APPENDIX D February 24, 1975 TO: "hr? Coaoittce on Recombinant DXA ?blecriles Assen'l.y of LiEe Sciences National Research Council Natiopal Academy of Sciences Vashington, D.C., U.S.A. Paul Eerg, Chairman FROX: Working Party on potential biohazards associated with expsrimentation involving genetically altered microorganisms, with special reference to bacterial plasmids and phagzs Royston C. Clowes Stanley N. Cohen Roy Curtiss 111 Stanley Falkow Richard Novick Sirs: We have the pleasure to transxit herewrth for your consideration proposed Guidelines OR Potential Biohazards Associated with Experiments Involving Genetically Altered Nicroorganisxs. in final form during two meetings, the first held in New York City, Noveiaber 7 - 10, 1974, the second in Palo Alto, California, February 20 - 23, 1975. This report was written PROPOSXD GIifDELINES OX POTENTIAL BLO'r!AZAi?DS ASSOCIATED WITS EXTERLXENTS INVOLVISG GENETICALLY ALTEPZD F!ZCftOU-RG~IS>iS Prepared by: Working Party on potential biohazards associated with experimentation involving gentically altered microorganisns, with special reference to bacterial plasmids and phagas Royston C. Clnwes Department of Biology University of Texas Dallas, Texas 75230 Stanley N. Cohen Departrnent of Medicine Stanford University Nedical School Stanford, California 94305 Roy Curtiss 111 Department of Microbiology University of AZsbama Birmingham, Alabama 35294 Stanley Falkow Department of Microbiology University of Washington School of Medicine Seattle, FTashington 96155 Richard Noviclc Department oE Microbiology Public Health Research Institute of the City of New York New York, New York 10016 February 24, 1975 Contents -- I. Introduction A, Scope and Purpose 13. Background C. Principles D. Experimental Systems and T'neir SaEety 11. Classification of Experinents A. Considerations for the Assessment of Potential B. Classes of Experiments C. Summary of Classification Biohazards 111. Containxent Principles and Procedures A. Introduction and General ReCOm2ndatiOnS 3. Levds of Containnent C . References IV. Recommendations for Implemantation of Guidelines V . Conclusions VI. Appendices A. B. C. Guidelines for Ninimizing Biohazards D. The Ecology of Plasmids and Bacteriophages Illbstrative Examples of Experiments in Each Class Guidelines for Monitoring and Reassessaent of Biohazards Associated with RecomSinant DEA Molecules Introduced into Plicroorganisms 2 A. Scone and Purcrosc 1. Scop-. . These guidc1ice.s cover the nodific,:-tion or' prokaryotic ~il ti:. r 00 qpn i s P.S by t1w introduction oE foreigr! gmetic inforinnziol-. i?'Ltiiough tii.is docunc2nt has been prepared in r-c3s?ozs~ to E? rccorxcnlatioz by `ch? Cormittee ox Reconbbinznt DXA P!olecdes (Ezrg et. 2%. , Proc. 1:a.t. Acad. Sei. ~ 'iJ3sh. 71, 2533, 1374) that guidelines 50, devised for ezperirricnts in- vol-iiing "potnntially hazardous recombinant DXh =o1ecules", it is our view tha; there are certain oth2r types of genetic nanigulatisn and reconstruction ehzt have so strong a logical klnship to the above that it Imu1d be artifi- cial to omit them. At its broadest, thei?, this docuixnt will dsal with all genetic manipulations involving the introduction into a prokaryotic species of genetic material that may or may not be native to that species afid nay be unlikely to be acquired by it in the natural ecvironaent. -- - For the purpose of this discussion, we xi11 refer to a microorganism whose genome has been artificially modjfied by the addition of genetic infar- xation that is foreign to the species and unlikely to be acquired by it in nature asa novel recor;binant iio typ2 (or nicroorganlsns) . As current tech- nology involves prir-arily the use of bacterial and phge genoses as carriers of foreign DXA, this terz refers prinarrly to bactsria carrying foreign phages or plasinids or to native phages: or plasmids that have had ganisrrs with foreign DXA carried chronosozally, it excludes organisms produced from pre-existing ones by simple Rutation. _I__L segments or' foreign DSA added in vitro. rr \;nile it includes, also, Kicroor- -II_ Thz lizitation of 0x11 rzcormendations to prokaryotic organisrns is a practLcal one t5at is 1Lc:ated by current lixits of technology and of avail- able infornation. These guiclelines can and should be extfnded to eukaryotic nicroorganisxs if and :*iheZ those nodificatians along similar lines become feasible. 2. Purpose, The purposs of this docuisnt is two-fold: first to explore and detail the potential biohazards poscd by a i.7ide variety of classes of experiments involving recozd~-Lnant nicroorganisms so as to raise the general levei of awareness of these biohazards; and second, to make availabls sugges- tions for dmling with potencia1 biohazards so that the individual need not rely en-tircly upon his or her owm judgment. Thus, it is hoped that the principle will be established that an open ewluation of biohazard potential and the adoption of an appropriate biohazard nininization procezure will be an integral part of experimnts dealing with genetically altered microorganisms. Once this principle is accepted, a set of guidelines developed by an open, collective process that has taken iwto considcration the gamut oE potentially conflicting interests vi11 serve to eniinnce the safety and eflectiveness of this line of rese2-rch rather than to intlerferc with fleedon of scientific inquiry, as has bsen feared. hive involved the attaclmsnt of a DXk segnent to a functional extrachromo- somal replicon of bacterial origin (a plasxid or i: bacteriophage genome) ad the introduction of the recombinant moleculs into 2 sui table bacterial host cell where lit replicates autonomously, serving to clone the added DNA segriieilt. It is already certain that DNA from eukaryotic as ::ell as fro2 prokaryotic sources can thus be replicated and transcribed in bacterial hosts. it is not yet known whether or not eukaryotic DSA- can be faithfully translated in bacteria, the consensus is that any barriers to rranslation codd be by- passed by relatively straightforward manipulations. Although This new technology thus constitutes a major breakthrough in molecular biology and gives rise to the possibility of important advances in at least four areas: (1) fundamental knowledge of gene structure, organization, and function; (2) genotypic modificat5on of plants or anicals to icprove their usefulness to man (e.g., the development of nitrogen-fixing non-leguminous plants); (3) construction of bacteria or other scch organisms &le to produce rare and medically valuable biological substances such as insulin, growth hor- mone, etc.; and (4) genetic restitution of hcman hereditary diseases. As with other major technological and scientific advances, gene grafting entails (along with its great: potential benefits) at least the potential of serious and often unpredictable adverse consequences. hong these are bio- hazards that might result from the intentional or unintentional release into the environment of microorganisms carrying novel combinations of genes that have never existed before and are very unlikely to arise in th2 course of natural evolution. These biohazards would result, basically, fron modifica- tion of the relationship between the organism and its environment - the gene- tically modified organism might be able to occupy new ecological niches or to function in a novel way within its noma1 environment, or both. One important subclass of these biohazards would involve an increase in the ability of a microorganism to cause human disease, including enhanced pathogenicity as well as increased resistance to eradication or treatment. These possibilities have given rise to a significant level of concern among the general public as well as within the scientific con-aunity as there is ample precedent for the fear that the accidental introduction of organisms into new environments may have uncontrollable and sometir;l.es dramztic untoward consequences. As examples of this, one might point to fire ants, killer bees, mudfish, snails, Xenopus toads and to Chestnut blight and Dutch elm disease. More germane, perhaps. to the present document is the serious biohazard inhe- rent in the astonishing spread in the space of a mere 30 years of bacterial plasmids carrying resistance to antibiotics consequent to the vast overuse and misuse of these valuable therapeutic agents.