PART 1 (COUNCIL DECISION 2002/813/EC)

Transcription

1 PART 1 (COUNCIL DECISION 2002/813/EC) SUMMARY NOTIFICATION INFORMATION FORMAT FOR THE RELEASE OF GENETICALLY MODIFIED ORGANISMS OTHER THAN HIGHER PLANTS IN ACCORDANCE WITH ARTICLE 11 OF DIRECTIVE 2001/18/EC A. General information 1. Details of notification (a) Member State of notification The Netherlands (b) Notification number B/NL/03/02 (c) Date of acknowledgement of notification 04/02/2004 (d) Title of the project Artificial colonization of clumping factor B deficient Staphylococcus aureus in the nose (e) Proposed period of release From 01/05/2004 until 30/03/ Notifier Name of institution or company: Erasmus MC, University Medical Center Rotterdam 3. GMO characterization (a) Indicate whether the GMO is a: bacterium (X) specify phylum, class eubacterium (b) Identity of the GMO (genus and species) Strain of Staphylococcus aureus in which the gene encoding ClfB, encoding a putative cytokeratin receptor, has been insertionally inactivated by introduction of a tetracyclin resistance cassette through homologous recombination. (c) Genetic stability according to Annex IIIa, II, A(10) High 4. Is the same GMO release planned elsewhere in the Community (in conformity with Article 6(1)), by the same notifier? 5. Has the same GMO been notified for release elsewhere in the Community by the same notifier? 6. Has the same GMO been notified for release or placing on the market outside the Community by the same or other notifier? 7. Summary of the potential environmental impact of the release of the GMOs. This release involves the application of an attenuated, genetically modified strain of the human commensal bacterial species Staphylococcus aureus in the nose of human volunteers. S. aureus is a ubiquitous bacterial species persistently or intermittently colonizing the vestibulum nasi of approximately half of mankind. The current GMO lacks an important adhesion factor, one that normally facilitates its stay in the human nostrils. The gene involved is replaced by an antibiotic resistance gene that has been frequently identified in natural Page 1 of 11

2 isolates of S. aureus as well. This gene cannot be disseminated through horizontal transfer since it is firmly anchored in the staphylococcal genome. Lack of the adhesion factor is thought to limit its possibilities to colonize. The mutant is considered less fit than wild type bacteria and, hence, is supposed to be a less able colonizer of the human nasal cavity. This implies that the impact of the GMO on its normal environment is anticipated to be low. This lack of impact is even further reduced by the fact that we have shown that resident strains are hardly ever replaced by incoming bacteria: once a person is colonized it is not often seen that replacement by a (less fit) competitor strain occurs. Finally, the GMO is broadly susceptible to a variety of antibiotics, rendering it susceptible to therapy and, therefore, easy to remove once established in our volunteers. The bacteria that are swallowed will not survive in gastric juice and the GMO has lowered expression of the sigma factor involved in the control of expression of virulence genes. This results in limitation of the capacity for environmental survival. B. Information relating to the recipient or parental organism from which the GMO is derived 1. Recipient or parental organism characterisation: (a) Indicate whether the recipient or parental organism is a: bacterium (X) (specify phylum, class) eubacteria 2. Name (i) order and/or higher taxon (for animals) eubacteria (ii) genus Staphylococcus (iii) species S. aureus (iv) subspecies n.a (v) strain NCTC (vi) pathovar (biotype, ecotype, race, etc.) n.a. (vii) common name n.a 3. Geographical distribution of the organism (a) Indigenous to, or otherwise established in, the country where the notification is made: Yes (X) No (.) Not known (.) (b) Indigenous to, or otherwise established in, other EC countries: (X) Yes (.) If yes, indicate the type of ecosystem in which it is found: Staphylococci are associated with human carriage and S. aureus can be encountered in the nostrils (either persistently or intermittently) of approximately 50% of mankind. (c) Is it frequently used in the country where the notification is made? not applicable Yes (.) No (.) (d) Is it frequently kept in the country where the notification is made? not applicable Yes (.) No (.) 4. Natural habitat of the organism (a) If the organism is a microorganism other, specify: in association with humans (b) If the organism is an animal: natural habitat or usual agroecosystem: Page 2 of 11

