Final Screening Assessment

Pseudomonas sp. ATCC 13867

Environment and Climate Change Canada
Health Canada
May 2016

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Table of contents

List of Tables

List of Figures

Synopsis

Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA), the Minister of the Environment and Climate Change and the Minister of Health have conducted a screening assessment on Pseudomonas sp.ATCCFootnote 1 13867.

Pseudomonas sp. ATCC 13867 belongs to a group of strains that are currently without a validated species name. Prior to 1982, the species was referred to as "Pseudomonas denitrificans" before that name was officially rejected. For the purposes of this assessment, the name "Pseudomonas sp. ATCC 13867" will be used when information pertains specifically to this strain.

Pseudomonas sp. ATCC 13867 is a bacterium that can proliferate in soil and water. It has properties that make it of potential use in the production of vitamin B12, coenzyme Q, other biochemicals and biofuels; in denitrification products for use in soil improvement, in treatment of activated sludge and wastewater and oil degradation.

No adverse effects in terrestrial or aquatic plants, invertebrates or vertebrates or infections in humans have been attributed to Pseudomonas sp. ATCC 13867 or its close relatives.

This assessment considers the aforementioned characteristics of Pseudomonas sp. ATCC 13867 with respect to environmental and human health effects associated with consumer and commercial product use and industrial processes subject to CEPA, including releases to the environment through waste streams and incidental human exposure through environmental media. To update information about current uses, the Government launched a mandatory information-gathering survey under section 71 of CEPA, as published in the Canada Gazette, Part I, on October 3, 2009 (section 71 notice). Information submitted in response to the section 71 notice indicates that Pseudomonas sp.ATCC 13867 was not imported into or manufactured in Canada in 2008.

Considering all available lines of evidence presented in this screening assessment, there is low risk of harm to organisms and the broader integrity of the environment from Pseudomonas sp. ATCC 13867. It is concluded that Pseudomonas sp. ATCC 13867 does not meet the criteria under paragraph 64(a) or (b) of CEPA as it is not entering the environment in a quantity or concentration or under conditions that have or may have an immediate or long-term harmful effect on the environment or its biological diversity or that constitute or may constitute a danger to the environment on which life depends.

Based on the information presented in this screening assessment, it is concluded that Pseudomonas sp. ATCC 13867 does not meet the criteria under paragraph 64(c) of CEPA as it is not entering the environment in a quantity or concentration or under conditions that constitute or may constitute a danger in Canada to human life or health.

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Introduction

Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA), the Minister of the Environment and Climate Change and the Minister of Health are required to conduct screening assessments of living organisms added to the Domestic Substances List (DSL ) by virtue of section 105 of the Act to determine whether they present or may present a risk to the environment or human health (according to criteria set out in section 64 of CEPA)Footnote 2. Pseudomonas sp. ATCC 13827 was added to the DSL under Section 105(1) of CEPA because it was manufactured in or imported into Canada between January 1, 1984, and December 31, 1986 and it entered or was released into the environment without being subject to conditions under CEPA or any other federal or provincial legislation.

This Screening Assessment considers hazard information obtained from the public domain and from unpublished research data, as well as comments from scientific peer reviewers. Exposure information was obtained from the public domain and from a mandatory CEPAsection 71 Notice published in the Canada Gazette Part I on October 3, 2009. Further details on the risk assessment methodology used are available in the document "Framework on the Science-Based Risk Assessment of Micro-organisms under the Canadian Environmental Protection Act, 1999" (Environment Canada and Health Canada, 2011).

In this report, data that are specific to the DSL-listed strain Pseudomonas sp. ATCC 13867 are identified as such. Strain-specific data was obtained from several sources: the Nominator, the American Type Culture Collection (ATCC), and unpublished data generated by Health CanadaFootnote 3 and the scientific literature. Where strain-specific data were not available, surrogate information from the literature was used. When applicable, literature searches conducted on the organism included its synonyms and superseded names. Literature searches were conducted using scientific literature databases (SCOPUS, CAB Abstracts, and NCBI PubMed), web searches, and key search terms for the identification of human health and environmental hazards. Information identified up to August 2014 was included in this Screening Assessment Report.

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Decisions from Domestic and International Jurisdictions

The Public Health Agency of Canada (PHAC) assigned the species known as Pseudomonas sp. to Risk Group 1 (low individual and community risk) for both humans and terrestrial animals (personal communication, PHAC 2014).

