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Update of the list of QPS‐recommended biological agents intentionally added to food or feed as notified to EFSA 10: Suitability of taxonomic units notified to EFSA until March 2019

Abstract The qualified presumption of safety (QPS) procedure was developed to provide a harmonised generic pre‐evaluation to support safety risk assessments of biological agents performed by EFSA's Scientific Panels. The taxonomic identity, body of knowledge, safety concerns and antimicrobial resistance were assessed. Safety concerns identified for a taxonomic unit (TU) are, where possible and reasonable in number, reflected by ‘qualifications’ which should be assessed at the strain level by the EFSA's Scientific Panels. During the current assessment, no new information was found that would change the previously recommended QPS TUs and their qualifications. The list of microorganisms notified to EFSA from applications for market authorisation was updated with 47 biological agents, received between October 2018 and March 2019. Of these, 19 already had QPS status, 20 were excluded from the QPS exercise by the previous QPS mandate (11 filamentous fungi) or from further evaluations within the current mandate (9 notifications of Escherichia coli). Sphingomonas elodea, Gluconobacter frateurii, Corynebacterium ammoniagenes, Corynebacterium casei, Burkholderia ubonensis, Phaeodactylum tricornutum, Microbacterium foliorum and Euglena gracilis were evaluated for the first time. Sphingomonas elodea cannot be assessed for a possible QPS recommendation because it is not a valid species. Corynebacterium ammoniagenes and Euglena gracilis can be recommended for the QPS list with the qualification ‘for production purposes only’. The following TUs cannot be recommended for the QPS list: Burkholderia ubonensis, due to its potential and confirmed ability to generate biologically active compounds and limited of body of knowledge; Corynebacterium casei, Gluconobacter frateurii and Microbacterium foliorum, due to lack of body of knowledge; Phaeodactylum tricornutum, based on the lack of a safe history of use in the food chain and limited knowledge on its potential production of bioactive compounds with possible toxic effects.


Introduction
The qualified presumption of safety (QPS) approach was developed by the EFSA Scientific Committee to provide a generic concept to prioritise and to harmonise risk assessment within the European Food Safety Authority (EFSA) of microorganisms intentionally introduced into the food chain, in support of the respective Scientific Panels and Units in the frame of market authorisations (EFSA, 2007). The list, first established in 2007, has been continuously revised and updated. The publication of the overall assessment of the taxonomic units (TUs) previously recommended for the QPS list is to be evaluated every 3 years through a scientific Opinion by the Panel on Biological Hazards (BIOHAZ). Intermediate deliverables in the form of a Panel Statement are produced and published for periods of around 6 months, should an assessment for a QPS classification of a microbiological agent notified to EFSA be requested by the Units dealing with feed additives, food enzymes, food additives and flavourings, novel foods, or plant protection products. These Panel Statements also include the results of the assessment of the relevant new papers related to the TUs with QPS status.

1.1.
Background and Terms of Reference as provided by EFSA 1.1.1. Background as provided by EFSA A wide variety of microorganisms are intentionally added at different stages into the food and feed chain. In the context of applications for market authorisation of these biological agents, used either directly or as sources of food and feed additives, food enzymes and plant protection products, EFSA is requested to assess their safety.
Several taxonomic units (usually species for bacteria and yeasts, families for viruses) have been included in the qualified presumption of safety (QPS) list either following notifications to EFSA or proposals made initially by stakeholders during a public consultation in 2005, even if they were not yet notified to EFSA (EFSA, 2005). 1 The EFSA Scientific Committee reviewed the range and numbers of microorganisms likely to be the subject of an EFSA Opinion and published in 2007 a list of microorganisms recommended for the QPS list. 2 In 2007, the Scientific Committee recommended that a QPS approach should provide a generic concept to prioritise and to harmonise safety risk assessment of microorganisms intentionally introduced into the food chain, in support of the respective Scientific Panels and EFSA Units in the frame of the market authorisations. The same Committee recognised that there would have to be continuing provision for reviewing and modifying the QPS list and in line with this recommendation, the EFSA Scientific Panel on Biological Hazards (BIOHAZ) took the prime responsibility for this and started reviewing annually the existing QPS list. The first annual QPS update 3 was published in 2008 and EFSA's initial experience in applying the QPS approach was included. The potential application of the QPS approach to microbial plant protection products was discussed in the 2009 update. 4 Also in 2009, bacteriophages were assessed and were not considered appropriate for the QPS list. After consecutive years of reviewing the existing scientific information, the filamentous fungi (2008 to 2013 updates) and enterococci (2010 to 2013 updates) were not recommended for the QPS list. The 2013 update 5 of the recommended QPS list included 53 species of Gram-positive non-spore-forming bacteria, 13 Gram-positive spore forming bacteria (Bacillus species), one Gram-negative bacterium (Gluconobacter oxydans), 13 yeast species, and three virus families.
In 2014 the BIOHAZ Panel, in consultation with the Scientific Committee, decided to change the revision procedure: the overall assessment of the taxonomic units previously recommended for the QPS list is no longer carried out annually but over 3-year periods. From 2017, the search and revision of the possible safety concerns linked to those taxonomic units start to be done every 6 months period. The revision of the 2013 update (EFSA Biohaz Panel, 2013)  and the next update will be published in a scientific Opinion of the BIOHAZ Panel after its adoption in December 2019. 6 The QPS list of microorganisms has been maintained and frequently checked, based on the evaluation of extensive literature searches. In the meantime and every 6 months, a Panel Statement, compiling the assessments for a QPS status of the microbiological agents notified to EFSA requested by the Feed Unit, the Food Ingredients and Packaging (FIP) Unit, the Nutrition Unit or by the Pesticides Unit, has been produced and published. In the follow up of the 2013 update 5 the Scientific Committee agreed to exclude some biological groups (filamentous fungi, bacteriophages and Enterococcus faecium 7 ) notified to EFSA from the QPS assessment because it was considered unlikely that any taxonomical units within these groups would be granted QPS status in the foreseeable future. Thus, the assessment of members of these biological groups needs to be done at a strain level, on a case-by-case basis, by the relevant EFSA Unit. The QPS provides a generic safety pre-assessment approach for use within EFSA that covers risks for human, animals and the environment. In the QPS concept a safety assessment of a defined taxonomic unit is considered independently of any particular specific notification in the course of an authorisation process. The QPS concept does not address hazards linked to the formulation or other processing of the products containing the microbial agents and added into the food or feed chain. Although general human safety is part of the evaluation, specific issues connected to type and level of exposure of users handling the product (e.g. dermal, inhalation, ingestion) are not addressed. In the case Genetically Modified Microorganisms (GMMs) for which the species of the recipient strain qualifies for the QPS status, and for which the genetically modified state does not give rise to safety concerns, the QPS approach can be extended to genetically modified production strains (EFSA BIOHAZ Panel, 2018a). 8 Assessment of potential allergenicity to microbial residual components is beyond the QPS remit; if there is however, science-based evidence for some microbial species it is reported. Where applicable these aspects are assessed, separately by the EFSA Panel responsible for assessing the notification. Antimicrobial resistance was introduced as a possible safety concern for the assessment of the inclusion of bacterial species in the QPS list published in 2008 QPS Opinion (EFSA, 2008) 3 . In the 2009 QPS Opinion (EFSA BIOHAZ Panel, 2009) 4 a qualification regarding the absence of antimycotic resistance for yeasts was introduced.