3 5. (a) Detection techniques The microorganisms can be cultured on synthetic media. The organism can be identified on the basis of microscopic, biochemical and serological characteristics. (b) Identification techniques These systems involve Gram staining, Api identification systems (biomerieux; Vitek technology (biomerieux) and slide agglutination. The cultivation system can be made more specific by addition of the antibiotic encoded on the genetic element used to inactivate the adhesin gene. The sensitivity of culture based detection is in the order of 1-10 colony forming units. Furthermore, the organism can be detected by specific molecular diagnostics involving PCR. 6. Is the recipient organism classified under existing Community rules relating to the protection of human health and/or the environment? 7. Is the recipient organism significantly pathogenic or harmful in any other way (including its extracellular products), either living or dead? Yes (X) No (.) Not known (.) If yes: Like many other organisms, S. aureus can produce toxins and superantigens associated with significant disease. In addition, several of its cell wall components can be harmful if injected. The strain that we use in the current experiments, however, has been shown not to produce the main toxins and superantigens. (a) to which of the following organisms: humans (X) animals (X) plants (.) other (.) (b) give the relevant information specified under Annex III A, point II. (A)(11)(d) of Directive 2001/18/EC Pathogenicity (infectivity, toxigenicity, virulence, allergenicity, carrier, vectors, host range); Possible activation of latent viruses; Ability to colonize other organisms. Among normal, immunocompetent humans, S. aureus is not considered a dangerous pathogen. It can cause boils or other skin diseases, but these are usually well treated by antibiotics. S. aureus is frequently found to colonize the nose of humans, this occurs either persistently or intermittently in approximately 50% of all people. In healthy humans, carriage is not associated with invasive disease. Some strains of S. aureus are capable of producing toxins or superantigens. This is not the case for the GMO here under consideration. S. aureus can also be carried by other organisms, including pets and cattle. This is usually considered to be transient, since the staphylococci normally found on these organisms are belonging to genetically distinct lineages. The GMO does not contain activatable viruses, at least as far as is currently known. Bacterial viruses are, however, not considered to be pathogenic to man. 8. Information concerning reproduction (a) Generation time in natural ecosystems: The precise generation time in its natural human niche is not known. Since it has not been documented in detail whether or not the cells reside in solitary, clumped or biofilm-like constellation data on reproduction strategies and efficiency are currently Page 3 of 11

4 lacking. In vitro this mesophylic organisms replicates every 20 to 30 minutes, depending on nutritional availability. (b) Generation time in the ecosystem where the release will take place: See 8a (c) Way of reproduction: Asexual Factors affecting reproduction: At best, the organism will colonize the human vestibulum nasi. The organism is expected to reside at that location in numbers between 1000 and bacteria in a steady state-like fashion. Reproduction rates and efficiency are currently not known (see 8a). The reproduction time under environmental conditions has not been recorded. It is known that S. aureus has the capacity to survive outside of the host, as is the case with many Gram positive microorganisms. 9. Survivability (a) ability to form structures enhancing survival or dormancy: None (b) relevant factors affecting survivability: The GMO has been genetically engineered in order to modify one of its major adherence factors. Hence it is anticipated that its fitness is diminished resulting in lesser survivability. Lesser fitness is further promoted by the reduced sigb expression, which leads to lowered expression of several virulence factors that might be associated with colonization potential. 10. (a) Ways of dissemination The organism can be shed by the inoculated volunteers. It has been shown that S. aureus can be disseminated through aerosols and direct contact (hand-nose contact). (b) Factors affecting dissemination Cases of upper respiratory tract infection by other pathogens can contribute to the numbers of cells that are released in fomites. 11. Previous genetic modifications of the recipient or parental organism already notified for release in the country where the notification is made (give notification numbers) None C. Information relating to the genetic modification 1. Type of the genetic modification (i) insertion of genetic material (X) (ii) deletion of genetic material (X) (iii) others, specify allelic replacement by homologous recombination 2. Intended outcome of the genetic modification Inactivation of a gene encoding an adherence factor (clumping factor B, clfb) involved in cytokeratin binding. 3. (a) Has a vector been used in the process of modification? Yes (X) No (.) If no, go straight to question 5. (b) If yes, is the vector wholly or partially present in the modified organism? Yes (X) No (.) Page 4 of 11