The Canadian Food Inspection Agency (CFIA) does not consider the species known as Pseudomonas sp. to be an animal pathogen (personal communication, CFIA 2014). Some members of the Pseudomonas genus are considered to be important plant pathogens; however, in the case of Pseudomonas sp. ATCC 13867, it was not possible to determine whether or not it is a pathogenic strain. Therefore, if the organism was to be imported, plant pest containment 1 (PPC-1) would be required for work with this organism (personal communication, CFIA 2014).

No other regulatory decisions by other international governments or organizationsFootnote 4 were identified for Pseudomonas sp. ATCC 13867 despite the numerous current and potential uses identified.

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1. Hazard Assessment

1.1 Characterization of Pseudomonas sp. ATCC13867

1.1.1 Taxonomic identification and strain history

Binomial name  Pseudomonas sp.

Taxonomic designation:

Kingdom:  Bacteria

Phylum:  Proteobacteria

Class  Gammaproteobacteria

Order:  Pseudomonadales

Family:  Pseudomonadaceae

Genus:  Pseudomonas

Species:  Pseudomonas sp. (Doudoroff et al. 1974; JCICSB, 1982)

Strain:  ATCC 13867

Other strain designations of Pseudomonas sp. ATCC 13867 include 926, NCIB 9496, NCIMB 9496 BCRC 14386, CCRC 14386, CCT 5425, CCUG 1783, CIP 104375, DSM 1650, DSM 1650/5040, Hugh 926, IAM 12573, IFO 13302, JCM 20650, LMD 84.60, LMG 7983, NBIMCC 1625, NBRC 13302 (Delwiche, 1959; Doudoroff et al. 1974; Sacks and Barker, 1952)

1.1.1.1 Synonyms and superseded names

Pseudomonas denitrificans (Lysenko, 1961; Peix et al. 2009), Pseudomonas nitroreductans Iizuka and Komagata 1964 emend. Lang et al. 2007 (DSMZ, 2014); Pseudomonas multiresinivorans (Mohn et al. 1999; DSMZ, 2014), Bacillus denitrificans fluorescens Christensen, 1903 (Lysenko, 1961).

Strain ATCC 13867 has historically been referred to as "P. denitrificans"; however, the species name was officially rejected in 1982 as nomen ambiguum based on a phenotypic and limited genotypic characterization (Doudoroff et al. 1974; JCICSB, 1982). In spite of the rejected status of the species name, the DSL strain (ATCC 13867) and another strain (ATCC 19244) are often still referred to as "P. denitrificans" in the literature. However, these two strains represent different species that can be differentiated by DNA composition and at least 40 phenotypic characteristics (Doudoroff et al. 1974). Therefore, information specific to the DSL strain will be identified with the name "Pseudomonas sp. ATCC 13867" (regardless of the name used by the source). Other strains historically named "P. denitrificans" may be distantly related to the DSL strain, and are therefore not considered reliable as surrogates; however, to be protective of the environment and human health, reports of adverse effects attributed to "P. denitrificans" were considered in this assessment where there is a possibility that the implicated strain could be a close relative of the DSL strain.

1.1.1.2 Strain history of Pseudomonas sp. ATCC 13867

Pseudomonas sp. ATCC 13867 was isolated from soil after enrichment with succinate-nitrate medium (Sacks and Barker, 1949). Originally named Pseudomonas denitrificansBergey et al. strain 926 or Hugh 926, it was deposited in to the American Type Culture Collection (ATCC) by R. Hugh who had received the strain from C. Delwiche (Table 1-1 ).