* The recent -- de novo appearance of such plasmids in Hemophilus influenzae and Streptococcus species suggests that their spread may by now have encompassed bacterial species to which they were never native before the present era. The worry over possibilities such as these is not new; it has been ex- pressed through legislation to prevent the transportation of certain plant and -X For documentation see, for example, the Report of the Joint Cornnittee on the Use of Antibiotics in Animal Husbandry and Veterinary Sfedicine (Chairman: Sir M. PI. Swann) HNSO London, 1969 4 Concern over pote?;ltial biohazards of novel ni croorgacisms produced by in vitro genstic reconstructioa was first articulated publicly in a repart by a group of distinguished scientists, the Comittez on Recox3inant DSA zoteculss, pub1ished in the Proceecl_ings 05 the I'htioral Acad. of Sei. U.S. (71:2593, -- 1974), . in the sumer of 1974. In this report, the Committee urged that a set of guidelines 3e developed to aid individual scientists to perfo-m safely experiments involving the production and study oE novel reconbi- nant microorganisns. These guidelines s~oulii help in the assessxat of Zhe degree of danger involved ar~d \%iould recomxend cox-?znsurate precautions. As a preliminary o.ove, the Cornittee recormended a voluntary termporary deferral for two types of experiments and recorxended that a third be performed with cailtion, until thn, appropriace guidelines were developed. -- It appears that this deferral was lergely successful and that the letter had the intended effect of setting in notion a nuiaber of inde?endznt inquiries to deal with the problem. One of these has already come to fruition in the fora of a report, dated DZC. 13, 1974, to the British Parliament by a "working party on the experimental manipulation of the genetic composition of micro- organisms" under the chairmanship of Lord Ashby. This report contains a very thoughtful analysis of the potential benefits and hzzards attendant upon gene grafting research and outlines vzry briefly a set of broad recomendations. The present document is in agreement with the philosophical position of the British report and is offered as a sone;ahat more detailed analysis of ex- perimental systems intending to provide an explicit set of working guidelines for experinentation in this field. The two documents will thus be seen as conplementary to one another, and their joint effect will be to replace the moratorium with specific recom.endations as urged in the NAS Cornittee letter. C. Principles _I_ The philosophical position underlying this proposal and its contents is best expressed in the form of a set of basic principles, some of which are clearly established as facts, while others nay be regarded as assumptions: 1. Since man hes some Lieasure of control over his actions, there is an operational dichotomy between the activities of man and the processes of the natural world. The distinction between "man-made'' and "natural" is therefore meaningful and control of the former is both worthwhile and possible. 2. It is possible to modify profoundly the genome of a (micro) organism by artificial Ineans involving the -1_1_ in vitro joining of unrelated DXA segments. Such modifications may Find expression in the organism's pheno+,& LvDe as well as in its gcnetic constitution. 3. Mod-ified (micro) organisms may behave in an unpredictable manner with 5 In view of the foregoing, a set of basic questiox zay be posed, L~UC~ this proposal is a rathsr elzborate attenpt to ansrrer: Is it or T.5; i.t not possible to evaluate 2. potentid. bio'nazard'? i.e., Em? likely is it in :my particular case that foreseeable or unf:orzeeable adverse consequscces will follo~ the re- lease o f a nov?l recoin':,inar,c organism into the envLrox?.ent? Or, alternntively , grantin2 the possibillty of zdverse consequences, how likely La it that a po- tentially hazardous but scientifically useful experimental systcn! car. be co~.- t a in e d ? In general terms, the vinii to be develoged hzre is that (a) it is often possible tc evaluate to a greater or less-r extent (but rarely, if ever, fii1l.v) the potenti21 biohazard associzted vith c3p.l; zovel biotype; possible to ensure absolute contzinF.ent; but (c) it is often possible to reduce a potep-ti.70Lild consti tutr-. a seriorrs blohazerd, should not be a"iep2ted. t'r-le experinent 6 (e) Finali.;;, ii nust be stressed that whilc this set of guidelines is des.i-gned to help tlie i.nves tigator perfom responsibly and with confidence tiioS2 experiments deemed s:if ficiently important to justify s$hatever risk may 5c involved. These guidelines are not intended as a lic-znse to do unrestrictsd e-i,periraectat-ioir in thfs area. ExperiEents involving th-2 constructix of poten- r-inl:Ly ha.zar&us novel recombinant biotypes should IiOt be u%dertak.cn casually even rjith-in the containment framework appropriate for thc level of risk in-dolved. After dcciding to Cons'irLict a gsnezically altnr2d microorganism, an i.nvzs- tigiltor sboulcl consider each of thz follos-ririg pints in decidhg on an app-ropriaj-e classification Tor the experim2n.t to de termins the type 02 containnent necessary. 2 o Specific Considerations a. Yotentid for Alteration of Pathogenicity. - For cur purposes, pathogenicity and virulence are deEined similarly as the (I capacity to cause disease". involved? genicity? city is used to construct a recombinant DEL4 molecule, then it is pertinent tu ask: Hot7 great is the knom pathogenicity of thhe organisnls Will the genetic manipulation contemplated cause an increase in patho- If gemtic infomation specifying traits thaL coztribute to patIrogeni- i) ii) Is the ecology or reservoir of the virulence gen2s being changed? Do these virulence genes occur naturally in the 2onor and recipient species in the general environnent, in the local environment or in both? \&.at is the potential for the transmission of these virulence genes frorn the modified organisn to other microorganisms? iii) b .) Potential for Dissemination. If the genetically altered microorganis3 is pathogenic, can growth be con- If anti- trolled by aEtibiotics customarily used against the recipient strain? biotic resistance is specified by ths recodinant DNA, is this resistance to a drug of choice for treatment of infec"tons by ths microorganism? Is it a drug for which resistance is commonly expressed by the recipient organism? Is this drug resistance phenotype comon locally Do the dor.or and recipient species naturally exchange genetic information? Ifhat is the potential for intercellular spread of the DNA chizllera? DNA to construct recombinant molecdes, do plasmids specify conjugal gene trans- fer? Are the recombinant DNA rcolecules normally restricted to an intracellular existence (as with plasmids) or do they normally persist extracellularly as en- capsulated phage particles? Is the recipient lysogenic? Does the recipient possess plasmids (cryptic, conjugative or non-conjugative, acltononous or inte- grated)? Are the chimeric DNA molecules likely to reconbin2 by natural means with other genetic material present in the recipient species? Is the reconbi- nant DNA likely to undergo genetic alteration in its new host that may affect its biological potential? among microorganisms of this type? Ifhen using plasmid c. ' Potential for Alteration of Ecolo~. For our purposes, ecological potential is defined as the ability to occupy ecological habitats and the ability to alter the local ecosystea. Do the donor and recjpient organisms share a comon habirat? phenocypic properties which, if expressed in the recipient, might substantially alter the ecological potential of the recipient? 13511 the genetically altered microorganism possess any unique metabolic properties that will alter the local ecosystein? Does the donor organism possess Is it: likely that the normal ecological habitat of the recipient will f Availab-ifity of Genetic InformatLon About OrganisEs Ix-;~ l-z.:J. -_-- HOW well characterized are the organisms? EIave they beer, ~so !;rtCfLf recently or are they well-studied laboratory strains? 3. General Considerations €3. Classes o€ Experiments _I following conditions miisC be fulfilled: a. b. C. d. e. The psthogenicity of the donor and recipient organism is minimal and is krio-m to be uac'nanged by the procedure in question, and It is known that dissemination of the organisms involved is fully and easily controllable, and All DNA species involved are xell characterized and their genetic properties are v:.;ell understood, and The experiment does not alter the ecological potential of the recipient compared to other strains of the sane species, and The genotypic and phenotypic properties under study occur naturally in the recipient species or can be readily trans- mitted to strains of the recipient species. Exaqles of Class I Experiment: Gene transfer or genetic recombination between laboratory strains of Escherichia coli such as S-12, B, C and 15. This includes conjugal transfer by I?, F'-containing and Hfr donors. See Appendix B for additional exanples. 2. Class I1 experiaent: Class I1 includes experiments in which the biohazards can be reasonably assessed and from what is known about then one can expect them to be minimal. More specifically, all of the follo-cring conditions must be ful- filled : a. The species used to construct the genetically altered micro- organism have either low or moderate pathogenicity similar to that expressed by Salmonella ty-phimur&in, Staphylococcus aureus or HaemoDhilus influenzae. and b. The genetic material used to construct the altered microorganism is derived from organfsms known to be capable of transmitting genstic infornation to the recipient, and The genetically altered microorganism should not have ecological potentials greater than can be conferred as a consequence of normally occurring genetic exchange processes, and The genetically altered microorganism does not contain genetic information that would prevent effective treatment of infections caused by it. e. d. It should be noted that in sone imtances an organism serving as a DXA donor may have a greater potential either to exhibit pathogenicity or to occupy unique ecological habitats than the recipieot organisms and hence poses a greater poten- tial biohazard than the recipient. In this event it is the potential biohazards associated with the donor of the DNA that determines the classification of the experiment. Examples of Class I1 Experiment: The construction of rccoabinant molecules either in vitro or -- in vivo between R and F' plasiaids, between Col and F' plasmids, between Col and F' plasmids or between bacteriophage X and a Col or R plasmid when introduced into E. -- coli. -___ See Appendix B for additional examples. Classes 111, IV and V Experiments include: (i) recipient organisms