5 If no, go straight to question If the answer to 3(b) is yes, supply the following information (a) Type of vector plasmid (X) (b) Identity of the vector pt181, containing a tetk resistance gene from S. aureus (c) Host range of the vector Staphylococci (d) Presence in the vector of sequences giving a selectable or identifiable phenotype Yes (X) No (.) antibiotic resistance (X) Indication of which antibiotic resistance gene is inserted Tetracyclin (e) Constituent fragments of the vector Not relevant, only the TetK cassette is ultimately introduced into the host organism (f) Method for introducing the vector into the recipient organism (i) transformation (X) 5. If the answer to question B.3(a) and (b) is no, what was the method used in the process of modification? Not relevant 6. Composition of the insert (a) Composition of the insert Tetracyclin resistance gene (b) Source of each constituent part of the insert pt181 (c) Intended function of each constituent part of the insert in the GMO Resistance gene (c) Location of the insert in the host organism - on a free plasmid (.) - integrated in the chromosome (X) - other, specify (d) Does the insert contain parts whose product or function are not known? If yes, specify D. Information on the organism(s) from which the insert is derived 1. Indicate whether it is a: bacterium (X) (specify phylum, class) eubacteria 2. Complete name (i) order and/or higher taxon (for animals) (ii) family name for plants (iii) genus Staphylococcus (iv) species S. aureus (v) subspecies Page 5 of 11

6 (vi) strain (vii) cultivar/breeding line (viii) pathovar (ix) common name 3. Is the organism significantly pathogenic or harmful in any other way (including its extracellular products), either living or dead? Yes (X) No (.) Not known (.) If yes, specify the following: (c) to which of the following organisms: humans (X) animals (X) plants (.) other.. (b) are the donated sequences involved in any way to the pathogenic or harmful properties of the organism Not known (.) If yes, give the relevant information under Annex III A, point II(A)(11)(d): not relevant 4. Is the donor organism classified under existing Community rules relating to the protection of human health and the environment, such as Directive 90/679/EEC on the protection of workers from risks to exposure to biological agents at work? 5. Do the donor and recipient organism exchange genetic material naturally? Yes (X) No (.) Not known (.) E. Information relating to the genetically modified organism 1. Genetic traits and phenotypic characteristics of the recipient or parental organism which have been changed as a result of the genetic modification (a) is the GMO different from the recipient as far as survivability is concerned? Yes (.) No (.) Not known (X) Specify It is currently not known whether the inactivation of the ClfB gene affects the viability or survivability of the GMO. It is anticipated that both are diminished because of the fact that an important adhesin gene, involved in maintenance of colonization, is inactivated. (b) is the GMO in any way different from the recipient as far as mode and/or rate of reproduction is concerned? Yes (.) No (.) Unknown (X) Specify See 1a above is the GMO in any way different from the recipient as far as dissemination is concerned Yes (.) No (.) Not known (X) Specify Again, due to the inactivation of an adhesin gene dissemination is supposedly diminished. (d) is the GMO in any way different from the recipient as far as pathogenicity is concerned? Yes (.) No (.) Not known (X) Page 6 of 11