Table 1-1: Major culture collections where Pseudomonas sp. ATCC 13867 was deposited
Culture collectionStrain designationYear deposited
American Type Culture CollectionATCC13867Not available
National Collection of Industrial, Food and Marine BacteriaNCIMB 94961963
Deutsche Sammlung von Mikrooganismen und ZellkulturenDSM 1650 (historical number: DSM 50405)Not available
Institute for FermentationIFO 13302 (=NBRCFootnote Table 1-1a 13302)Not available
IAM Culture CollectionIAM 12573Not available
Japan Collection of MicroorganismsJCM 206502007
1.1.1.3 Phylogeny of the genus Pseudomonas

Members of the Pseudomonas genus are Gram negative, diverse, widely distributed and dominated by non-pathogenic, saprophytic colonizers of soil, water, and rhizosphere ecosystems and non-pathogenic commensal colonizers of healthy human skin (Cogen et al. 2008; Li et al. 2013a). Historically, the genus Pseudomonas (sensu lato) included members from the alpha, beta, gamma-beta and gamma proteobacteria, many of which have been or are likely to be reclassified based on modern taxonomic methods. A subgroup of Pseudomonas species within the gamma proteobacteria are considered to represent the genus (sensu stricto). These include the P. aeruginosa, P. chloroaphis, P. fluorescens, P. putida, P. stutzeri and P. syringaespecies groups.

A phylogenetic tree was constructed using publicly available 16S rRNA gene sequences and sequences obtained from Anzai et al. (2000) (Appendix 1). The DSL strain (named Pseudomonas denitrificans ATCC 13867 in Figure 1-1 ) is distinct from "P. denitrificans" neotype strain ATCC 19244 (named Pseudomonas denitrificans IAM 12023 in Figure 1-2) as a separate species and clusters differently in phylogenetic analysis (Appendix 1). Pseudomonas sp. ATCC 13867 does not fit within the P. aeruginosa group (Figure 1-1), whereas "P. denitrificans" ATCC 19244 does (Figure 1-2 ).

Figure 1-1: Phylogenetic subtree tree using 16S rRNA gene sequences showing species and strains that are closely related to Pseudomonas sp. ATCC 13867

Figure 1-1 (See long description below)

Long description for figure 1-1

Figure 1-1: Phylogenetic subtree tree using 16S rRNA gene sequences showing species and strains that are closely related to Pseudomonas sp. ATCC 13867: S000003555 Pseudomonas alcaligenes (T); IAM12411;D84006, 98 (S000395038 Pseudomonas jinjuensis (T); Pss 26; AF468448, 88 (71 (55 (S003614267 Pseudomonas sp. SP2(2012); JX298094, S003710837 Pseudomonas denitrificans ATCC 13867; CP004143), S000626936 Pseudomonas nitroreducens (T); IAM 1439; AM088473), 65 (S000824948 Pseudomonas panipatensis (T); Esp-1; EF424401, 52 (S000427988 Pseudomonas knackmussii (T); B13; AF039489, 100 (S000006645 Pseudomonas citronellolis (T); DSM 50332T (type strain); Z76659, S000639965 Pseudomonas delhiensis (T); RLD-1; DQ339153)))).

Figure 1-2: Phylogenetic subtree tree using 16S rRNA gene sequences showing species and strains that group closely with the pathogenic species P. aeruginosa

Figure 1-2 (See long description below)

Long description for figure 1-2

Figure 1-2: Phylogenetic subtree tree using 16S rRNA gene sequences showing species and strains that group closely with the pathogenic species P. aeruginosa: 10 (77 (90 (S000010427 Pseudomonas aeruginosa (T): DSM50071; X06684, S000514601 Pseudomonas otitidis (T): MCC10330; AY953147), S000007012 Pseudomonas resinovorans (T): LMG 2274T (type strain); Z76668), 82 (S000390990 Pseudomonas indica (T); AF302795, S000567747 Pseudomonas azotifigens (T); 6H33b; AB189452)), 4 (70 (100 (S000008928 Pseudomonas oleovorans (T); IAM 1508; D84018, S000416841 Pseudomonas psychrotolerans (T); type strain; C36; AJ575816), 93 (S000010364 Pseudomonas luteola (T); IAM 13000; D84002, S000903111 Pseudomonas duriflava (T); HR2; EU046271)), 19 (S000639962 Pseudomonas pohangensis (T); H3-R18; DQ339144, 80 (S000495962 Pseudomonas pachastrellae (T); KMM 330; AB125366, 57 (S000968494 Pseudomonas xinjiangensis (T); J64; EU143352, 56 (S000942753 Pseudomonas sabulinigri (T); J64; EU143352, 68 (S001156249 Pseudomonas pelagia (T); CL-AP6; EU888911, 99 (76 (S000001098 Pseudomonas pertucinogena (T); IFO 14163T; AB021380, S000558609 Pseudomonas xiamenensis (T); C10-2; DQ088664), 38 (S003283193 Pseudomonas denitrificans; KH-1; JQ612512, 100 (S000013307 Pseudomonas denitricans IAM 12023; AB021419, S002233769 Pseudomonas denitrificans; CL-11.3; HQ113222))))))))).