that ordinarily do not - exchange genetic information and all. constructions of genetically altered microorganisms which use donor and a. Ti? recorrbincnt: CN~~ :Jill cot contribute signlficzntly irizreased paEhogenizity to the recipienz, nor signifi- cantly dter irs ecological potential, and b. Pathogeniclry of tt.5 genetically altered nicroorganisia or its pzrents is minimal (e,g. - E. -- coli) or moderate (e.g., 5. - typhinurius), but not severe (e.g., Y, pestis), and ?Tie genetically altered microorganism does cot contain information that +Todd prevent effective treatment of infections caused by it. - B. su3tilis), 1x7 (e.g. , - -- C. Exarriyles of Class III Exgeriment: Construction of a hybrid p.Lasmid or phage that includes an antibiotic resistance gens &rived fro= S- aureus when intraduced into E. coli so long as genes conferrirrg resistance to that antibiotic are found in - E. coli. genes from Xenopus laevis or random framents of Drosophila Eelangaster DXA when introduced into E. coli. See Appendix B for additional- examples. I -~ - -9 Construction of a hybrid plasmid or phage that includes ribosoml -- 4. Class LV Experinent: Class IV, like Glass 111, includes eqeriments in which the biohazards are usually unknown, and cannot be accurately assessed, but because of the known genotrypic and/or phenotypic properties of the DNA and/or organisms used to construct the genetically altered microorganism, they are judged to be potentially significant in affecting either the ecologic potential or pathogeni- city of the recipient organism. --- Exazples of Class IV Experiment: Construction oE a hybrid bekween random Constructionof hybrids between random DNA f ragmnts from normal human DNA fragwnts fron S. py~genes and an F'lac plasmid and its introduction into E. coli. fibroblasts and an E. coli plasmid or phage when introduced izito E. coli. Con- struction of a hybrid between either J. or plasmid DXA and the genes specifying synthesis of cellulase and/or ligninasz from Polyporus annosus and its introduc- tion into E. coli. See Appendix B for additional examples. - ~ -__ -- -- 5. Class V Eqerimznt: Class V also includes eiqeriments in which the biohazards are usually uDknuwn, but because of the known genotypic and/or phenotiypic proper- ties oE the DSA and/or the organisms used in the construction of the genetically altared microorganism, they are judged to be severe in affecting either the ecolo- gical potential or pathogenicity of the recipient organism. Examples of Class V Experiment: The construction of a recombinane DLZ nole- cule between the plasmid from - S. -- aureus determining exfoliative toxin and an R plasmid or X and its introduction into E. -_I coli. -I__ E. coli phage or p1.asni.d DXA, and unknown genes fron x. pestis, E. nthracis, or - H. _--- abortus, when the hybrid is introduced into K. coli. additional exanples. Construction of hybrids between See Appendis B for 6. -I_ Class VI Experiment: Class VI includes experiments in which the biohxzards are jurlczii to lie oE such great potentizl severity as to preclude perforr?ance of ttie expariment at tile present time undes any circunstanczs, alzd regardless of containxnt conditions. A natural tendency is to consider changes in pathogenicity 3s the primary biohazcrd coclcern since these coroe to nicd most readily ;::?en considering micro- organism; other changes which mzy affect the EundamenZal ecological potential, adaptability, metabolism, etc. of a recipient organist, may be more subtle and much Eore difficult to assess than pathogznicity . EIowi.v=.r, thwe alterations may potenelally present an equd or greater biohazard. relatively few guidelirres to help ari investigator iix deter7ining ;he class assignment of an experivent 5n Classes 111, IV or V; perhaps the most critical is the extent of charact-rization ol the genztic riaterial being employzd in the expzrirre-nt sFnce we believe that the potential biohazards of a purified aild well- characterized donor- DM4 species are rore easily assesszd th?n t'h~ biohazclrds inherent in the introduction of a xandoa assortment of DNA fragzents. \,?e can offer orly a A. Introduction and C3neral Recommendations _- -__--- Biological. safety and environmentaL control prograrns €or dialing with pathogenic bacteria have been implemented in clinicrrl and biorncdical resznrch laboratories for nany years (refs. 1-12). Once a potential b-iohazard h,zs been defir.2d and the risk has been assessed, thc naj,r thrust oE the procedures einployed to minimize the biohazard involves steps to linit risk to the labora- tory worker and to prevent the escape of potentially hazardous biological nxterial. Many of the bask problems oE containment that face an investigator srudying A clinical specimen received for microbiological analy- ricoxbinant DNA in a microbial species are similar to those faced in every xedical microbiology laboratory. sis ray contain an etiologic agent ranging from those of ordinary potential hazard to those which may require the most stringent conditions for their containroent. One cannot be certain until the etiologic agent is isolated and its known patho- genicity (i.e. its potential hazard) zssessed. By the sane token, an investigator who employs a randon assortment of DNA nolecules for construction of recombinant DNA -n?olecules could, at least in theory, isolate a variety of novel transforroant bacterial clones which range in their potential biohazard. The following safety considerations are applicable to all procedures involving etidogical agents in the clinical laboratory. As such they may be considered as prudent standard pro- cedures for those working with bacteria containing recombinant DNA molecules. Obviously, those investigators working with anirnal or plant viruses will need to satisfy rhe special containment problems inherent in the laboratory manipulation of these agents. The procedures listed below are a reiteration of long-standing microbiologi- cal practices and simply reenforce the concept that microbiological safety is a matter of good working habits. All of the general recornendations listed below are desirable €or all classes of ewerimnts, although we recognize that they are not specifically needed for the safe handling or containmnt of all agents. 1. Consequently, our primary recommendation for containmnt of potential bio- hazards is that all individuals planning research with recombinant DNA molecules in bacteria receive adequate training in microbiology. Such training should not be construed to mean that one needs to learn only aseptic techniques or th2 pro- cedures for handling potentially infectious material. Rather, investigators cannot afford to ignore the basic biology of the microorganism -- irs ecology, innate pathogenicity, physiology, growth requirements, etc. In short, an inves- tigator must try to think in microbiological terms before initiating experiments that could potentially affect the basic ecology and/or pathogenic potential af an organism that serves as a carrier for a recombinant DXA nolecde. The microorganism is not simply a ''warn body" to house a recombinant DNA molecule of interest. It is axiomatic that no safety facilities or equipment (no matter now sophis- In terms of ticated) can take the place of an investigator's responsibility. biological safety, the principal investigator cannot delegate, reassign, abandon or ignore his or her responsibility that adequate safety training be given to all laboratory personnel. which deal with the general topics of laboratory safety, biohazards in biological research and the handling of specific bacterial agents which may prove useful as a source of specific information. We have appended a list of books and other publications 2. dous material is handled should be kept closed. As a general principle , doors to laboratories in which potentially biohazar- 3. which potentially biohazardous material is handled should be specifically forbidden. Eating, drinking or smoking in the laboratory is undesirable and in areas in 15 4. infections are accidenral ora1 aspiration of infectious material tbrough a pip:-.tte, xcickntal inoc1rl.3- tion with syringe I?eedles and animal bites (10,11). A iurtkr iraportxant cause of both laboratory acquired inFections arid contankasion of the environment is clerosols f roa cenLriZugation, blendicg, loose nzedfes on syringzs and even the impropzr flacie steril2zation of contsninatcd inoculating Loops and needles. (sse chaptax by DiriGck et .al., ref. 1) :is minim1 recormendattons, handwashing by lab0 ratory personnel shorrld be encourapd and direct =out5 pipetting shDuld be discouraged. The use o€ cotton plugged pipettes my be accepcable for agents of low or moderate hazard but a mechanical pipet Ling de-Jice is pre€erable. Special aerosol precautions are generally not required for most bacterial species, but thdr iisc deserves careful consideration. 