Terms of Reference as provided by EFSA
The Terms of Reference, as provided by EFSA are as follows: ToR 1: Keep updated the list of biological agents being notified in the context of a technical dossier to EFSA Units such as Feed, Pesticides, Food Ingredients and Packaging (FIP) and Nutrition, for intentional use directly or as sources of food and feed additives, food enzymes and plant protection products for safety assessment.
ToR 2: Review taxonomic units previously recommended for the QPS list and their qualifications when new information has become available. The latter is based on a review of the updated literature aiming at verifying if any new safety concern has arisen that could require the removal of the taxonomic unit from the list, and to verify if the qualifications still efficiently exclude safety concerns.
ToR 3: (Re) assess the suitability of new taxonomic units notified to EFSA for their inclusion in the QPS list. These microbiological agents are notified to EFSA and requested by the Feed Unit, the FIP Unit, the Nutrition Unit or by the Pesticides Unit.

Interpretation of the Terms of Reference
The absence of acquired genes conferring resistance to clinically relevant antimicrobials is a qualification 9 applied to all QPS bacterial TUs. The verification of such qualification is under the remit of the Unit conducting the safety assessment of the organism notified to EFSA for market authorisation, and is therefore done at strain level (EFSA BIOHAZ Panel, 2017a).
In June 2017 (EFSA BIOHAZ Panel, 2017b), the BIOHAZ Panel has agreed to exclude Escherichia coli and any species of the genus Streptomyces from QPS evaluation within this mandate.
In June 2018(EFSA BIOHAZ Panel, 2018b, the BIOHAZ Panel clarified that the qualification 'for production purpose only' implies the absence of viable cells of the production organism in the final product and can also be applied for food and feed products based on microbial biomass.

2.
Data and methodologies

Data
Only valid TUs covered by the relevant international committees on the nomenclature for microorganisms are considered for the QPS assessment.
In reply to ToR 3, (re)assessment of the suitability of TUs notified within the time period covered by this Statement (from October 2018 to March 2019) is carried out. The literature review considered the identification, the body of knowledge, the potential safety concerns, and the knowledge on acquired antimicrobial resistance (AMR). Relevant databases, such as PubMed, Web of Science, Cases Database, CAB Abstracts or Food Science Technology Abstracts (FSTA) and Scopus, were searched. More details on the search strategy, search keys, and approach are described in Appendix A.
In reply to ToR 2, concerning the revision of the TUs previously recommended for the QPS list and their qualifications, an extensive literature search (ELS) was conducted as described in Appendices B and C.

Evaluation of a QPS recommendation for taxonomic units notified to EFSA
In response to ToR 1, the EFSA Units were asked to update the list of biological agents being notified to EFSA. A total of 47 notifications were received between October 2018 and March 2019, of which 32 were for a feed additive, 2 for food enzymes, 10 for novel foods and 3 for plant protection products (Table 1).
In response to ToR 3, out of the 47 notifications, 19 were related to TUs that already had QPS status and did not require further evaluation. Of the remaining 26 notifications, 20 were related to TUs not evaluated for a QPS status for the following reasons: • eleven notifications related to filamentous fungi, which were excluded from QPS evaluations in the follow-up of a recommendation of the QPS 2013 and 2016 updates (EFSA BIOHAZ Panel, 2013, 2014, 2016 • nine notifications related to E. coli, which were recently excluded from the current mandate by the BIOHAZ Panel.
The notifications received by EFSA, per risk assessment area, by biological group from October 2018 to March 2019, are presented in Table 1. To explore the potential application of a machine learning technique (MLT) for screening papers in the context of the QPS project the performances of such technique were assessed against the previous batch of papers retrieved for the Bacillus and Yeasts taxonomic units.
To that purpose, the DistillerAI Toolkit included in the DistillerSR online software was used. DistillerAI 'Preview and Rank' function was used mapping the papers from 'Title screening' to 'Article evaluation'. The SVM algorithm with 100% training set and 100% references to preview was used and the references were subsequently tagged. The algorithm was trained on the combined results of the 2 reviewers in the QPS rounds from 1 June 2016 to 31 December 2017. This is considered a conservative approach since, in the case of conflicts among the experts, the algorithm considers the paper as relevant.
The MLT predicted screening results on the batch of papers corresponding to the period January-June 2018 were obtained and compared with the results obtained by the two reviewers in the real exercise.
The results of the exercise showed that, in the case of yeasts, MLT had around 88% and 80% of sensitivity and specificity, respectively, while, in the case of Bacillus, MLT had 100% and about 82% of sensitivity and specificity, respectively. Moreover, it was found that in case of using the MLT algorithm as a reviewer in parallel with a human reviewer, in both projects no information relevant for the QPS status would have been missed.
On the basis of these results and considering the high number of papers retrieved for both yeasts and Bacillus in the context of the QPS exercise, it was decided to use the MLT in parallel with a human reviewer to screen the current batch of papers in these two TUs.
As expected, considering its specificity, the application of the MLT algorithm resulted in a high number of potentially relevant papers at the end of the screening phase. On the other hand, the algorithm did not miss any paper identified as potentially relevant by the human reviewer.