1.1.1.4 Phenotypic and molecular characteristics

The purpose of this section is to describe characteristics that can be used to distinguish between Pseudomonas sp. ATCC 13867, a closely related strain (SP2, see Figure 1-1) and Pseudomonas aeruginosa PAO1, as a representative pathogenic relative (Table 1-2 to Table 1-4 ). Observations by Health Canada scientists for ATCC 13867 were consistent with those reported in the literature for most morphological and growth characteristics. Although growth curves were not generated at Health Canada, it was observed that colony size of Pseudomonas sp. ATCC 13867 varied depending on the temperature in which it was incubated when plated on tryptic soy broth for 48 hours (1-2 mm at 28°C, 1 mm at 32°C, 3 mm at 37°C and 0.5 mm at 42°C). This finding could suggest an optimal growth rate closer to 37oC.

Table 1-2: Morphological and growth characteristics of Pseudomonas sp. ATCC 13867, Pseudomonas sp. SP2 and P. aeruginosaPAO1
CharacteristicFootnote Table 1-2aATCC13867Strain SP2Strain PAO1
Cell size (L × W)1.05 x 0.8 μm2.0 x 0.5 μm1.3 to 3.0 x 0.5 to 0.8 μm
FluorescenceNon fluorescentNon fluorescentFluorescent
ColoniesEntire, smooth, glistening, translucent, off-white/tanb, raisedFootnote Table 1-2bLight cream colourEntire, smooth, glistening, convex, and tan in colour with a fried egg morphology
Pigment productionNone to pinkPinkDiffusible, green
Optimal growth temperature25°CNo data37°C
Growth at 40°CNegativeNo dataPositive
Growth at 42°CNegativebNo dataPositive
Table 1-3: Biochemical characteristics of Pseudomonas sp. ATCC 13867, Pseudomonas sp. SP2 and P. aeruginosaPAO1
CharacteristicATCC13867Footnote Table 1-3aATCC13867Footnote Table 1-3bStrain SP2bStrain PAO1b
Gelatin liquefactionNot observedNegativeNegativePositive
Utilization of maltoseVariablePositivePositiveNegative

The complete genome of Pseudomonas sp. ATCC 13867 has been sequenced; it is 5.7 Mbp in size and contains 5,135 genes (Winsor et al. 2011), 2,567 operons, and 5,059 protein-encoding genes (Ainala et al. 2013). No plasmids were identified in ATCC 13867 using the Pseudomonas Genome Database (Winsor et al. 2011).

Table 1-4: Molecular characteristics of Pseudomonas sp. ATCC 13867, Pseudomonas sp. SP2 and P. aeruginosaPAO1
CharacteristicaATCC13867Strain SP2Strain PAO1
G+C content65.2%Footnote Table 1-4aNot available66.6%a
Genome size and accession number5.7 Mb (CP004143)Not available6.2 Mb (AE004091)
16S rRNANR_102805JX298094AB626118

Characteristics such as cell morphology, total lipid content and sensitivity to certain antibiotics could permit distinction between Pseudomonas sp. ATCC 13867 and Pseudomonas sp. strain SP2 (Arasu et al. 2013). The differences highlighted in Table 1-5 can be used to distinguish between the DSL strain and other strains of Pseudomonassp. Fatty acid methyl ester analysis of Pseudomonas sp. ATCC 13867 was conducted by Health Canada scientists (Appendix 2).

