Category Three

indentThis category consists of strains useful in undergraduate University and College studies. The Centers for Disease Control in Atlanta, GA can make suggestions as to which non-pathogenic strains should be used. Some exotoxin producers and opportunistic pathogens are included, and adherence to appropriate handling procedures for each type according to Appendices B and C of the NIH Guidelines (Federal Register 05 July 1994) is recommended.

indentList 3a. Bacteria. Alcaligenes (Hydrogen cycle, and 2,4D degradation), Clostridium, E. coli (see Appendix B; students should not use those highly pathogenic types possessing the serotype O150 or O157, which may only be used in a professional laboratory which normally studies these), Klebsiella, Mycobacterium phlei, Mycobacterium smegmatis, Proteus.

All Bacillus spp. are permitted EXCEPT for the following four which are NEVER exempt from review: Bacillus alvei (bee pathogen), Bac. anthracis (anthrax in mammals), Bac. popilliae (bee pathogen), and Bac. larvae (bee pathogen).

indentList 3b. Fungi (Use hood or other air purification method to obviate problems when working with sporulated fungi. While normal people have little sensitivity to fungal spores at usual environmental levels, adverse allergic and bronchiopulmonary reactions can be triggered if lab cultivation produces very high levels of spores.) Exempt from review are Aspergillus (except fumag. and flavus), Asperg. oryzae (soy sauce), and Rhizopus stolonifor (soy bean tempeh).

indentList 3c. Miscellaneous

indentEnvironmental Micro-organisms that are isolated from normal places such as the ocean, soils, swamps, food stuffs are exempt to university students and higher who have had a minimum of 50 hours of lab training covering aseptic techniques, and the functioning of sewage treatment facilities. Work must be done under the direct supervision of a trained microbiologist.

indentMicrobes derived from normal human, primate and canine sources are recommended to be treated as members of Class 3 until identification places them in lower classes. Exemption is granted for all cases of isolates from human sources when the donor is the sole handler of the material up until it is properly decontaminated and discarded.

ACKNOWLEDGMENTS

indentWe wish to thank Thomas Shih of the NIH Office of Recombinant DNA Activities for initial guidance, and to the following, alphabetically, for assisting in the microbial content of this document: Alan S. Cross, M.D. (formerly Colonel, USA, and Head of Infectious Diseases at the Walter Reed Army Institute of Research; now at the Univ. of Maryland Cancer Institute), Larry Hale, Ph.D. (Head of Enteric Diseases, WRAIR), Charles Hoge, MD (Captain USA, Enteric Diseases, WRAIR), John Madden, M.D. (Duke Univ. Medical Center), John B. Robbins (NIH [NIAID], Antibacterial Vaccine Development), Richard Warren, Ph.D. (formerly at WRAIR, and the Department of Defense's representative to the Recombinant DNA Advisory Committee of the NIH Director; now at SmithKline Beecham), Frank Witebsky (NIH, Head of Clinical Mycology), S. R. Zaki (Head of Infectious Diseases at the National Centers for Disease Control and Prevention). We also wish to thank Ronald Giess (Prof. of Science Education at the College of William & Mary) for help in the pedigogical content.

Appendix A

Micro-organisms for the Immuno-compromised

indentIt is very difficult to draw the line with regard to absolute and universal safe micro-organisms. Perhaps a good rule of thumb would be to consider whether or not the person in question would deem it safe to work with non-sterile garden soil. If they would feel safe doing so, then all of Categories One and Two should be considered safe.

indentHowever there is the special case of persons with weakened immune systems. Immunocompromised persons should refrain from contact with most micro-organisms and their handlers. Immunocompromised persons include those who are undergoing antibiotic therapy, or who have undeveloped immune systems (i.e. newborns), or who are taking immunosuppressive drugs, such as during tissue or organ implantation.

indentNevertheless, there are large numbers of different microbes that remain non-infectious for even the most immunologically compromised persons. Microbes that cannot survive and grow above 33°C simply cannot successfully infect most warm-blooded animals (among the exceptions are the sloth, platypus, naked mole rat, and hibernating mammals). Similarly, microbes that cannot grow below 45°C should not be able to infect any warmblooded animal even in its most feverish state (normal body temperature of small birds is 43°C). Further, it is reported that all microbes isolated from tomatoes have so far been found to be innocuous to even the most advanced AIDS patients. Thus there are large numbers of microbes usable by persons who either are immuno-compromised or come into frequent contact with those who are.