Monitoring of new safety concerns related to the QPS list
The aim of the ELS carried out in response to ToR 2 (review of the recommendations for the QPS list and specific qualifications) was to identify any publicly available studies reporting on safety concerns for humans, animals or the environment caused by QPS organisms since the previous QPS review (i.e. publications from July to December 2018). For a detailed protocol of the process and search strategies, refer to Appendices B and C. After removal of duplicates, 3,710 records were submitted to the title screening step, which led to the exclusion of 3,179 of them. The remaining 531 records were found eligible for the Title and abstract screening step, which led to the exclusion of 39 of these. Of the 492 articles that finally reached the Article evaluation step (full text), 85 were considered to be relevant for the QPS project.
The flow of records from their identification by the different search strategies (as reported in Appendix C) to their consideration as potentially relevant papers for QPS is shown in Table 2.  et al., 2000) and later on renamed as B. ubonensis (Anonymous, 2000).

Body of knowledge
There are only a limited number of scientific papers published on this TU. B. ubonensis as well as other Burkholderia species, produce lipases, which are used in a broad range of industrial applications.

Safety concerns
B. ubonensis is considered to be non-pathogenic, although its virulence potential has not been extensively tested. It appears to be innocuous after a subcutaneous challenge to mice (Price et al., 2017).

Antimicrobial resistance aspects
Different features, namely the external membrane structure and an inducible class A b-lactamase contributes to the intrinsic resistance to diverse antibiotics (Rhodes and Schweizer, 2016) Conclusions on a recommendation for the QPS list B. ubonensis cannot be recommended for the QPS list due to its ability to generate biologically active compounds and limited body of knowledge.

Identity
C. ammoniagenes is a species with Standing in Nomenclature. It was first described by Cooke and Keith (1927) as Brevibacterium ammoniagenes, an urea-splitting bacterium isolated from the human intestinal tract. It was transferred to the genus Corynebacterium by Collins (1987).

Body of knowledge
C. ammoniagenes is used for the industrial production of nucleotides, nucleosides and riboflavin (Koizumi et al., 2000;Serrano et al., 2017).
C. ammoniagenes derived single-cell protein 10 can also be used as a non-conventional protein source in animal diet such as in broilers  and growing pigs (Wang et al., 2013), without any negative effects on blood, bone characteristics or meat quality .

Safety concerns
No information was found in relation to pathogenicity of the organism, including when used for production of SCP for feed (Oliveira et al., 2017).

Antimicrobial resistance aspects
No information was found in relation to the AMR.
Conclusions on a recommendation for the QPS list C. ammoniagenes can be recommended for QPS list with the qualification 'for production purposes only'.

Corynebacterium casei
Identity C. casei was first isolated from the surface of a smear-ripened cheese (Brennan et al., 2001) and is a valid species according to the list of prokaryotic names with Standing in Nomenclature.

Body of knowledge
There are only a limited number of articles published on this TU. Most of the relevant articles described the microbial communities and activities in ripened cheese where C. casei contributes as a spontaneous contaminant to the production of the desired organoleptic properties (Brennan et al., 2002;Hannon et al., 2004;Mounier et al., 2005;Rea et al., 2007;Bockelmann, 2011;Cogan et al., 2014;Bertuzzi et al., 2017).

Safety concerns
One article described the identification of C. casei among 57 bacterial strains isolated during the screening of blood samples of patients with cardiovascular diseases and not having any active infection (Dinakaran et al., 2012), so no proven connection with the ongoing diseases was identified.

Antimicrobial resistance aspects
No information was found in the literature.

Conclusions on a recommendation for the QPS list
C. casei cannot be recommended to the QPS list due to lack of body of knowledge.

Identity
It is a species with Standing in Nomenclature (Mason and Claus, 1989). It was separated from Acetobacter in 1989. It has been classified as a separate cluster in the Gluconobacter genus according to 16S rRNA gene sequence analysis (Sievers et al., 1995) and to restriction analysis of 16S-23S rDNA internal transcribed spacer regions (Malimas et al., 2006).

Body of knowledge
There are only a limited number of articles published on this TU. It can produce high amounts of fructans and can efficiently produce glyceric acid from raw glycerol (Poljungreed and Boonyarattanakalin, 2018). It is an obligate aerobic acetic acid bacterium that is found as a spontaneous contaminant as part of the microbial community in some food processes (e.g. vinegar production and kefir, cocoa and coconut water fermentation) (Korsak et al., 2015;Ho et al., 2018). Although these foods are consumed and exposure of humans and animals to this microorganism is expected, the scientific knowledge on this species is limited.

Microbacterium foliorum
Identity M. foliorum is a valid species name according to the List of Prokaryotic Names with Standing in Nomenclature. It was first isolated as a plant associated bacterium (Behrendt et al., 2001).

Body of knowledge
There are only a limited number of articles published on this TU. M. foliorum is a saprophytic organism that may become endophytic and allow cruciferous plants to grow on oil seep soils, due to its capacity to process hydrocarbons such as catechol, toluene, naphthalene, octanol and others (Lumactud et al., 2016(Lumactud et al., , 2017. In addition, it is, as a spontaneous contaminant, part of the microbiota involved in production of flavour and aroma in surface-ripened cheeses, such as Limburger and Munster, due to its caseinolytic, aminopeptidase and deaminase activities (Deetae et al., 2007(Deetae et al., , 2009. Although these foods are consumed and exposure of humans to this microorganism is expected, the scientific knowledge on this species is still limited.