Table 1-5: Differentiation of Pseudomonas sp. ATCC 13867 and SP2 using cell morphology, fatty acid composition and antibiotic sensitivity adapted from Arasu et al. (2013)
CharacteristicPseudomonas sp. ATCC 13867Pseudomonas sp. SP2
Total lipid (%)48%50%
Cell morphologyShort cell shape with smoother surfaceElongated cell shape with rougher surface
Nalidixic acidSensitiveIntermediate
SparfloxacinSensitiveIntermediate
ColistinIntermediateResistant

1.1.2 Biological and ecological properties

1.1.2.1 Growth parameters

Pseudomonas sp. ATCC 13867 is a colourless sulphur bacterium with a diverse metabolism (Robertson et al. 1989). It is facultatively anaerobic, facultatively chemolithotrophic, and capable of both heterotrophic nitrification and aerobic denitrification (Kornaros and Lyberatos, 1998; Robertson et al. 1989). It is able to use a variety of carbon sources and will switch from a preferred one to a secondary source of carbon in a pattern of diauxic growth (Casasus et al. 2005; Hamilton et al. 2005). A maximum growth rate of 0.043 mg dry biomass/mL per hour was achieved for Pseudomonas sp. ATCC 13867 in 4 g/L glucose (Apel and Turick, 1993). The DSL strain is also able to use succinate, yeast extract, ethanol and pretreated sewage sludge as carbon and energy sources (Dasu et al. 1993; Nilsson et al. 1980; Wu et al. 2001). Pseudomonas sp. ATCC 13867 does not grow at 4°C or 40°C (Doudoroff et al. 1974). Growth kinetics and growth in different media of Pseudomonas sp. ATCC 13867 are presented in Appendices 3 and 4, respectively.

1.1.2.2 Persistence and survival in the environment

In one study, amplified fragment length polymorphisms (AFLP)-derived strain-specific DNA markers were developed to detect Pseudomonas sp. ATCC 13867 and other Pseudomonas species (Xiang et al. 2010). Using these markers in combination with quantitative real-time PCR, estimated concentrations of Pseudomonas sp. ATCC 13867 cells in soil could be tracked over time. Cell suspensions of Pseudomonas sp. ATCC 13867 were added to agricultural soil in the laboratory to a final concentration of 108 to 1010 CFU/g dry weight. Pseudomonas sp. ATCC 13867 persisted in clay loam soil (22°C, pH 5.8, 60% water holding capacity) for at least 181 days, indicating a high capacity for colonization. The authors suggest that the long-term persistence observed for Pseudomonas sp. ATCC 13867 (longer than P. aeruginosa, and as long as P. stutzeri) may be attributed to the metabolic versatility of the species, and particularly its capacity for aerobic denitrification (Xiang et al. 2010).

In another study, Pseudomonas sp. ATCC 13867 formed biofilms in pressurized flow-through columns. The columns contained diatomaceous mudstone and sandstone, synthetic groundwater and were supplemented with sodium acetate. Cell numbers increased over the experimental period of 28 days (Harrison et al. 2011; Wragg et al. 2012).

On the basis of these studies, introduced populations of Pseudomonas sp. ATCC 13867 are expected to persist and proliferate under certain environmental conditions and where nutrients are available.

1.1.2.3 Transformation of nitrate and nitrite

The DSL strain was reported to grow more rapidly with both nitrate and oxygen together compared to either electron acceptor separately (Robertson et al. 1989). The DSL strain was able to rapidly reduce N2O to N2; maximum removal N2O rate of 0.017 mM/hr/mg dry biomass occurred at 35°C and an initial N2O concentration of 0.9 mM (Apel and Turick, 1993). Above this concentration the reduction rate decreases (Apel and Turick, 1993). When Pseudomonas sp. ATCC 13867 was grown in nitrate or nitrite, N2 gas was immediately produced (Robertson et al. 1989). The ability of Pseudomonas sp. ATCC 13867 to reduce nitrates and produce gas was also observed by Health Canada scientists.

1.1.2.4 Resistance to antibiotics, metals and other chemical agents

Pseudomonas sp. ATCC 13867 contains multiple genes for resistance to antibiotics, metals and other chemical agents (Winsor et al. 2011). These genes include:

The antibiotic susceptibility profile of the DSL strain was recently reported (Arasu et al. 2013). When tested using the disk diffusion method, Pseudomonas sp. ATCC 13867 was sensitive to representatives of the major classes of antibiotics including aminoglycosides, β-lactamase inhibitors and fluoroquinolones; resistance was reported for carboxypenicillins and variable susceptibility was reported for cephalosporins (Table 1-6). The zone of inhibition was measured after incubation at 37°C for 17 hours.