'I" tit mst frrqclcnt cr?uses of laboratory acq:lirsd - -- 5. sterilized by autoclaving. contamtnated and all contaminated naterid placed in discard pans (preferably covered) containing a suitable disinfectant or autoclaved at thz end of the day. The us2 of specific disinfectants cannotr be recornended here, since they will. vary from bacterial species to bacterial species and, ac?ditionally, must be capable of rendering nucleic acid solutions l'non-infectious" . One should riot accept ranufac- turer's claims for disinfectant effectiveness -- there is no substitute for a use- test evaluation performed Elgainst the nicroorganisrn and nucleic acid solutions pro- cessed in the laboratory. Bacterial cultures and potentially hazardous DNA should be disinzected or Tile laboratory should be cleaned, work surfaces de- 6. should have an emergency plan, including a clean-up procedure to follow if an accident contaminates personnel or environnent. Eere again, the principal inves- tigator aust insure that everyone in the laboratory is familiar with both the potential hazards of the work and the eillergency plan. Any research group working with agents with a known or potential biohazard 7. vaccine is available, all workers should be vaccinated. ever, a license for procedural short-cutsnor a substitute for safe la3oratory practice, If a research group is working ~~ith a knovm bacterial pcithogen for which a Imunization is not, how- B. Levels of Containrnent -- The containnent procedures proposed are designed to patch the previously defined classes of experimnts involving novel recornbinant bacteria. I Since containnent cannot be absolute, the rationale underlying these contain- ment recomendations is that the greater the potential biohazard, the more stringent should be the containment. In our judgir.ent, each l2vel of containment implies an acceptable level of protection for laboratory t~orkers and an acceptably low proba- bility of esczpe for the organisms involved. Class I Experiments: Requires no special containment other than practice of stan- dard aseptic technique (i.e. use of procedures to maintain pure cultures and dis- infection oE discarded materials). Class TI Experiments: operating procedures employed in a clinical microbiology laboratory. The basic criteria €or this category are those minimal These. are: 1. Eating, drinking and smoking are forbidden in the laboratory. 2. Laboratory coats are required during handling of biohazardous mntcrial.. Tiiese should not be w~rn outside the work area. 3. 4. Cotton-plugzed pipettes o;r x~clinnical pipe ttixlg d2vi~ces are required. The larrer arc preferable. Routine disinfection of work surfaces am! prompt disinfection or sterilization of all corltaninated material should be carried out. Ixiaanization of personnel is required for experinenting with S. typhi, V. cholera2 C. diphthzriae and C. tetani. SpnciCic aerosol precautions are required (see below, 111, 3) wh~n large voimes (6 or rmre liters). of bzohazardous materials art3 centrlf.ugsd. 5. 6. - - - --'- - Class III Espzriments: applicable with the added provisions that : Eo Eouth pi?ztting oE potentially biohzzardous material is permitted, controlled access, which no other work is concurrently bein;: conduc red. the intent oE this containment feature is to exclude estra- IEOUS persons from the area and, hence, reduce the number of exposed individuals should a laboratory spill or other Zccident occur. Appropriate biohazard signs will be posted on the doors of laboratories during biohazardous experimentation as w.?.ll as on the doors of storage areas or cabinets containing potentially hazardous materials. Visitors to these tjork areas are prohibi- ted unless they have pemission frocl the investigator in charge who is responsible for the visitors while they are in the area. Specific aerosol precautions are nandacory (see for exaatple, R.L. Dimmick, ?.T.F. Voge and Pf.8. Chatigny. Potent5.d for accF- dental PEcrobial Aerosol Transmission in the Biological Lahora- tory In Biohazards in Biological Research ed A, Hillman, M.N. Osmnand R, Pollack. Cold Spring Harbor Laboratory, 1973, pp. 246-266). Thus, syringes to which the needle is firmly fixed (e.g. Luer-Lok) should be used. Screw-capped safety cups on centrifuge tubes are required when centrifuging bio- hazardous materials. Operation of centrifuges in hoods or other enclosed areas is desirable. Safety equlpaent to pre- veat the dissemination of aerosols generated by blending, soni- cation, centrihgaeion, etc. is commercially available (I), The same minimal srandards described for Class 11: are 2. 2. T'ne experiwnts are perlormd in labsratories that are under Tois doss not require a separate room in Pkchaaical pipetzing devices are required. Rather, 3. Class IV Experiments: ments are applicable with the added provisions that: The saxe minim1 stanciark required €or Class 111 experi- 1. At the minimum, a partial containment cabinet (see W.E. Barkly, ref. 1) or its equivalent should be used for experiments in this category. Tiiis is a local exhaust ventilation hood with a limi- ted front opening in which air entering through is subjected to high efficiency particulate air (Ffspa) filtratior, or incinerated before bein;: exhausted from the area. Special aerosol precautions are mndatory for experiments in this class, creating aeroso Is sIiouLd be operated in sepriratc isolation rooms or hoods (see Dimiikk, -- et.al. 2nd Bonn, ref. I). biological hazard sign used Eur highly infectious agents (op. - cit. p. 1-22) will be posted Oil cabinets, frzezerj:, refr igera tors, and/ or work arca rzherc biohazardocr; materials crre kept or are bein:; uried. 2. Ccntrifuges, blenders and othzr equipment capabl-e oE The standard Only p2rsonnel who wa-~rk in the laborstory r~y enter the 16 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Eanual of Clinlical Xicrobiology, 2nd edition. E.H. Lennette, E.H. Spaulding and 3.2'. Truant (ecl.) . Anericm Soceity for Pficrobiology, T:'~~:;hifi~.to~~, U D.C. 1374. Classificaciou of Etiologic Bg:eilts on the Basis of Hazard, 3rd edition. U.S. kpartnent oLC Health, Education and Lklfare. Lealth Services and llental Health Addnistration, CevLer for Disease Control, Atlanta, Georgia. June 1972. Lab Snfety at the Center for Disease Control. DepartmeiIt 05 .Health, Education and Welfare Pu5lication No. HSK 72-8118. Hzndbook of Laboratory Safety, 2nd edition. N.V. Steere (4.). The Cherrrical Rubber Co., Cleveland. 1971. Disinfectioa, Sterilization and Freservation. C.A. Lawrence and S.S. Block (ed.) . Lea and Ferbiger, Philadelphia- 1971. Disinfection. M. Benarde (ed.). EIarcel Ikkker, Inc., New Pork. 1970. Darlow, H.M. Safety in the nicrobiological laboratory. In J.R. Norris and D.W. Robbins (ed.) , Hethocis in Nicrobiology. New York. pp. 169-204, 1969. AcademiTPress, linc. Safety in FEcrobiology. D.A. Shapton and R.G. Board (ed.). Acadeinic Press, Inc., New Yorlc, 1972. Sulkin, S.E. and R.N. Pike. Prevention of la3oratory infections, In E.H. Lennette and N.J. Schinidt (ed.), and rickettsial infections, 4th edition. Aqerican Public Health Association Inc., New York. pp. 66-78, 1969. Diagnostic procedures for vizl Chatigny, M.A. laboratory-devices and procedures. Advan. Appl. Pficrobiol. - 3:131-192, 1961. Protectlon against infection in the microbiology FEcrobial Contamination Control Facilities. R.S. Bunkle 2nd G.B. Phillips (ed.). Van Eostrand Reinhold Environmental Engineering Series. Van Eostrand Reinhold Go. , New Yorlc. 1969. Lechevclier, H .A. and M. Solotorovsky. Three Centuries of Microbiology. McCraw-Hill , New York. 1965 . B. All invsstisators wishing to carry o:it e-qeri:?.eiits ir,vol.vi.ng poss-ible bio- haza.rds would be required to subait a pcoposal to the institutional comiitte;!, i.ndicat.ing the purpose of the esp?rinect, the explicit ben2:its to he deri;.&, and an assessrcent of t?s potential b-io?iizards arrd precaukions ECI~ containzent that are pro?osed. C. The responsibilities of the ccrciinittee xould be to famiii?rize themselves with the extent of potential bjohazards anJ the necessdry Tzasures for their iainiaization and conCain3ent. It should ensure that no experimnts of this riciture are carried oct unless the invzstigator had subrlitted such a pro2osal. It would ensure that the invzstigator \?as fadliar with 2pprOpriate gclidc F ines and that a thorough review and assessnent of the biohazards and their contain- rent had been carried out. It would ther, evaluate the proposal. and any supporting evidence and would make its recomeodation on the proposed research. D. The subattted proposal and the cornnittee's rsviet~ would be filed as ptxblic documnts in a biohazards repository at the irstitution. Tnis fife would be submitted x.7ith all grant proposals and applications related to the experiments. Any subsequent modifications to rhe research program which materially affected the extent of the biohazards would require a ne77 proposal and a further review. Pragress reports would bs requirsd a': year?Ly iritervals to erisuce that the proposed experiments, precautions 2nd con"Lair!!erit wsre adhered to. A coz;?lete Eile of all approval programs under study would also be kept in a federal repository and would be available for publicztion. The file of documents on each proposal would be nade available by the investigator to those journnls where publication policy required appropriate docuzzntation. C. mal eyuiprcnt, of chi? type generally available at most acadeaic institutions, it is recognized that in the absence of continual supetvision or monitoring, the responsibility to pursue such a program of research rests f iilally with che investigaror . the ultimite responsibility Eor the experiments. ceeding with an experirent should not be shifted fron the PI to a local camittee, absolving the PI frox responsibilities. mendations and provide advice but cannot approve a program. Thus, even in face of a favorable review by the connittee, an imestigator would need to ensure that a program satisfied the rcquirexents of the guid?