Safety concerns
M. foliorum has been associated with clinical samples. Laffineur et al. (2003) reported the isolation of 30 Microbacterium spp. specimens 'during the past decades' one of which was classified as M. foliorum, with no further indication of its source or the conditions of the patient. Gneiding et al. (2008) identified 50 isolates as belonging to the genus Microbacterium in a survey of bacteria obtained during the previous 5 years from pathological samples and processed in a German Coryneform reference laboratory. Unfortunately, only the origin of the samples was reported, with no indication of the patient conditions or the procedures to which they were subjected. Of these isolates, seven were ascribed to M. foliorum, their origin being wound swabs (5), pleural fluid (1) and blood (1).
A toxicological study was made on lysed cultures from a single strain without adverse effects, but the results cannot be extrapolated to the species (Kim et al., 2018a).

Antimicrobial resistance aspects
One paper (Gneiding et al., 2008) reported susceptibility profiles of M. foliorum but without reference to transmissibility of resistances.

Conclusions on a recommendation for the QPS list
M. foliorum cannot be recommended for the QPS list due to lack of body of knowledge.

Body of knowledge
Not applicable.

Safety concerns
Not applicable.

Antimicrobial resistance aspects
Not applicable.

Conclusions on a recommendation for the QPS list
S. elodea could not be assessed for a possible QPS recommendation because it is not a valid species.

Euglena gracilis
Identity E. gracilis belongs to the genus Euglena, phototrophic euglenoid flagellates possessing complex chloroplasts. The taxonomy of E. gracilis and closely related species has not been amended so far based on molecular phylogenetic analyses (Zakry s et al., 2017). The whole genome of the E. gracilis strain Z1 was recently sequenced (Ebenezer et al., 2019).

Body of knowledge
There are an extended amount of scientific papers published on this TU. E. gracilis is found in many freshwater habitats, especially in shallow eutrophic ponds. The species is able to synthesise biotechnologically relevant compounds such as polyunsaturated fatty acids, vitamins, b-glucans and tyrosine used in cosmetics and food supplements. E. gracilis is also used for bioremediation of heavy metals in contaminated water and as a toxicity bioindicator (Kraj covi c et al., 2015).
E. gracilis biomass, generally based on dried cells, is used as a feed additive in aquaculture and in animal feed as well as in human food (Suzuki, 2017). Food products containing E. gracilis are marketed in Japan as cookies, cereal bars and nutritional drinks.

Safety concerns
Using dried preparations of non-viable E. gracilis, no genotoxicity was observed in bacterial reverse mutation and mammalian micronucleus tests. Moreover, subchronic toxicity tests in rats did not show any adverse effect and a no-observed-adverse-effect-level (NOAEL) of 50,000 ppm was determined. (Simon et al., 2016).
Literature searches did not provide any evidence for a safety concern for human or animal health for any use of E. gracilis.

Antimicrobial resistance aspects
Not applicable.

Conclusions on a recommendation for the QPS list
E. gracilis may be recommended for the QPS list with the qualification 'for production purposes only'.

Identity
The unicellular, photosynthetic alga P. tricornutum was first isolated and described as a new species by the end of the 19th century (Bohlin, 1897). It is a pennate diatom (class Bacillariophyceae) and the only known species in the genus. It is an unusual diatom since it is pleiomorphic (fusiform, oval or triradiate morphology) and production of a silica frustule is facultative. Identification has recently been made by 18S rRNA gene sequencing (Demirel, 2016;Haro et al., 2017). The genome of one isolate has been sequenced (Bowler et al., 2008;Rastogi et al., 2018).

Body of knowledge
There are an extended amount of scientific papers published on the TU. P. tricornutum has been isolated from marine or brackish locations, often in benthic habitats, and appears to have a global distribution (De Martino et al., 2007). However, it is not a dominant species of algal communities and relatively little is known about its ecology.
It is of biotechnological interest, for studies within algal physiology and metabolism and as a model test species in toxicology. The potential of P. tricornutum as a source of biomass for biofuels, antimicrobial agents, health-promoting substances and food or feed components in general is subject to intensive study (Bajpai, 2016;Garcia et al., 2017;Haro et al., 2017;Shah et al., 2018). For example, the alga produces many compounds of high nutritional value, such as essential fatty acids and carotenoids (Zhang et al., 2015;Garcia et al., 2017). It has also been shown that P. tricornutum biomass can be used as a feed supplement (Skrede et al., 2011;Cerezuela et al., 2012;Sørensen et al., 2016).
Although several scientific studies are available, there appears to be no history of use of P. tricornutum in food or feed in practice, since no evidence was found that P. tricornutum, or substances produced by it, have yet been commercialised and/or widely consumed.

Safety concerns
The literature search did not reveal any studies reporting infection or intoxication by P. tricornutum. Niccolai et al. (2017) tested in vitro toxicity of water-or methanol extracts of P. tricornutum (strain F&M-M40) and found them to be toxic to fibroblasts. Neumann et al. (2018a) showed cytotoxic effects on human peripheral mononuclear cells.
In another study, Neumann et al. (2018b) investigated the bioavailability and toxicity of disrupted and freeze-dried cells of P. tricornutum in a mouse model and no adverse effect was reported.
Recent studies suggest that P. tricornutum can produce b-N-methylamino-L-alanine (BMAA) (R eveillon et al., 2016;Lage et al., 2019), which is a neurotoxin produced by certain cyanobacteria, diatoms and dinoflagellates. BMAA occurs ubiquitously in marine environments and can be transferred in food-webs to fish and seafood (Salomonsson et al., 2015;Lance et al., 2018).

Antimicrobial resistance aspects
Not applicable.

Conclusions on a recommendation for the QPS list
P. tricornutum is not recommended for QPS status based on the lack of a safe history of use in the food chain and on its potential for production of bioactive compounds with toxic effects.

Monitoring of new safety concerns related to organisms on the QPS list
The summaries of the evaluation of the possible safety concerns for humans, animals or the environment caused by QPS organisms described and published since the previous ELS (i.e. between January and June 2018, as described in Appendices B and C) and the references selected as potentially relevant for the QPS exercise (Appendix D) for each of the TUs or groups of TUs that are part of the QPS list (Appendix E) are presented below.
A search for papers potentially relevant for the QPS consideration of Bifidobacterium spp. and Carnobacterium divergens 11 provided 206 references. The analysis of their title left 21 articles; the rest were discarded because they did not deal with safety concerns. Three articles were found relevant for the QPS consideration of Bifidobacterium spp. at the level of title and abstract screening. For one article, the full text was not available (conference proceedings) (Magistrelli et al., 2018). For one of the two other articles (Suzuki et al., 2018), the study was not considered because of study design shortcomings in relation to QPS assessment. For the third article (Kim et al., 2018a,b), no safety concern was identified for both Bifidobacterium bifidum and Bifidobacterium longum.
Based on the available evidence as described above, the QPS status of Bifidobacterium spp. is not changed.