Table 1-6: Antibiotic susceptibility patterns of Pseudomonas sp. ATCC 13867 (using the disc diffusion method), adapted from Arasu et al. (2013) et al. (2013)
Antibiotic (µg)Diameter of inhibition zone (mm)StatusFootnote Table 1-6a
Amikacin (30)23Sensitive
Gentamicin (10)25Sensitive
Kanamycin (30)34Sensitive
Streptomycin (30)17Resistant
Tobramycin (10)31Sensitive
Penicillin (50)0Resistant
Ampicillin (50)0Resistant
Augmentin (30)28Not availableFootnote Table 1-6b
Imipenem (10)30Sensitive
Ticarcillin (75)25Sensitive
Ciprofloxacin (5)39Sensitive
Gatifloxacin (5)32Sensitive
Levofloxacin (5)37Sensitive
Moxifloxacin (5)30Not available
Nalidixic acid (30)35Sensitive
Norfloxacin (10)38Sensitive
Ofloxacin (5)34Sensitive
Sparfloxacin (5)32Sensitive
Cefpodoxime (10)0Resistant
Ceftriaxone (30)30Sensitive
Colistin (10)10Intermediate
Co-Trimoxazole (25)25Not available

Antibiotic susceptibility testing was conducted by Health Canada scientists (Table 1-7). Most of the tested antibiotics effectively inhibited growth with the exception of trimethoprim.

Table 1-7: Antibiotic susceptibility of Pseudomonas sp. ATCC13867
AntibioticPseudomonas sp. ATCC 13867 (MIC, µg/mL)Footnote Table 1-7aResultFootnote Table 1-7b
Amoxicillin12N/AFootnote Table 1-7c
Aztreonam15 ± 6IFootnote Table 1-7d (SFootnote Table 1-7e Less Than or Equal To 1, RFootnote Table 1-7f greater than 16)
Cefotaxime/cephotaxime7.5 ± 3N/A
Ciprofloxacin0.37S (S Less Than or Equal To 0.5, R greater than 1)
Colistin0.37S (S Less Than or Equal To 4, R greater than 4)
Doxycycline1.5N/A
Erythromycin1.2N/A
Gentamicin0.37S (S Less Than or Equal To 4, R greater than 4)
Meropenem0.37S (S Less Than or Equal To 2, R greater than 8)
Trimethoprim24N/A

A strain obtained from C. Delwiche (original depositor of the DSL strain to ATCC), that is likely the DSL strain Pseudomonas sp. ATCC 13867, was used in a study that compared the inhibitory effect of heavy metals on pseudomonads. This strain was reported to be much more resistant to the effect of heavy metals compared to another isolate identified as Pseudomonas sp. and a strain of P. aeruginosa(Bollag and Barabasz, 1979).

No information was identified on the resistance of Pseudomonas sp. 13867 to disinfectants such as chlorine or quaternary ammonium compounds.

1.1.2.5 Pathogenic and toxigenic characteristics

Virulence factors of P. aeruginosa include pili; flagella; siderophores; pyocyanin; elastase; proteases; rhamnolipid; alginate; other polysaccharides; and lipopolysaccharides (reviewed in Hay et al. 2014; Nelson et al. 2002; Palleroni, 2005). The Pseudomonas Genome Database listing for Pseudomonas sp. ATCC 13867 confirms the presence of some of the same virulence factors (Winsor et al. 2011), including:

A search of the literature and of the Pseudomonas Genome Database listing for Pseudomonas sp. ATCC 13867 confirms the absence of extracellular toxins, such as rhamnolipids, pyocyanin, pyochelin and hydrogen cyanide (Winsor et al. 2011)

The secreted exopolysaccharide alginate helps bacteria to adapt and survive in many habitats. It has also been implicated in biofilm and capsule formation and may increase resistance to antibiotics and bactericides and therefore enhance its ability to evade the host immune system (Govan and Deretic, 1996; reviewed in Hay et al. 2014; Rezaee et al. 2002). Several genes associated with alginate biosynthesis and regulation have been identified in Pseudomonas sp. ATCC 13867 including alg8, alg44, algK, algE, algG, algX, algL, algJ, algF, algR and algB (reviewed in Hay et al. 2014; Winsor et al. 2011). However, other genes required for alginate biosynthesis and regulation (such as algD(promoter), algA, algU, algC and amrZ) were not identified (reviewed in Hay et al. 2014; Winsor et al. 2011).