7 Specify See 1a and 1b: probably less virulent 2. Genetic stability of the genetically modified organism High, mutation solidly embedded in the bacterial chromosome. 3. Is the GMO significantly pathogenic or harmful in any way (including its extracellular products), either living or dead? Yes (X) No (.) Unknown (.) (a) (b) to which of the following organisms? humans (X) animals (X) plants (.) other give the relevant information specified under Annex III A, point II(A)(11)(d) and II(C)(2)(i) Annex III A, point II. (A)(11)(d) of Directive 2001/18/EC Pathogenicity (infectivity, toxigenicity, virulence, allergenicity, carrier, vectors, host range); Possible activation of latent viruses; Ability to colonize other organisms. Among normal, immunocompetent humans, S. aureus is not considered a dangerous pathogen. It can cause boils or other skin diseases, but these are usually well treated by antibiotics. S. aureus is frequently found to colonize the nose of humans, this occurs either persistently or intermittently in approximately 50% of all people. In healthy humans, carriage is not associated with invasive disease. Some strains of S. aureus are capable of producing toxins or superantigens. This is not the case for the GMO here under consideration. S. aureus can also be carried by other organisms, including pets and cattle. This is usually considered to be transient, since the staphylococci normally found on these organisms are belonging to genetically distinct lineages. The GMO does not contain activatable viruses, at least as far as is currently known. Bacterial viruses are, however, not considered to be pathogenic to man. Annex III A, point II (C)(2)(i) of Directive 2001/18/EC Considerations for human health: The GMO is not considered to be toxic or allergenic, since it is not fundamentally different from S. aureus strains occurring in the wild. Little is known on the toxigenic or allergenic effects of metabolic product. The GMO is free of several of the commonly recognized toxins and superantigens. With respect to the parental organism, the GMO is considered to be even less pathogenic since it has been modified in a gene which is considered to encode an important adhesin, involved in maintaining the colonization state. Consequently, the capacity to colonize is most probably reduced. The GMO is not highly pathogenic to man. Other strains of S. aureus can be transferred from person to person and examples of S. aureus outbreak related disease have been described in literature. Strains possessing exfoliative toxins, for instance, have been implicated in outbreaks of impetigo. The infective dose for such events to occur are unknown. S. aureus can survive n inanimate surfaces, as most other gram positive organisms, the duration of survival is unknown, but depends on the availability of nutrients and water. There are no biological vectors, besides direct physical contact or phomites, described for S. aureus. The mutant is considered to be genetically stable, which is supported by the fact that S. aureus seems to be an organisms of limited genetic variability. The resistance pattern of the GMO reflects Page 7 of 11

8 susceptibility to most antibiotics, rendering many adequate options for (multiple independent) therapies. We are not aware of any obvious product hazards. 4. Description of identification and detection methods (a) Techniques used to detect the GMO in the environment See B5a and B5b above (b) Techniques used to identify the GMO See above F. Information relating to the release 1. Purpose of the release (including any significant potential environmental benefits that may be expected) The purpose of the release is to identify the relevance of the ClfB protein in the colonization of the human vestibulum nasi by S. aureus. This is highly relevant to substantiate the relevance of the ClfB receptor, which is keratin-10, in the colonization process. Identification of cytokeratin-10 as an important receptor could form the lead to a new generation of agents that could interfere with S. aureus colonization. 2. Is the site of the release different from the natural habitat or from the ecosystem in which the recipient or parental organism is regularly used, kept or found? 3. Information concerning the release and the surrounding area (a) Geographical location (administrative region and where appropriate grid reference): The greater Rotterdam area, Rotterdam, The Netherlands (b) Size of the site (m 2 ): Since 20 humans will be inoculated and since these humans will be at liberty to move around, this is hard to define. (c) Proximity to internationally recognized biotopes or protected areas (including drinking water reservoirs), which could be affected: Not relevant (d) Flora and fauna including crops, livestock and migratory species which may potentially interact with the GMO Humans and cattle 4. Method and amount of release (a) Quantities of GMOs to be released: 10 9 bacteria per individual (b) Duration of the operation: 6 months (c) Methods and procedures to avoid and/or minimise the spread of the GMOs beyond the site of the release None, the GMO is either eliminated from the site of inoculation within 2-3 weeks or establishes itself in numbers ranging between 1000 and colony forming units per individual. 5. Short description of average environmental conditions (weather, temperature, etc.) In The Netherlands the summers are of average temperature (20-25 o C) with occasional showers. Winters are mild, rainy and with lots of wind. Page 8 of 11