-Lines. In the event tlot the investisator decided to ignore reco-mmendat ions of the local comit ~CP, supporting evidence for procezding xiith the experiments shoirld be obtainecl Fron outside the institution iThich wodd justify the ultimate coLirse oE actiun. Since tile types of experirnents under discussion usually require only mini- We believe therefore thpt the Principal Investigator must shoulder Thus, respons ibility for pro- The local comaittee should make recom- G. All individuals enbarking upon expe-rinznts categorized as Cl,.rss I1 to V, should receive tr;rinirrg in the handling of potential or infectious inaterial :md must be faniliar xcittl tha NIH acd ASX guidelines (Sec refs 2 and 3; also 9, 10 and IS) of experinonral use oE such naterials. [An eeperiJenter who has beer. vel1 trained in working rrith pathogenic micro- organiszz 2nd .rrfio is Eacpiliar with the hS?f Handbook oE Clinical Microbiology Guidelines should have sufficient expzrtise to bs able to make appropriate judgements regarding the classification of individual experiments in the laboratory s ituation. Familiarity with this in€ornation should enable him to prescribe appropriake conrainment procedures for that particular type of experiment and will also enable him to make correct judgment about the type of training required $or technical personnel that w3y participate in the experiment.] 11. In those countries where experinants of the type referred to in this proposal are being carried out, it would seem necessary that national bodies sould be constituted to cstabiish, monitor an2 promulgate guidelines. h inrernational body should also be established 1. to consult with and advise natior,al organizations on th2 2. 3. to coordirate and periodically review the efficiency and 4. developzent artd implexentation of guidelines; to encourage the maintainance of uniform standards through- out the xorld; applicability of international guidelines; and to authorize any dissexization ineo the environment of new recombinant types that ars Likely to prodace signifi- cant ecological effects. I. 3 We believe that considerable bene L i-ts zre likely to result f~on exper- j- meats involving thz genetic alteration of microorgmisxs. The range of possible benefits exterlds €-cox the USE oE the;-? tcchniques to add to our knowledge of basic biologic-71 phenoxena, to possijle p.ractiza1 applica- tions in the areas of agriculture and aedicine. Ve bzlieve also thaL a scale of risks exists in the construction of genetically alter& microorganisms, and we ilre unconiortable Aout our inability to assess precisely the extent: of such risks for nany types of experinents. Hovever, t~c bilieve that the contaiment procedures described in this p~oposal will reduce any risk to laboratory workers ancZ to the envirorznent to a level that is acceptably low and :;'nich will allow invzs- tigators to carry out research in this area. We believe that certain experinants should presently not: be carried out under any circuxstances (i.e. Class VI), but that Dost experiments can be dono if containment facilities appropriate to the risk are utilized. We recornend that specific steps be taken as soon as possible to develop cloning vehicle-host systems which will further reduce biohazard potential, will minimize the necessity of elaborate containment facilities; and will obviate judgeitents which must necessarily be based oa little or no data at the present tine. Specifically, we recornend that special sporsored program be instituted immediately for the development and testing of such system. We recornend also the proiqt establishment 0.E experimental pro- grams intended to evaluate more fully the potential hazards that may be involved in the genetic alteration of microorganisms. \?e believe that perhaps the greatest: potential for biohazards involving genetic alteration of nicrcorganisns relates to possible military applica- tions. We believe strongly that construction of genetically altered micro- organisms for any military purpose should be expressly prohibited by inter- national treaty, and we urge that such prohibition be agreed upon as expeditiously as possible. Other recornendations for implementation or' the guidelines proposed in this report are contained in Section IV. The microbial geneticist was atcractec! to the sttidy cf I: plasmids not only from the starrdpoint oLC thcir siailarity 20 the classical F transfer system, but also frorr, the standpoi.nt of public health, end ths nnriqi.ic opportuniry to r.onitor the extent of change a the gece'iric basis of chang:e in natural bacterial po;i?:ilat;ions I The iiicreasld atttntion to nitaral bacteri.al populations ha Icd to a broad v.iew of the ecology of Sactzrial plasmids. For exzzple, €&ly %ne- third of Escherichia coli from aspp tonatic humazl and ciomestic animal popularions possess at least one self-transmissible (conjugativ3) plasnid that confers few or no known Thenotypic tra.its other than cc:zJugal fertility Bacterial plasmids confer a fa-r greater diversity of phenotypic traits upon the bacteria that pos- sess them than 'simply' antibiotic resistance or gene:; (such as enterotosin bio- synthesis) that contribute to bacterial. pathogenic2.tjr. PLassids have been idsntified in a variety of bacterial. genera and associated with such diverse functions as the control oE lactose fermzntation in ---____-__I_ Streptococ.cus lactis, sporulatioc in Bacillus piirn~.lus, and camphor degradation in species of --- Pseudo- I_- nonas. Tnere his been a growing appreciation of the fact that the genes for antibiotic resistance, toxin biosynthesis and other genes such as lactose Eer- ent tat ion, which are of 'transient' evolutionary advz.ntage nay be carried by vi.rtually identical molecular vehicles. Thus, it is possible to isolate conju- gntivr! plasmids tdkich are identical in over 80 percent of their molecular lengths but which carry on the 0p.e hand antibiotic resistance genes, on the other hand genes for the biosynthesis of enterotoxin and, in yet another instance, genes wllich control the utilization of lactose; there are numerous other exanples to suggest: that the same plasmid rJealri.ng diEferent phenotypic garb is often isoilated irrdepzndently in several laboratories. extcnt it appears that the genetic information which controls essential plas- rniti functions such as replication, the dis tribut-ion of progeny replicas and, to n somelqhat Ses..xr degree, transfer functions is conserved; indeed, plasmids, regardless of phenotype, can be 'spec.iated' by genetic and molecular studi.es. .- ~ To a great --~. 2. The "Infcctivity of E. coli K-12" - ___ Thus far, the lcloni~~p,' of recorbihant DXP. molcxules has been restricted to subsstrains of Esc.herichla coli E;-12, B or gen?t?c hybrids of r'ne tzo. Both -__ coli K-U and B are long established laboratory strzlns rz.hic'n were initially E. isolated fron man. One of the first qliestions to be asked, th%refore, is 'now comoniy these E. coli substrains can colonize the hrrzin or animal intestine. Although this precise question has not beer, scudled extensively, ir has been shom~ that E. coli E;-12 is a very poor color,izer of the norzal bowel. For example, after feeding of between 5 x 10" to I x 10l2 E. coli K-12 cells to calves, only about lo7 cells can be recovered per grarl~ of feces 5.n 24 hours and by 72 hours cannot be ideatifie6 at 21.1 ( < LO cells) (3). Siiiilar3.q- in ncm, ingestion of lo4 cells ds-2 xot norxlly lead to colonization, indeed, the detec- tion of imre than 100 X-12 cells/gia after 24 hours is rare. appears that E. coli K-12 has ve:ry little inhereit capacity to colonize man. c. .- I_- - -- --I_ -- Consequeotly, it - -- There are, however, exceptions to this general rule, If the noma1 flora of wan or animals is disrupted, for exampTe, by therapzutic levels of anti- biotics, the ingestion of - E. -- coli K-12 bearing the resistance determinats to thest3 antibiotics leads to colonization at easily detectable levels (about IO5 per gm of feces). Slrnilarly, individuals who have had surgical treatment for stomach or bowel disorders are far more easily colonized by all. enteric species (including - E. -- coli K-12). Finally any substance xLiich 'proiTecCs' an ingested organism from the acidity of the stonach leads often to a higher level of I:-12 excretion (although subsequent colonization of the nonal bowel does not occur, the length of timz of excretion may be increased by a few days). Tnerefore, a few simple rules appear to be prudent with regard to handling E. -- coli K-12, par- titularly when they contain either recombinant DYA molecules or naturally occurring plasmids for that matter: a. The usual laborarory procedures enployed in dealing with enteric b. Individuals who are receiving antibiotic therapy should not work pathogens should be flollow2d as described above. with tile strains during the period they are rc!ceiving therapy and for seven days after the cessation of t'nzrapy. 23 C. I~-cl-tviduds who I;civ~ functional intcsti.nal Ciisoxd2J-s ailtl thor;? who have hac! surgical removal of part of the stonnch or bowel should no L work 1,iith these strairs. d. Individuals S.T~O take large affiomts of' antacids should bc avare thar they are m.c)~s readily colonizec! by ing2s Lsc! bacteria. Obviously, the usual laboratory precant-ior: of no e,aticg in the laboratory should be followed. 