Appendix B

PATHOGENIC STRAINS OF Escherichia coli

indentTens of thousands of E. coli strains have been identified, the vast majority of which are common necessary intestinal inhabitants. Fewer than 1% of the known strains are pathogenic and produce toxins. Clinical laboratories often test for toxigenic strains. Those which are not toxigenic are categorized as Category Two. One benign strain, O75:K100:H5, has even been fed in bulk to humans with no adverse effects, and persisted in their colons for about three weeks.* A few reference laboratories in the world have looked at E. coli in a different way. These labs are interested in the surfaces of the bacteria. This applies to vaccine production and to how the bacteria resist being killed by normal, non-specific immune functions in our bodies. The various surfaces are categorized as serotypes which are preceeded by the letters O (an underlayer of anionic lipopolysaccharide), K (exterior capsule of acidic polysaccharide), and H (flagella). Experiments using O150 and O157 ("Jack-in-the-Box") are never exempt from review, since they produce the same toxins as do the dysentery bacteria Shigella. Those studies using the other strains of E. coli which are pathogenic are not exempt from review for children below ninth grade. High school students may use these if they work directly under the supervision of a trained microbiologist. Decontamination of cultures and equipment after use is strongly advised. See Appendix C for suggested methods of decontamination.

_____ * Schneerson, R., and J. B. Robbins. 1975. "Induction of serum Haemophilus influenzae type b capsular antibodies in adult volunteers fed cross-reacting Escherichia coli O75:K100:H5. New England Journal of Medicine 292: 1093-1096.

Appendix C

DECONTAMINATION

indentHere are several suggested inexpensive and highly effective methods for decontaminating cultures, equipment, hands and the work area. Decontamination is advised for organisms in Categories 2 and 3:

indent1. Used cultures and some equipment. Autoclaving at 1 atm for 15 minutes is effective. Note: a kitchen pressure cooker is a manual autoclave. Heat-resistant plastic bags are easy to use in autoclaves for decontamination of used plastic petri plates. These bags can be purchased at many supermarkets.

indent2. Hands, equipment and counter tops. Most handsoaps are ineffective in killing bacteria. Alcohol washes used on hands, equipment and counter tops pose a fire/explosion hazard in microbiology labs where open flames are used to sterilize wire transfer loops. Household bleach diluted 30-fold with water is highly effective and very inexpensive. None of the above organisms can survive a few seconds of this strong oxidizer. This is safe for hands, although a short rinse with tap water shortly after disinfection with the bleach is recommended.

Amending these lists of exempt micro-organisms

indentThe ultimate approval for additions to these lists comes from the NIH Director. There are thousands of different species of microörganisms and viruses which would qualify for inclusion in these lists. These lists are thus far from comprehensive or complete. Because many additional species are expected to be nominated for inclusion, an intermediate method for expediting approval must be devised. With millions of grade and high school scientists scattered across this country, and thousands of others of college age and above, the influx of applications for exemption to one office would be overwhelming and time delays too long to be tolerable for the advancement of science. Also because the decision as to whether the organism is safe to health or the environment would usually not be difficult, anything more complex that an expedited preliminary approval route would be an exercise in the paper-shuffle. We would like to suggest that candidate safe microbes be initially presented in a brief postcard-type scan-tron form to be countersigned by two federal microbiologists. These persons might be infectious disease workers at a local Veterans Administration Hospital, or a U.S. Public Health Service microbiologist. There are many other possible federal experts in microbiology who are scattered widely across the nation. The card would be sent to the NIH Director for consideration of inclusion into the above lists. If bacteriologically oriented, the card then could be sent to the editors of Bergey's Manual of Systematic Bacteriology for proper notation to be printed in the next edition. In any event, once that card has been signed by two federal microbiologists, then that particular researcher would have exemption rights until the NIH Director reached a final decision. (A duplicate card signed by the reviewers would be retained by the researcher.