Carnobacterium divergens
A search for papers potentially relevant for the QPS consideration of Bifidobacterium spp. and Carnobacterium divergens provided 206 references. The analysis of their title/abstracts left 21 articles; the rest were discarded because they did not deal with safety concerns. No article arrived to the final stage for this TU. Consequently, the QPS status of C. divergens is not changed.

Corynebacterium glutamicum
A search for papers potentially relevant for the QPS consideration of Corynebacterium glutamicum provided 45 references. No paper reached the final selection phase, therefore no new safety concerns were identified.

Lactobacillus spp.
A search for papers potentially relevant for the QPS consideration of any of the 35 Lactobacillus species included in the list provided 555 references. Analysis of their title left 22 articles; the rest were discarded because they did not deal with safety concerns. Inspection of their abstracts allowed the selection of 12 papers that might raise safety concerns. After analysing the full texts, nine were considered to be not relevant, while the other three described safety concerns linked to L. paracasei (Kao et al., 2018) and L. rhamnosus (Naqvi et al., 2018;Zeba et al., 2018). In one case (Kao et al., 2018), no indication is provided on how the identification of the pathogen was done, while in Zeba et al. (2018) an automated phenotypical method was used, known not to reliably identify lactobacilli. Patients in the three studies (Kao et al., 2018;Naqvi et al., 2018;Zeba et al., 2018) presented with clear predisposing conditions.
Based on the available evidence as described above, the QPS status of the lactobacilli involved in the reported cases and, by extension, of all others included in the QPS list, is not changed.

Lactococcus lactis
A search for papers potentially relevant for the QPS consideration of Lactococcus lactis provided 173 references. Analysis of their title/abstracts left six articles; the rest were discarded because they did not deal with safety concerns. Analysis of the abstracts allowed selection of four papers that might raise safety concerns. After analysing the full texts, one was found not to deal with safety concerns (Kato et al., 2018) while the other three Kabor e et al., 2018;Shimizu et al., 2019) did. In two of them Kabor e et al., 2018) phenotypical methods for identification of the etiological agent were used, which are known not to be reliable for this species. In addition, two of the papers Shimizu et al., 2019) described cases in which clear predisposing conditions were identified. Shimizu et al. (2019) describes a patient suffering from a cholangiocarcinoma that blocked bile evacuation, reason why a bilioduodenal catheter was inserted. This might have been the source of the L. lactis cholangitis (predisposing condition) that appeared just 2 days later. The last reference (Kabor e et al., 2018) described the microbiota associated to 125 cases of endodontitis, out of which five contained L. lactis as part of a polymicrobial community, which is suggestive of colonisation of the oral cavity rather than aetiology of the infections.
Based on the available evidence as described above, the QPS status of Lactococcus lactis is not changed.
3.3.1.6. Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides A search for papers potentially relevant for the QPS consideration of Leuconostoc spp. and Microbacterium imperiale 11 provided 68 references. The analysis of their title left five articles; the rest were discarded because they did not deal with safety concerns. One paper lacked information on the identification procedures used to identify the infectious agents. Four papers arrived to the full text phase. Three were immediately excluded as they were not dealing with a safety concern or with these TUs. In the article of Menegueti et al. (2018), a 67-year-old female patient with chronic Chagas disease was submitted to several surgical interventions, and presented complications such as hypotension and hypoxemia. The identification of cultures was performed using automated phenotypical method known not to reliably identify Leuconostoc spp.
The safety concern identified was linked to severe underlying health conditions and the identification of strain was not reliable due to the use of phenotypic tests. Consequently the QPS status of Leuconostoc spp. is not changed.

Microbacterium imperiale
A search for papers potentially relevant for the QPS consideration of Leuconostoc and Microbacterium imperiale 11 provided 68 references. The analysis of their title left five articles; one paper lacked information on the identification procedures used to identify the infectious agents. The rest were discarded because they did not deal with safety concerns. None of the remaining four articles dealt with this TU. Consequently, the QPS status of M. imperiale is not changed.

Oenococcus oeni
A search for papers potentially relevant for the QPS consideration of Oenococcus oeni and Pasteuria nishizawae 11 provided 24 references. The analysis of their title/abstracts left one article for consideration. No paper reached the final selection phase, no new safety concern was found. Consequently, the QPS status of O. oeni is not changed.

Pasteuria nishizawae
A search for papers potentially relevant for the QPS consideration of Oenococcus oeni and Pasteuria nishizawae 11 provided 24 references. The analysis of their title/abstracts left one article for consideration. No paper reached the final selection phase, no new safety concern was found. Consequently, the QPS status of P. nishizawae is not changed.
A search for papers potentially relevant for the QPS consideration of Pediococcus spp. provided 146 references. The analysis of their title left two articles Singla et al., 2018). The latter does not describe a safety concern. Chen et al. (2018) describes an infectious endocarditis caused by L. lactis subsp. Lactis and Pediococcus pentosaceus. P. pentosaceus was identified after levofloxacin treatment in blood cultures, but the phenotypical method used for its identification is known not to be reliable for this species. In addition, the papers describe a case in which clear predisposing conditions were identified.
Consequently A search for papers potentially relevant for the QPS consideration of Propionibacterium spp. provided 27 references. Following the analysis of their title/abstracts, no articles were selected for the final selection phase, thus no new safety concerns were identified. Consequently, the QPS status of Propionibacterium spp. is not changed.