Biofilms have been extensively reported as a mechanism of pathogenicity in pseudomonads. Biofilms contribute to the persistence of infections and cells within them are up to 1,000 times more resistant to the effects of antimicrobial agents than their planktonic counterparts (O'Toole and Kolter, 1998; Costerton et al. 1999; Mah and O'Toole, 2001). Pseudomonas sp. ATCC 13867 is known to form biofilms (Harrison et al. 2011; Wragg et al. 2012).

Strong hemolytic activity (as well as lecithinase activity) may indicate the presence of cytotoxic phospholipases that may facilitate invasion and are associated with virulence (Rowan et al. 2001; Sorokulova et al. 2008). Pseudomonas sp. ATCC 13867 did not demonstrate any hemolysis when tested by Health Canada scientists.

Catalase activity can enable a micro-organism to protect itself from reactive oxygen-induced killing from immune cells, potentially making it a more effective pathogen. Catalase activity was determined by Health Canada scientists to be positive for Pseudomonas sp. ATCC 13867.

In testing conducted by Health Canada scientists, the cytotoxicity of Pseudomonas sp. ATCC 13867 was assessed in two cell lines, HT29 (human colonic epithelial cells) and J774A.1 (macrophage cells), with and without gentamicin. No toxicity was observed in HT29 cells. Some toxicity was observed in J774A.1 cells in the presence of gentamicin. In the absence of antibiotic, J774A.1 was also more sensitive compared to HT29 (in general, J774A.1 cells are more sensitive to toxic substances). However, overall the toxicity is limited (maximum 30% bioreduction at 24 hours) suggesting that the structural components of the bacteria were able to cause a limited toxic response. Similar marginal responses were observed with some Acinetobacterspecies studied with the same MTT assay (Tayabali et al. 2012). In comparison, strains of the known pathogen, P. aeruginosa, caused a 60-90% drop in MTT formazan production.

1.1.3 Effects

To be precautionary and ensure that all cases of infection possibly involving Pseudomonas sp. ATCC 13867 or its close relatives would be identified, the following section also includes information on cases of infection attributed to "P. denitrificans".

1.1.3.1 Environment

One study reported "P. denitrificans" as being widespread in populations of the plant nematode, Xiphinema americanum, in West Virginia. However, it was unclear whether the relationship was symbiotic or pathogenic in nature (Adams and Eichenmuller, 1963).

No data were identified specifically implicating Pseudomonas sp. ATCC 13867 or its close relatives Pseudomonas sp. SP2, P. nitroreducensor P. citronellolis in adverse effects in aquatic or terrestrial invertebrates, vertebrates, or plants.

1.1.3.2 Humans

Similarly, no information was identified implicating Pseudomonas sp. ATCC 13867 or its close relatives Pseudomonas sp. SP2, P. nitroreducensor P. citronellolis in adverse human health effects.

One fatal case implicated a strain of "P. denitrificans" as the etiological agent in a serious infection in an individual predisposed by underlying disease (Fischer et al. 1981). Pure cultures of "P. denitrificans" were isolated from cerebrospinal fluid in a case of fatal meningitis in a patient with systemic lupus erythematosus, chronic leg ulcers, mitral insufficiency, seizure disorder, and mild dementia. The authors proposed that an old healing ulcer led to bacteremia which ultimately resulted in the meninges becoming infected as well. Treatment in this case was compromised by resistance to a number of antibiotics. It is unclear whether the isolated micro-organism was closely or distantly related to the DSL strain.

A therapeutic bronchoscopy yielded "P. denitrificans" and S. aureus in a cystic fibrosis patient with a two-year history of bilateral recurrent lung abscesses (Canny et al. 1986). Despite treatment with tobramycin, ticarcillin, and cloxacillin, the patient developed septic shock with disseminated intravascular coagulation and compromised renal function resulting in death. "P. denitrificans"was co-isolated with S. aureusfrom the lung abscess of a patient with cystic fibrosis at autopsy, but blood cultures yielded only "P. denitrificans" with an antibiotic susceptibility identical to the organism cultured from the lung abscess.

No cases of allergic reactions related to Pseudomonassp. ATCC13867 or its close relatives Pseudomonas sp. SP2, P. nitroreducens or P. citronellolis were found in the literature. Like all micro-organisms, the DSL strain contains or produces components, such as lipopolysaccharides and enzymes, which may act as immune stimulants, allergens or sensitizers. Sensitization or allergic reactions to micro-organisms could occur via dermal and respiratory routes in frequently exposed or susceptible individuals (Martel et al. 2010; Ring et al. 1992).