9 6. Relevant data regarding previous releases carried out with the same GMO, if any, specially related to the potential environmental and human health impacts from the release. G. Interactions of the GMO with the environment and potential impact on the environment, if significantly different from the recipient or parent organism 1. Name of target organism (if applicable) (i) order and/or higher taxon (for animals) mammalia (ii) family name for plants (iii) genus Homo (iv) species H. sapiens (v) subspecies (vi) strain (vii) cultivar/breeding line (viii) pathovar (ix) common name Man 2. Anticipated mechanism and result of interaction between the released GMOs and the target organism (if applicable) Transient or persistent colonization of the vestibulum nasi 3. Any other potentially significant interactions with other organisms in the environment The strains have been derived from humans and are generally human specific. In case of very intimate contact colonization of animals cannot be excluded, be it that it is generally accepted that animals are colonized with specialized staphylococcal lineages, distinct from the human lineages. 4. Is post-release selection such as increased competitiveness, increased invasiveness for the GMO likely to occur? Yes (.) No (X.) Not known (.) Give details The GMO has been modified in one of its major adhesion determinants. Consequently, the GMO will be at a disadvantage when compared to wild type S. aureus strains. In addition, individuals standing an elevated chance of acquiring staphylococcal infections are excluded from the study. This involves people suffering from boils or eczema, with antibiotic sensitivities or allergies, with endocarditis, being pregnant or suffering from sinusitis or diabetes. 5. Types of ecosystems to which the GMO could be disseminated from the site of release and in which it could become established None 6. Complete name of non-target organisms which (taking into account the nature of the receiving environment) may be unintentionally significantly harmed by the release of the GMO Not relevant 7. Likelihood of genetic exchange in vivo (a) from the GMO to other organisms in the release ecosystem: Page 9 of 11

10 (b) (c) Exchange of genetic material between S. aureus and other microbial species has only been demonstrated in the laboratory setting under forced conditions. Natural exchange is only likely to occur with coagulase negative staphylococci, although this is restricted: only limited numbers of examples are known and most of these involve the dissemination of resistance cassettes including methicillin resistance. This is a very low frequency event. from other organisms to the GMO: Again, only exchange with coagulase negative staphylococci cold possibly occur given optimal environmental circumstances. This will be a low frequency event. likely consequences of gene transfer: Seen the ubiquity of the resistance marker used to construct the GMO it is unlikely that exchange events will generate deleterious events. 8. Give references to relevant results (if available) from studies of the behaviour and characteristics of the GMO and its ecological impact carried out in stimulated natural environments (e.g. microcosms, etc.): No data available. 9. Possible environmentally significant interactions with biogeochemical processes (if different from the recipient or parental organism) Not relevant H. Information relating to monitoring 1. Methods for monitoring the GMOs The GMO can be detected by specific cultivation or biochemical testing. The GMO and its diagnostic resistance can also be identified on the basis of DNA testing (see also B5a and B5b above). 2. Methods for monitoring ecosystem effects 3. Methods for detecting transfer of the donated genetic material from the GMO to other organisms 4. Size of the monitoring area (m 2 ) The GMO will be applied intranasally. Surface of colonization will be restricted to a few cm 2. During the experiments volunteers will also be samples at the perianal region, the throat and the arm pits to follow eventual dissemination from the nasal cavity to other body sites. 5. Duration of the monitoring 6 months after which therapy will be provided, assuming that the GMO can still be detected. Monitoring will continue until the GMO cannot be detected anymore in the volunteers. 6. Frequency of the monitoring Monitoring will be performed at weekly intervals spacing for a period of three months. After the initial frequent screening, the intensity of screening will be reduced to once a month. I. Information on post-release and waste treatment Page 10 of 11

11 1. Post-release treatment of the site Ultimately, the nasal cavity will be treated with antibiotics (mupirocin, once daily for five consecutive days). Treatment will be offered to all volunteers.. When colonization at other body sites is detected, volunteers will be offered systemic antibiotic treatment courses. 2. Post-release treatment of the GMOs See (a) Type and amount of waste generated (b) Treatment of waste J. Information on emergency response plans 1. Methods and procedures for controlling the dissemination of the GMO(s) in case of unexpected spread Antibiotic treatment of volunteers and their close relatives. 2. Methods for removal of the GMO(s) of the areas potentially affected See Methods for disposal or sanitation of plants, animals, soils, etc. that could be exposed during or after the spread No specific measures will be required. The GMO is not pathogenic and fully susceptible to antibiotics. 4. Plans for protecting human health and the environment in the event of an undesirable effect Any undesirable effects of the colonization experiments will be noted in the early phases of the experiment. Volunteers will be closely monitored by high-frequency consultation and culturing. All volunteers will be under close supervision of an independent medical doctor for the duration of the experiments. In addition, there will be a doctor on call (24 hours a day, seven days a week) during the entire experiment. Molecular testing will be performed in addition. Antibiotic treatment will be provided immediately in case of any unexpected sideeffect. In case of such events occurring, the trial will be stopped immediately. Page 11 of 11