Streptococcus thermophilus
A search for papers potentially relevant for the QPS consideration of Streptococcus thermophilus provided 81 references. The analysis of their title/abstracts left one article that did not reach the final selection phase; thus, no new safety concern was found. Therefore, the QPS status of S. thermophilus is not changed.
A search for papers potentially relevant for the QPS consideration of Bacillus spp. and Geobacillus stearothermophilus 11 provided 804 references. The analysis of their titles left 199 articles. A first round has been conducted using the MLT as a co-assistant; the second round of analysis of the 199 articles by two experts left two articles for more in-depth analysis. The remaining articles were discarded because they did not deal with safety concerns. The paper of Osman et al. (2018) concerns the enterotoxinogenic potential of Bacillus pumilus strains, a topic covered by the current qualification for Bacillus spp. The paper of Tsonis et al. (2018) concerns the description of a cerebral abscess due to Bacillus subtilis in an immunocompetent patient. For both papers there were methodological shortcomings on the identification methods of the bacterial strains and therefore the data presented were not included for further assessment.
The ELS did not come up with any information that would change the status of the Bacillus species included in the QPS list.

Geobacillus stearothermophilus
A search for papers potentially relevant for the QPS consideration of Bacillus spp. and Geobacillus stearothermophilus 11 provided 804 references. The analysis of their titles/abstract left 199 articles. None was dealing with this species. Consequently, the QPS status Geobacillus stearothermophilus is not changed.

Gram-negative bacteria 3.3.3.1. Gluconobacter oxydans
A search for papers potentially relevant for the QPS consideration of Gluconobacter oxidans and Xanthomonas campestris 11 provided 115 references. The analysis of their titles left one article; the rest were discarded because they did not deal with safety concerns. No paper reached the final selection phase for this TU. Consequently, the QPS status of G. oxydans is not changed.

Xanthomonas campestris
A search for papers potentially relevant for the QPS consideration of Gluconobacter oxidans and Xanthomonas campestris 11 provided 115 references. The analysis of their titles left one article; the rest were discarded because they did not deal with safety concerns. No paper reached the final selection phase for this TU. Consequently, the QPS status of X. campestris is not changed.

Yeasts
A search for papers potentially relevant for the QPS consideration of the yeasts' species included in the QPS list provided 1,358 references. The analysis of their titles left 268 articles. A first round has been conducted using the MLT as a co-assistant; 229 of these were immediately excluded because they were not in English or because they were not dealing with safety concerns. Thus, the ELS identified 30 articles relevant for different yeast species with QPS status (please refer to Appendix D for the complete list of references).
For the other yeast species with QPS status, no relevant studies were identified through the ELS. Methodological problems were identified in 30 out of those 45 studies (corresponding to 22 articlessee Table 3). In 17 of those 30 studies (corresponding to 11 articles: de Paula et al., 2018;Hasan and Yassein, 2018;Kaur et al., 2018;Okmen et al., 2018;Ruelle et al., 2018;Soman et al., 2018;Treguier et al., 2018;Simi et al., 2019), a problem was found in respect to the methodology used for identity confirmation of the microorganism, therefore the value of these results and conclusions were very limited. In 2 of those 30 studies (corresponding to 1 article, , the problem was due to a lack of information regarding the source attribution. In 19 studies (corresponding to 15 articles: Afsarian et al., 2018;Diba et al., 2018;Das et al., 2018;Hasan and Yassein, 2018;Jung et al., 2018;Kara et al., 2018;Khedri et al., 2018;Kaur et al., 2018;Nagy et al., 2018;Omran and Mansori, 2018;Sadrossadati et al., 2018;Teblick et al., 2018), the problem was due to predisposing factors in the exposed subject.
In short, the ELS did not identify any information that would change the status for the yeast species included in the QPS list.  (2018) A search for papers potentially relevant for the QPS consideration of Alphaflexiviridae and Potyviridae 11 provided 39 references. No paper reached the final selection phase, thus no new safety concern was found.

Potyviridae
A search for papers potentially relevant for the QPS consideration of Alphaflexiviridae and Potyviridae 11 provided 39 references. No paper reached the final selection phase, thus no new safety concern was found.

Baculoviridae
A search for papers potentially relevant for the QPS consideration of Baculoviridae provided 69 references. No article reached the final selection phase, thus no new safety concern was found.
The ELS did not come up with any information that would change the current QPS status of any of the above virus families.

Conclusions
ToR 1: Keep updated the list of biological agents being notified, in the context of a technical dossier to EFSA Units (such as Feed, Food Ingredients and Packaging (FIP), Nutrition Unit and Pesticides Unit), for intentional use in feed and/or food or as sources of food and feed additives, enzymes and plant protection products for safety assessment: • Between October 2018 and March 2019, the list was updated with 47 notifications that were received by EFSA, of which 32 were for feed additives, 2 for food enzymes, food additives and flavourings, 10 for novel foods and 3 for plant protection products.
ToR 2: Review taxonomic units previously recommended for the QPS list and their qualifications when new information has become available: • In relation to the results of the monitoring of possible new safety concerns related to the QPS list, there were no results that justify removal of any TU from the QPS list or changes in their respective qualifications.
ToR 3: (Re)assess the suitability of taxonomic units notified to EFSA not present in the current QPS list for their inclusion in that list: • The TUs corresponding to 19 out of the 47 notifications received, already had a QPS status.
• Of the 28 notifications without a QPS status, 11 notifications related to filamentous fungi which were excluded from QPS activities in the follow-up of a recommendation of the QPS 2013 update (EFSA BIOHAZ Panel, 2013, 2014, 2016, 9 notifications related to E. coli, which was recently excluded from the current mandate by the BIOHAZ Panel (EFSA BIOHAZ Panel, 2018a).

Recommendations
• Burkholderia ubonensis cannot be recommended for the QPS list due to its ability to generate biologically active compounds and limited of body of knowledge.
• Corynebacterium ammoniagenes can be recommended for QPS list status with the qualification 'for production purposes only'.
• Corynebacterium casei cannot be recommended to the QPS list due to lack of body of knowledge.
• Euglena gracilis may be recommended for the QPS list with the qualification 'for production purposes only'.
• Gluconobacter frateurii cannot be recommended to the QPS list due to lack of body of knowledge.
• Microbacterium foliorum cannot be recommended to the QPS list due to lack of body of knowledge.
• Phaeodactylum tricornutum cannot be recommended for QPS status, based on the lack of a safe history of use in the food chain and on its potential production of bioactive compounds with toxic effects based on the lack of a safe history of use in the food chain and a limited knowledge on its potential production of bioactive compounds with toxic effects.
• Sphingomonas elodea could not be assessed for a possible QPS recommendation because it is not a valid species. Appendix A -Search strategy followed for the (re)assessment of the suitability of TUs notified to EFSA not present in the current QPS list for their inclusion in the updated list (reply to ToR 3)

Burkholderia ubonensis
A literature search was performed in PubMed database using the search term: "Burkholderia ubonensis": 18 citations were identified and screened.