3. Genz Transfer in the Gut Although E. coli ic-12 2nd derivatives do not usually actively RLlltipiy -- and colonize the norml anirral bowel, the organisma that survive the acidity of the stomach and other natural host defenses, remain viEble and can act as genetic recipients or genetic donors under the propsr circu:Ps*Laiices. Irr so Ear as we are aware all of the recombinant DNA molecules that have been pre- pared thus far zre nonconjugative, that is, they do not inherently have the ability to initiate transfer of DNA, Nonetheless, these nonconjugative plas- mids can be mo3ilized by a transfer plasmid (such as the classical F plasmid) residing in the same cell. A possible scenario for extension of the reservoir of a recoinbimnt DXA molecule could be as follows: A research worker ingests -- coli K-12 containing a reconbinant DNA plasmid. E. The surviving cells while in the gut, engaged in conjugation with a meolber of the normal flora containing a transfer plasmid. (Note: about 38X of all E. coli strains froill asymptomatic animals and ~nan harbor at least one transfer piasr;lid.) The converted K-12 organism containing both the tracsfer plasmid and the recorihinant plasmid Eates with a maher of the norm1 gut flora and the reconb-inant plasn-id is transferred. The latter strain is fully capable of survival in the gut and can, in turn, mate with other strains. This hypothetical sequence of events has a certain probability thar can be calculated on the basis of lzboratory experiments at 1 in - 1 in low8 .per bacterial cell. Experiments of this nature suggest, however, that the probabi- lity of this occurrence in the nornal gut is on the order of 1 in to 1 x lO-I4. that the best defense against R plasmid and other gene transfer is a nornal gut and gut flora. etc. are siaply not optimal for genetic transfer. logical conditions of the normal bowel provide us with one of the major natural defense mechanisms against infection by enteric pathogens. A major exception is, again, instances in which the normal flora has been nodified by antibiotic treat- ment or if there is a functional or pathological bowel disorder. Under these circumstances, the robability of -CI in vivo transfer increases to an merage of 1 x Thus, the parameters which affect the colonization oE -- E. coli K-12 likewise affect the probability of genetic transmission and the guidelines listed above apply to the prevention of in vivo genetic transmission. Of course, the probability of gene transfer by an ingested K-12 is exceedingly low particularly after the first 24 hours of ingestion. transfer from E. coli K-12 is not a significant hazard so long as normal pre- cautions of the bacteriology laboratory and the containment guidelines listed earlier are followed. This differential between laboratory and gut illustrates the concept Conditions in the bowel such as Eh, pH, fatty acid concentration, Indeed these sane physio- to 1 x lo-'. In our judgement, gene 4. Gene Transfer Outside the Gut There is one situation in which gene transfer might contribute to the dis- This situation could result from an semination of recombinant plasmid species. i~~:fortun it:~ly counoa practice in some Izboratori.zs, nzinely th?. dlscardin:; 02 culture supexiat~nt~ and even vi;ible ciilturcs 02 E. ~03.i EC-12 2nd otheir ''2.3n- patttogeni.c" bacterial sp2cies into the laboratory siiik ~htcfi eapt:tn,s into the coir3:inity s;?.'i.i?z system. On the face of the nattzr it night be imgined char virtually any forrn 01 sewage treatrr:ent X,,;o;.tld effectively destroy the i)act"~-.ia. This assu:erJ%ion i.s to talLy ug Founded, Iiowevt?r. For ei:alxple, in Ibshing::on, D.C., during periods of heavy water lis2 or during a period of heavy rainfail, it is qlclitz posstble chat a hi@1 proportion cf orgzni.sas disposed of do:m a di-aiu would reach the Potomi: Rivar whsre E. coli counts in excess oE 107/100 cii are not unCmmoi1. areas) . genetic exchange in water- iiwever, it is kco;Jn that fecal E. coli harboring R plasrnids have a very good survival potential 5n sewage and in river water, At any rate, it should be reemphasized that it is not a good practice to dis- pose of any viable bacterial cultlire into the comunity sewage disposal system, This is, of course, particularly critical with respect to cultures contaiding recombinant plasmid species or naturally occurring E: plzsnids for that matter. All such strains should bi considered to have at least sone ninixal degrce of. hazard and treated with the comqon sense experbental practica detailed in the section on containment. of gene transfer on bench tops, etc. which may be contaninated by spills. Again, one needs to reemphcrsize the basic methodology that is tarlght to every beginning student of microbiology. __ _-__ -_I (Kote that this sititation is found, of course, in most ttrban There is relatively little data availzble concerning the frequency oE -I_ Similarly, on2 does not know the potential hazards Roughly 10-15X of nomal, asyqtoxatic individuals harbor E. coli and other coloiforiu organisms in their nasophar-ynx. It is not know with any degree of certainty to what exteEt well-es tablished laboratory strains of -- coli such as K-12 may colonize this anatomical region. E. should be investigated. -- This possibility Re f erence s (1) Anderson, J.D., Gillespie, IJ.A., and Richmond, M.H. (1973) Jour. Med. (2) Microbiology. 6:461-73. Human Strains of Escherichia coli to Resident E. -- coli in the Alimen- tary Tract of Man. Lancet. i:1174-6. Smith, H.W. (19697 Transfer of Antibiotic Resistance fron Animal and (3) Falkow, S. , unpublished experinents. C. Sacteriophage Ecology The literature on bacteriophage is enornous and it would be obviously futile to attempt to summarize all that is known about their distribution in nature. Virulent bacteriophages are capable of only a productive life cycle in bacteria so that their propagation invariably leads to death and lysis of the bacterial host. the phage X of -- E. coli K-12, lead a sort of JekyL1-Hyde existence in bacteria. They are capable of productive growth (lysis) or ;nay become inserted into the bacterial chromosome and so assume a relatively passive role (lysogeny). The decision to lyse or insert is under the control of a conplex system of geneti- cally controlled biochemical 'switches' and it is possible for the inserted bacteriophage chromosome (called a prophage) to become induced to CL productive Temperate phages on the other hand, as exenrplified by grnsit:1 cycle after peacefully cocxis ting with t1;z bxrerial hos:. for many gcnrIations, Other temperate phages such AS PI, h3L-e propfiagc's that do not integrate -into the bacterial chroiuosoxe hut rather replicate while attached to thz hactcrial inner cell n:ai?brane. As such, th-se prophages arz plas.;;icls. One nzed only examine fiLtrates of fecal susp~.~?sloas, raw scwage, soil, vater, uapasteurized diary products or even diseased tissue to learn that both virulent and temperate phages are vary cmmon in nsture. The systematic starch of bacterial s?ecies for the presence oE a carried teiqxrcite phage is so often successful that some wrirers have been moved to rezzrk that it is difficult to believe that there are many bacterial cells that are not carrying at least onz temperate phage! Th-is certainly seems to be the c3se, for exarr,pl.e, when speaking oE staphylococci but for other bacterial species the reported inci- dence of carried phage varies from 22 to 94%. oE this document we are primarily interested in the strain E. coli I(-12 and the bacteriophage A and its derivatives, it is probabl;i best to simply focus on how often E. coli species of natural origin carry phages which can also infect E. coli K-12 and how many of these phages are `lambdoid'. Since for the major purpose -- -- -- Apparently phages resezr,bling A are not uncoznon in wild-rype E. coli. - For exaxple some 20 years ago Jacob and T?ollman found that 32 or 500 fecal E. coli carried temperate phages capable of propagation on E. coli K-12 deri- vative. and at lezst six others could recombine with X. carried by E. coli K-12 but were not related to X. Nore recent unpublished. observations from several laboratories have confirmed these findings and it is probab1y.fair to say that some 8% to 10% of all fecal E. coli harbor at least one phage capable of infecting E.col.1 K-12 and that from 1% to 2% of fecal E. coli carry a phage that is closely related to A. - Among these 32 phages, 3 were apparently identicalto All of the other phages could be effectively -- -- -- -- Sorce temperate phages alter profoundly the properties of bacteria that becoae lysogenized. responsible for the synthesis of a nuraber of clinically important bacterial products such as diphtheria toxin, (C. diphtheriae), fibrinolysin (S. aureus), erythrogenic toxin (S. pyogenes) , tetanus toxin (C. tetani) , bo tuliTum toxin (C, botulinum) , and Tor the serological specif ici'fy of the somatic antigens (endo toxins) of Salmonella species and enteropathogenic -- E - coli. the bacteriophage genome encodes the genetic inforaation for the synthesis of the specific protein product. This process has been tenet? phage conversion and is In each case, Phages are capable of transduction (phage-mediated gene transfer) and this is probably true €or all temperate phages as well as some virulent phages. Transducing phages can pick up DNA from prophages and/or plasmids in donor strains as well as chromosomal DNA and introduce it into appropriate recipient strains. genic donor and non-lysogenic recipient strains for both 5. aureus and -- E. coli. Transducing phages or their DNA are also taken up by marmalian cells in culture where they persist and/or replicate and in at least one instance express func- tional gene products. Transduction has been demonstrated to occur in mice by using lyso- In closing, it should be noted that there has been increasing evidence over the years to suggest specific relationships between temperate phages and plasmids. the chromosome but replicate and persist in bacterial cells as extrachromosomal DNA or plasmids, The generalized transducing phage! pflG of - Pseudomonas putida, in picking up the genes €or degradation of mandelatz, was found to acquire the ability to act as a conjugative plasmid and to pronote transfer of both Mutant derivatives of A have been found that fail to integrate into chronosornnl series and genes for nandzlare degradation to rzcipient strains. The discovery that inberiixnce of donor genetic rmrkers in intergeneric matings between E, coli donors and S. typhi recipients and between Yllebsiella pneumoiyjae domrs and E. -- coli recipients often results in the Eornation of new plasmids, -~ - --- --- - raises the question as to the origin oE the genes to perm5.t autonomous replica- tion of these elements. Yfie ubiquity of both defective and non-defective pro- phages in lysogenic bacteria that should contain such inforration leads us to believe that such defectivz and/or non-dezective integrated prophages night contribute the necessary information for the fornation aild replication of donor DXA fragments as autonomously replicating circular plasmid molecules in reci- pient strains as a consequence of intergeneric ratings. Ihe exaiqles given bel0:.7 are mtioly for illustrattvc purpuszs. ~op.2 of the experiments might not be possible, and thnre is little or no jiistificacion for the performance of certain others. A. EsaEples of Class I Zxperinent: 1. J, from E. coli K-12. 