival specifically for fermentation facilities using S. cerevisiae. Therefore, the potential worker exposures and routine releases to the environment from large-scale, conventional fermentation processes were estimated on information available from eight premanufacture notices submitted to EPA under TSCA Section 5 and from published information collected from non-engineered microorganisms (Reilly, 1991). These values are based on reasonable worst-case scenarios and typical ranges or values are given for comparison.
During fermentation processes, worker exposure is possible during laboratory pipetting, inoculation, sampling, harvesting, extraction, processing and decontamination procedures. A typical site employs less than 10 workers/shift and operates 24 hours/day throughout the year. NIOSH has conducted walk-through surveys of several fermentation facilities in the enzyme industry and monitored for microbial air contamination. These particular facilities were not using recombinant microorganisms, but the processes were considered typical of fermentation process technology. Area samples were taken in locations where the potential for worker exposure was considered to be potentially greatest, i.e., near the fermentor, the seed fermentor, sampling ports, and separation processes (either filter press or rotary drum filter). The workers with the highest potential average exposures at the three facilities visited were those involved in air sampling. Area samples near the sampling port revealed average airborne concentrations ranging from 350 to 648 cfu/m³. Typically, the Chemical Engineering Branch would not use areamonitoring data to estimate occupational exposure levels since the correlation between area concentrations and worker exposure is highly uncertain. Personal sampling data are not available at the present time. Thus, area sampling data have been the only means of assessing exposures for previous PMN biotechnology submissions. Assuming that 20 samples per day are drawn and that each sample takes up to 5 minutes to collect, the duration of exposure for a single worker will be about 1.5 hours/day. Assuming that the concentration of microorganisms in the worker's breathing zone is equivalent to the levels found in the area sampling, the worst-case daily inhalation exposure is estimated to range up to 650 to 1200 cfu/day. The uncertainty associated with this estimated exposure value is not known (Reilly, 1991).
B. Environmental and General Exposure
1. Fate of the Organism
S. cerevisiae is a normal inhabitant of soils and is widespread in nature. S. cerevisiae is able to take up a wide variety of sugars and amino acids. These traits enhance the organism's ability for long term survival. S. cerevisiae can be isolated from fruits and grains and other materials with a high concentration of carbohydrates (LaVeck, 1991).
2. Releases
Estimates of the number of S. cerevisiae organisms released during production are tabulated in Table 1 (Reilly, 1991). The uncontrolled/untreated scenario assumes no control features for the fermentor offgases, and no inactivation of the fermentation broth for the liquid and solid waste releases. The containment criteria required for the full exemption scenario assume the use of features or equipment that minimize the number of viable cells in the fermentor off-gases. They also assume inactivation procedures resulting in a validated 6log reduction of the number of viable microorganisms in the liquid and solid wastes relative to the maximum cell density of the fermentation broth.