Corynebacterium ammoniagenes
A literature search was performed in PubMed database using the search term: "Corynebacterium ammoniagenes": 112 citations were identified and screened.

Corynebacterium casei
A literature search was performed in Scopus database using the search term: "Corynebacterium casei": 125 citations were identified and screened from which, 40 were relevant for the assessment. • "E. gracilis" biotechnology 49 references.

Gluconobacter frateurii
A literature search was performed in Web of Science core collection and in Pubmed using the search term: "Gluconobacter frateurii": 74 and 45 citations were identified and screened respectively.

Microbacterium foliorum
A search using the species name "Microbacterium foliorum" in the Web of Science core collection provided 29 hits.

Phaeodactylum tricornutum
Literature searches were performed in the Web of Science Core Collection. Using only the species name "Phaeodactylum tricornutum" gave 2,917 references. Using refined search terms gave the following numbers of references, which were all scanned at title level: • "Phaeodactylum tricornutum" and (species characterization): 55 references. • "Phaeodactylum tricornutum" and taxonom*: 55 references. • "Phaeodactylum tricornutum" and ecology: 50 references.

Sphingomonas elodea
A literature search was performed in PubMed database using the search term: "Sphingomonas elodea": 26 hits were identified and screened, mainly dealing with the gellan gun production and the molecular characterisation of the enzymes and genes involved in this production.
Appendix B -Protocol for Extensive literature search (ELS), relevance screening, and article evaluation for the maintenance and update of list of QPS-recommended biological agents (reply to ToR 2) The following protocol for extensive literature search (ELS) will be used in the context of the EFSA self-task mandate on the list of QPS-recommended biological agents intentionally added to the food or feed (EFSA-Q-2016-00684).

B.1. Description of the process
An ELS of studies related to safety concerns for humans, animals, plants and/or the environment of microorganisms recommended for the Qualified Presumption of Safety (QPS) 2019 list will be performed.
The process will be performed according to the following main steps: • ELS for potentially relevant citations; • Relevance screening to select the citations identified by the literature search, based on titles and abstract and then full-text; • Evaluation of articles according to pre-specified categories of possible safety concerns; • Discussion between experts to come to collective expert evaluation of the outcome, reflected in the QPS Opinion and Panel Statements.
Considering the purpose of the QPS approach, a broad search will be performed.
The review questions will be broken down into key elements using the PECO conceptual model:

B.1.1. Objective
The aim is to identify any publicly available studies reporting on safety concerns for humans, animals or the environment caused by microorganisms on the QPS recommended list (see Appendix E).

B.1.2. Target population
The populations of interest are humans, animals, plants and the environment.

B.1.3. Exposure
Citations must report on at least one species included in one of the five groups of named species specified in the EFSA QPS recommended list of the QPS 2016 update ((see Table A.1 in Appendix A to (EFSA BIOHAZ Panel, 2017a)): a) Gram-positive non-spore-forming bacteria; b) Gram-positive spore-forming bacteria; c) Gram-negative bacteria; d) Viruses used for plant protection; e) Yeasts.
In more detail: a) Gram-positive non-spore forming bacteria: For the yeast species, as previously, the name of the teleomorphic form is used in the list of QPS species, when available. Important synonyms and older names were also included in the searches. For instance, names of the anamorphic growth forms were included, when such a form is known: • Xanthophyllomyces dendrorhous: anamorph Phaffia rhodozyma.

B.1.4. Comparator
It is expected that the prevalent study designs will be case reports or case series and studies based on surveys or isolate collections. The remaining study designs may include: studies using laboratory isolates; randomised controlled trials, field trials or experimental designs in the laboratory; experimental designs in live animals with a deliberate disease challenge; observational study designs; animal or insect models; investigations to identify or to understand the causes of safety concerns (e.g. identification, characterisation of toxic factors, virulence mechanisms); studies to demonstrate beneficial effects but with reporting of unwanted side-effects.
Since it is expected that in the majority of the study designs relevant for the review question, the comparator will not be available, the latter will not be included as a key element in the search strategy. B.1.5.

Outcomes of interest
The outcomes of interest to this ELS are: Question 1: • potential harms; • safety issues; • virulence or infectivity; • intoxication.
The QPS concept does not address hazards linked to the formulation or processing of the products based on biological agents added into the food or feed chain. Neither the safety of users handling the product nor the genetic modifications are taken into account. B.1.6.
Identification of the review questions The following research questions will be addressed: • Is there evidence of any safety concerns, including virulence features and toxin production, for humans, animals, plants and/or the environment associated with microbial species currently recommended for the QPS list since the previous QPS review (i.e. published from June 2016 until June 2019)?
• Is there evidence related to the presence or absence of antimicrobial resistance or antimicrobial resistance genes for the same microbial species published during the same time period?

B.2. Eligibility criteria for study selection
The selection of studies relevant to questions 1 and 2 will be performed applying the eligibility criteria described in Table B.1 below.

B.3.
Literature searches Searches will be conducted in a range of relevant information sources to identify any evidence of safety concerns and AMR regarding the target microbial species.
Considering the results of the previous QPS exercise, to handle the high number of studies identified in each group, 20 search strategies were prepared: three for yeasts, one for insect viruses, one for plant viruses, 13 for Gram-positive bacteria and two for Gram-negative bacteria according to named species specified by EFSA in the QPS recommended list of the QPS 2016 update (see Table A.1 in Appendix A to (EFSA BIOHAZ Panel, 2017a)).
The 20 subgroups of target microbial species will be searched separately. Each search strategy will comprise two elements: the search terms (Section B.3.1) and the information sources (Section B.3.2) to be searched.