2. OK 080 DNA. 3. Transformation, transduction, or transfection of Bacillus subtilts 168 with E. subtilis 168 chromsomal DNA or PES1 phage. 4: TransforEation of a well-established laboratory strain of Nzisseria catarrhalis bjj DM derived from the sann str2Ln. Transductional gene transfer to Escherichia coli using phages pi or Transformation of E. coli R-12 with E. coli E(-12 chronosonal, F plasmid -- -- - -- B. Examples of Class I1 Experiment: 1. lilurium 2. Conjugal gene transfer between Hfr and F- strains of Salmonella typhi- Conjugal gene transfer between 3fr and F- enteropathogenic E. coli LT2. -- strains. 3. Formation of a recombinant plasmid between the pSClOl (tetracycline resistance) and RSFlOlO (streptomycin and sulfonamode resistance) plasmids when introduced into E. coli strain I;-12. 4. 5. 6. Formation of a recombinant replicon between phage h and the ColEl Integration of the plasmid R64 into the chroEosome of - S. typhimuriun A survey oE the host range plasmid when introduced into E. coli K-12. LT2, and its excision to isolate an R' plasmid. isolated from nature when introduced into - E. coli K-12, - S. typhimurium LT2 and Shigella dysenteriae SK. Construction of a recombinant between phage P1 and an ampicillin resis- tance (Ap) plasmid, and the introduction of the recombinant P1-Ap molecule into -- of R plasmids found in S. -~ typhi strains 7. E. coli K-12. -- 8. Construction of a recombinant between bacteriophage Mu and the R plasmid Rldrdl9 and its introduction into E. -- coli K-12. (Fredericq) plasmid when introduced into -- E. coli. (It should be noted that a colicin V gene identical or similar to that on the Fredericq plasmid has been identified in a high proportion of bacterial strains involved in extra-intes- tinal infection.) 10. Construction of a recombinant DNA molecule involving the plasmid of B. pumilus (carrying genetic information for the inhibition of sporulation) and a temperate phage from B. subtilis when introduced into E. subtilis. 9. Construction of recombinant molecules between phage 080 and the Col 9 11. Intragenerictransformation of chroroosornal DXA in avirulent strains of Streptococci. 12. Intrageneric transformation of chromosonal DXA in Bacillus species except E. anthracis . - __- I. Construt-tion oE a recor25in~nt 172Jh molectlI.cn. buLveen t?Lc cryptic plasmid frorr, SI tvphimuriuG LT2 and the S taohylacoccca axweus ?lnsnid pI2.53 anr! its iutroductian into S. atireus. fibrinolysin into a S. albus strain. gaies and a plasrnid or bacteriophage replicon fron E. coli, ad their introduc- tion into E. coli. (specifies ctiioramphenicol resis Lance) from S . pnecmonlae and ColEl, and their introduction into E. coli. 5. Construction of a recombinant DNA molecule between X or pSClOl and a plasmid d2rived from Streptoxyces coelicolor and its introduction into E, coli. 6. Construction of recoxbinant DNA molecules betwem E. coli genes involved in histidine biosynthssis and a 'is. pumilus plasmid, and their introduction into B. subtilis. 7, genes froin Xombyx mori, wh~n ictroduced into E. coli. 8. gene and ColEl and its introduction into E. coli. 9. xosas putida and either phage ?, or the RSPlOlO plasmid, and its introduction into E, coli. 10, Construction of a DNA chiEzra between TilOuse mitochondrial DXA and phage h or the pSClOl plasnid vhen introduced into E. coli K-12. --c- --.__ - --_- __--_ - r It 2. ine iatroduction of a phage fron S. zur*?us thnt leads to production of 3. Construction OE reconbinant DNA molecules becweeil sea urchin histone - - __--- - -- - -- 4. Construction of recombinant DXA molectdes between the Ci3 plasnid - _. - --- I -~ -__I-II- -__I - - - Construction of a recoxbinant p1asmi.d or phage that ir?.cludes fibroin Construction of a recoEbinant DNA molecule between the chicken ovalbrmin Construction of a recoxbinant: rcolecule between the OCT plasmic! of Pseudo- -____ -- -- -I__ -- -- -- D. Exaqles of Class IV Eqerimeilt: 1. Construction of recombinant DNA molecules containing DXA from a phage of S. aureus that codes €or the production of fibrinolysin and either E. coli plasmid or phage DXA, 2nd their introduction into -- E. coli. derived from any prokaryotic or eukaryotic organism, and E. -- coli phage or plasmid DNA and their introduction into E. coli. Construction of a recombinant DNA noleccile between plasmid DXA (specify- ing the synthesis of kananycin) fron Streptomyces kanamyccticus and -- E. coli plas- i6.d or bacteriophage DNA, and its introduction into E. coli. 4. Construction of a reconbinant betwezn an 2. mutans cariogenic plasmid 1 and an E. coli plasmid and its introduction into E. coli. 5. Construction of a chimeric DNA molecule containing a single purified DNA fragnent derived from cucuaber mosaic virus and ColEl and its introduction into - -- 2. Construction of recombinant nzolecules between genes for photosynthzsis, I- 3. -- E. coli. -- 6. Construction of a recon3inant between the gene coding for the synthesis - of human growth h0rmor.e and the pSCl01 plasmid, and its introduction into E. __I_ coli. 1 .. Con.; truct ion OE a rccoritbincant betwenn thz - S. -..-_ axeus plas:nid that specifies exfoliative toxin production and an E. coli phzge or plasmid, and its introducrion into - E. coli. 2. Construction of rzcombinant DYA molecules betwen cryptic plasnid DNA from microoreanisms such as Yersinia psstis, I;. acthracis, or Brucella abortus and any other carrier molecule and th-ir introduction into 5. coli. 3. Construction oE 3 chineric DNA molecule which includzs the DNA o? 'Dane' particles of thz hepztitis 3 virus and bacteriophage A or plas~id DXA, and its introduction into E. coli. ---- -_._I_ - -- -- II F. Examples of Class VI Experimnr: 1. Construction of a recombinalk between the 9 phage of CorYneSacterfun diphtheriae that specifies toxin production and a phage or plaF~d from -- E. coli and its introduction into E. coli. 2. Construction of a recombinant conraining genetic information for toxin production from strains of Clostridiu botulinm - cr - C. -- tetani and -- E. coli phage or plasmid DXA and its introduction into E. -I__ coli. -- X - GeilerciL Cu5.d:ince Princinles Reoarding the Choice of Vnli-icl~)~ for DIJii. -___c__ ___-______ r---b ___I._----. __ Clo-iling hps rixents I. By selecting and/or genetically naniplil.eti2g vehicles used in cloning for- eign DNA, investigators n2.y minisize the possible biohazards involved in the c 0 ns t r u c t i o n c f g en e t i. c 2 1 l y a I t 2 re d 3.i c ?c 0 0 T 2 ail 2 s ins 1.: i t 11 out s 2 c r i f 2 c in g t h e ob - jec til-es of th;: experiixn?. LE ger-eral, no:~-c.onj ugative plas3ids are pref erabie to conjugative pl.as!n-i_ds as clonii~g vehicles. _1_---1_--_--11 2. bacteria are preferable to vehicles which ~ay oEfer such an advantage. Cloiiing vehicles which do not affer any biological. advantage to recipient 3. preferable to those existing as enczpsulated extracellular particles. ClOiliRg vehicles ~7htch ordinzrily have an jntracelluhr existence are 1;. Cloning vehicles that express genocypic or phenotypic properties that are already cozaon in the recipient bacterial specizs are preferable to those expressing less common properties. 5. A vehicle .r\-hich has not been subjected to experirental procedures, such as mutagenesis, vhich may alter its biological host range, is preferabl-. to ;? vehicle which has been subjected to such procedures. 6. gz :ion are preferable to rriId-t>-pe cloning vehicles. Cloning vehicles carrying genetic defects which nay restrict their propa- .7. Cloning veliicles that have bceri well. c,harzicterizsd r:ith regard to their- genctic and moiecular properties are preferable to those ~iilc!~ have not been ;!ii rv.iell studied. 3 .L 2. rare in exrrachror.csornal gene pools (e. 2. resistance to tricsthoprix and €TJsidic acid) should be avoided. The use of plasmids which carry antibiotic resistance gen?a tha'l are nonrzlljr 3. Certain antibiotic resistance genes are preferable to others €or use as selective agents ir? DNA cloning experiments; hence, tetracycline, sulfonamide, and streptomycin resistance are preferable for us2 because they occur naturally at high frequency among microorganisms present in both huzan and. ioeestic ani- nal populations. D. 1. Hosts that possess conjugative plasmids or prophages, which may facilitate dissemination of genetic material to other hosts, should be avoided if consis- tent with the objectives of the experimmt. Guidelines for Selection of BacterTa as DNA Donors and Recipients 2. perties of a donor or recipient strain, such strains should be avoided for construction of genetically altered Eicroorganism. Vhen little is known about the genetic, metabolic, and/or ecclogical pro- 3. Spore-forming nicroorganisns should not be used as donors or recipients of chimeric DXR molecules; mutant derivatives unable to form spores should be employed; restoration of sporogeny should not be a possible outcorn;? of theexperiment. E. Suggestions for Possible Genetic Modification of Recipient Strains Genetic modification of the recipient strains prior to introduction of recombinant DNA molecules nay contribute further to reducing or eliminating possible biohazards. The use of recipient strains that possess nutations that reduce pathogenicity, ability to survive and/or establish in a diversity of ecological habitats and/or transmit genetic information is therefore desirable.Exmples of genetic modifications that can be introduced into E. coli strains to accon- plish the above objectives are provided below: 1. 2. Use of a pur- mutant since purine-deficient mutants of many pathogenic Use of a dap- mutant since the amino acid diaminopinelic acid is not microorganisms are avirulent. very prevalent in natural environments and its absence will result in inability to synthesize the cell wall and thus lead to cell lysis. 3. would minimize the ability of the genetically altered microorganism to colonize animal hosts . 32OC. This would minimize the ability of the genetically altered microorganism to survive in soil, water and other natural environmznts. Use of a temperature-sensitive mutant that cannot grow at 37°C. This 4. Use of a cold-sensitive mutant that cannot grow at tezperatures be1Oi-7 5. Use of il Straiil th3t would be unable to fernmt or utilize a diversity oE carbohydratzs - e.g. a -- prs - mitarit, phosphotransferase system deftictive. This would contribute to rhe inability of tile genetically altzred nicmorganisn: to grow in a diversity of ecological k-bitats. 5. Use of a rrutant with =stations such as uvr, poU, etc. that VJOILL~ confer increased sensitivity to ultraviolet light, sir.ce this wor?ld contri- bute to inability of the genetically altered microorganism to survi~e in natural environments. 7. Use of a rec- mutant since this Eight reduce the exchange of genetic information by the recipient strain. 8. Use of a bacterial mutant that is deficient as a recipient of genetic information by conjugation. This would reduce the likelihooc! of introduction of conjugative plasmids from other bacteria in the nztural envirortrnents and thus reduce the likelihood of mobilization and traisniission of the informtion on the recombinant E4A molecule by conjugation. conjugation by bacteria nay also confer increased resfstance to a diversity of bacteriophages, and thus might reduce the likelihood of transmission of genetic information by transduction. ducing phages since this would ninirnize the likelihood of dissemination of gene- tic infornation from the genetically altered microorganism. - I_ Some mutations that inhibit 9. Use of a mutant that is resistant to a multitude of potential trans- 4. Introduction -_ After c3ns truction of a rccoin5i~~nt DXA molz~ul~ and Fts introduciion into r? r:j-crohizl host, it still he inpartant for the investisator to ;1ssoss thz b iohazards associared witE the formtion OE chis gecdtically altered Ricioorganism. In marly instances the informtion obtained fro% thzse studies wiLl require reclas- sification of the expmirrent into a ness class category. result in the experiment being designated in a class requiring less contein- nent, although in certain cizcumstances tfie determined biohazards may be Fore severe than originalty espscted which would require the reclassification of the expcriment into a class requiring a more sixingent lzvel of containment. ReclcssiFication right Cer tatn principles should be followed in obL=tntng informtion that might be useful in assessing the real biohazards associated with any given expet-i- ment. One should initially conduct specific experiments to dctermhe whether there are any alterations in the pathogenicity of the genetically altered micro- organism and any changes in its ecological potentials. If the altered Eicro- organism contains DNA specifying unknown gene products it will be difficult, if not impossible, to assess the biohazards associatcld with th2 distribution of this genetic inforination aaong microorganisms occupying the saoli? ecological niches as the recipient strain. In these instances it will not be possible to reclassify thz experiment to employ less stringent degrees of containment. In these evaluation exp2riments, thz cells containing recombinant DNA should be grown under the same conditions of contziment as were used in the experiments thzt produced them. If cell products are to be analyzed, the cells should be lysed or extracted under these saze conditions and these extracts tested for sterility prior to taking the material into a genercrl research laboratory where less containment is necessary. If the product is potentially toxic, then appropriate precautions need to be taken to protect the investigator from exposure, and special facilities shocld be utilized to house any animals and/or plants used for testing the product. are being evaluated for pathogenicity in aniroal or plant hosts, these animals or plants should be under containment facilities similar to those used for the construction of the genetically altered microorganism. Such animal or plant hosts must be disposed of in a way that will not permit dissemination of the organism being tested. organisins sliould be avoided if possible until there has been saxe assessment of the biohazard. ted under conditions of more stringent containm2nt. When the genetically altered microorganisms Tests requiring large numbers of altered micro- If this is not possible, then such experimenrs should be conduc- B. Infornation That \Jill Ce Helpful in Evaluatins Pathogenicity The following tests should not be considered to be all-inclusive si.nce the particular tests to be performed will be dictated by the nature of the geneti- cally altered microorganism, vith respect to both the origin of the genetic informarion on the recombinant DNA molecule and the particular attributes of the recipient host species. The design and conduct of specific experiments to evaluate the real biohazards will therefore reqirfre careful evaluation by the 34 1. 2, 3. 4 * 5. 6, 7. 8, Iilfectix..ity in appropriztc arrimts or plan~s. Colonization in the ipt, oral cavity, on tht.. skin, etc. of 3.1 hos;s or on the rmts, leav,~s, etc, of a22ro- priatte plants. Production of kerstoconjunctivi tis in guirxa pigs (thz Sereny tes'i) :\rhich would be an indicacion of tile capacity of the altered nicroorganisa to penetrp-te thc?. intestinal cxcosa. Xnvzsion and proliferation in cacrophages and/or fibroblasts. Production of such cell products as bacteriocins, hemolysins, fibrinolysins, collagenases, pectiaaszs, etc. that might ccm- tribute to colonizing ability and/or invasiveness and toxin; of various sorts and to test the potency of such toxins by using appropriate cell culttires of eukaryotic organisms, ligated intzstirsl loops oE appropriate ariitzal hosts or appropriate plant or animal species. Production of hypersensitivity and/or necrGsis by cells or extracts when injected ineradermally into the skin of appro- priate aninal hosts. Determination of the aicimal inhibitory concentrations of various antimicrobial agents useful in killing and/or inhibi- ting growth of the altered microorganism. Determination of whether or not the gene products specified by the recombinant DNA appear extracellularly, intracellularly or in the perlplasLzic space. C. Infomation That Will Be Helpful in Evaluating Ecological Potential The individual expgrirnents needed to assess ecological potential of the altered microorganism will of necessity be dictated by the properties of the strains used to construct it. The following types of experiments should there- fore only serve to illustrate the range of tests to determine the properties of the genetically altered microorganism: 1. Expression of the genetic traits that are specified by the recombinant DNA molecule. 2. Resistance to W, disinfectants, etc. 3. Survival in soil, water and the dry state or in say ecolo- gical habitat likely to be occupied. 4. Ability to form spores. 5. General metabolic activities and attributes including changes in growth rate, utilizable and preferred substrates, temperature and pH optima for growth, aerobic vs. anazrobic growth, photosynthetic and 82 fixing ability, etc. 6. Production of substances that displace or inhibit other micro- organisms that normally occupy the same ecological habitats. D. Other Information Needed to Evaluate the Severity of Biohazards It will be extremely important to test the ability of the recornbinant DNA contained in the altered microorganism to be transnitted by piiage and/or If one performs any or all of the above experiiaental tesrs to evaluate potential bio:,:zards of genetically altered microorganisws, it wj 11 bz neces- sary to inc1dz as controls the organisms used as donors of the genet5.c inor- mation to form the reconbinant DXA molecule as wdl as the rec-ipient host s t ra in.