B.3.1. Search terms
The search strategies used to identify studies are given in Appendix C. Each strategy will comprise two key elements: • Target microbial species as described in Section B.1.3 ('Exposure') • Safety issues as described in Section B.1.5 ('Outcomes').
In order to maximise the sensitivity of the search for the species for which the number of overall publications in the relevant time period is expected to be low, the search strategy will not include outcome-related terms.
The population of interest (humans, animals, plants or the environment) will not be included as a key element in the search strategies, as it is often not explicitly described within a title or abstract. It would also have been difficult to describe adequately such a broad population using title/abstract words and/or subject headings. Population information will be captured at the time of evaluating the articles (see Section B.1 above).
The 20 subgroups of target microbial species will be entered on separate search lines. The search line for each group will be combined with the safety terms individually.
The searches will not be limited by language or study design.
The review period will be from June 2016 to June 2019.

B.3.2. Information sources searched
The same information sources used for the previous QPS exercise (EFSA BIOHAZ Panel et al., 2017a) will be searched for studies reporting safety concerns regarding the target microbial species (see Table B.2 below).
Search results will be downloaded from the information sources and imported into EndNote ® X8 bibliographic management software. For each of the 20 species groups, within-group removal of duplicate entries will be done in EndNote ® X8. Following uploading of the species groups into the DistillerSR 12 online software, removal of duplicates will again be undertaken, using the Duplicate Detection feature.

B.4. Study selection and article evaluation
To identify potentially relevant studies to be included in the review the studies will be selected by a three -step procedure using the DistillerSR online software.
The results of the different phases of the study selection process will be reported in a flowchart as recommended in the PRISMA statement on preferred reporting items for systematic reviews and metaanalyses (Moher et al., 2009). Screening for potential relevance at title level Articles will initially be screened at title level in parallel by two Working Group (WG) expert reviewers and, if needed, EFSA staff.
If the information in the title is not relevant for the research objectives, the article will not proceed to the next step (Section B.4.2).
Articles that will be excluded during screening at this step will be stored in Distiller SR. In case of doubts or divergences between the reviewers, the paper will proceed to step 2.

B.4.2.
Screening for potential relevance at title and abstract level The articles passing the first step will undergo a screening at abstract level in parallel by two experts. If the information in title and abstract is not relevant for the research objectives, the article will not proceed to the next step (Section B.4.3).
Articles that will be excluded during screening at this step will be stored in Distiller SR. In case of doubts or divergences between the reviewers, the paper will proceed to step 3.

B.4.3. Article evaluation
The aim of this step will be to confirm that the article is relevant for the QPS project and, in case it is, to evaluate it. It will be carried out at full text level.
The articles passing the second step will undergo a validation procedure carried out by two experts. One reviewer will initially be tasked with the evaluation of a paper. The evaluation will be then forwarded to another reviewer for the validation of the appraisal received.
In case of disagreement with the initial appraisal, the second reviewer will write down their comments. The reviewers will initially try to solve the disagreement. In case this will not be possible, the conflicting information will be presented for collective expert evaluation of the ELS outcome (see Section B.5).
If the information contained in the article is not relevant for the research objectives, the article will not be evaluated. Articles that will not be considered relevant will be stored in Distiller SR.

B.4.3.1. Questions for study selection and article evaluation
STEP 1 (Screening for potential relevance): Question 1: Is the full-text available, in English and dealing with safety concerns?
• Yes: Include and continue to Article evaluation form; • Full text not available: Exclude; • Full text not in English: Exclude; • Full text in English but not dealing with safety concerns: Exclude.

STEP 2 (Article evaluation):
Question 2: Identification of the microorganisms • The article will be characterised in terms of the microorganisms involved; Single choice question: the Experts will identify the microorganism/s described in the article. In case more than one microorganism is described in the paper, the form will be repeated for each microorganism.
Question 3: Is there any "methodological" problem identified in the paper under consideration?
• No problems identified; • Yes some problems were identified.
Question 4: Which "methodological" problems were identified in the paper under consideration? (this question will appear in case in question 3 the option "Yes some problems were identified" will be selected) • Methodology used for identity confirmation of the microorganism; • Reliability of the source attribution; • Misuse of the microorganism (e.g. parenteral exposure); • Predisposing factors in the exposed subjects; • Others.
When one of the above options will be selected a dedicated free text box will appear to describe the problem identified.
Question 5: Is there any safety concern identified? (this question will appear in case in question 3 the option "No problems identified" will be selected) • No safety concerns identified; • Yes some safety concerns were identified.
Question 6: Which safety concerns were identified? (this question will appear in case in question 5 the option "Yes some safety concerns were identified" will be selected) • On human health; • On animal health; • On the environment; • On AMR; • On other aspects.
When one of the above options will be selected a dedicated free text box will appear to describe the safety concern identified. Question 7: Overall, is there any information that could potentially lead to a change in the QPS status of the microorganism? (this question will appear in case in question 5 the option "Yes some safety concerns were identified" will be selected) In case the option "Yes" will be selected a dedicated free text box will appear to describe the information that could potentially lead to a change in the QPS status of the microorganism.

B.5.
Collective expert evaluation of the ELS outcome and presentation in the QPS opinion The overall results of the searches and evaluations of individual articles will be presented in tabular format for each group/subgroup and species. These results will be further evaluated collectively by the working group and the outcome will be reflected in the QPS opinion.

B.6.
Update of the process The literature search, study selection and collective expert evaluation will be repeated every 6 months.
Appendix C -Search strategies for the maintenance and update of list of QPS-recommended biological agents (reply to ToR 2) Gram-Positive Non-Spore-forming Bacteria Bifidobacterium spp.

Yes No
Novel foods Yarrowia lipolytica A-101 As a novel food production of yeast biomass/Selenium enriched EFSA-Q-2018-00796 Application from same applicant as Yarrowia lipolytica biomass (See above)/Summary of application: NA Qualification: 'for production purpose only' implies the absence of viable cells of the production organism in the final product and can also be applied for food and feed products based on microbial biomass