1.2 Hazard Severity

The environmental and human health hazard severity for Pseudomonas sp. ATCC 13867 is assessed to be low because 1) Pseudomonas sp. ATCC 13867 can be discriminated from closely related Pseudomonas species found in the environment and from P. aeruginosa strains that are pathogenic towards human and environmental species; 2) although genes which may confer virulence were identified in the genome of Pseudomonas sp. ATCC 13867, there is no indication that the DSL strain acts as a pathogen. It is possible that the identified virulence genes are inactive, or require the expression of other or missing genes to cause harm; 3) no adverse effects in environmental species or humans, attributed to the DSL strain or its close relatives, have been reported; and 4) in the unlikely event of infection, veterinary and clinical antibiotics are available.

Hazards related to micro-organisms used in the workplace should be classified accordingly under the Workplace Hazardous Materials Information System (WHMIS)Footnote 5.

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2. Exposure Assessment

2.1 Sources of Exposure

This assessment considers exposure to Pseudomonas sp. ATCC 13867 resulting from its addition to consumer or commercial products or its use in industrial processes in Canada.
Pseudomonas sp. ATCC 13867 was nominated to the DSL in 2005 for its use in commercial and consumer products.

Responses to a voluntary questionnaire sent in 2007 to a subset of key biotechnology companies, combined with information obtained from other federal government regulatory and non-regulatory programs indicate that Pseudomonas sp. ATCC 13867 was in commercial use in 2006.

The Government conducted a mandatory information-gathering survey under section 71 of CEPA, as published in the Canada Gazette, Part I, on October 3, 2009 (section 71 Notice). The section 71 Notice applied to any persons who, during the 2008 calendar year, manufactured or imported Pseudomonas sp. ATCC 13867, whether alone, in a mixture, or in a product. No industrial, commercial or consumer activities using Pseudomonas sp. ATCC 13867 were reported in response to the section 71 Notice.

The 2007 and 2009 surveys differed significantly in target and scope. In this assessment, results from the 2009 survey were used to estimate exposure from current uses because it requested information on uses of the micro-organism strain that is listed on the DSL, whereas the 2007 survey asked about uses of the products that had been associated with the micro-organism at the time it was nominated to the DSL. Because product formulations may have changed, information from the 2009 survey may more accurately represent current uses. Uses reported in the 2007 voluntary survey were also considered in the assessment of potential uses.

Although no uses were reported for Pseudomonas sp. ATCC 13867 during the mandatory survey, it is available for purchase from the ATCC. As it is on the DSL, and so can be used in Canada without prior notification, it could be an attractive choice for commercialization. A search of the public domain (MSDS, literature and patents) revealed the following consumer, commercial and industrial applications of other strains of Pseudomonassp. To ensure that all potential uses of the DSL strain were captured, activities identified for "P. denitrificans" were also included:

2.2 Exposure Characterization

2.2.1 Environment

Based on the absence of industrial, consumer and commercial activity in Canada according to the section 71 Notice, the overall environmental exposure estimation for Pseudomonas sp. ATCC 13867 is low. Nevertheless, given the range and scale of known and potential applications listed in Section 2.1, there is potential for an increase in environmental exposure to products containing Pseudomonas sp. ATCC 13867, and therefore potential exposure scenarios arising from these products have been considered.

Should potential uses identified in Section 2.1 be realized in Canada, plant and animal exposure to Pseudomonas sp. ATCC 13867 will depend on its persistence and survival in the environment. However, the DSL strain is metabolically versatile and is expected to readily colonize new terrestrial environments. Xiang et al. (2010) investigated the persistence of Pseudomonas sp. ATCC 13867 in microcosm soil and reported that populations of this strain persisted in soil for at least 181 days, indicating a high capacity for colonization. The authors suggest that the long-term persistence observed for Pseudomonas sp. ATCC 13867 may be attributed to the metabolic versatility of the species, and particularly its capacity for aerobic denitrification. In another study, mentioned in section 1.1.2.2, Pseudomonas sp. ATCC 13867 formed biofilms in pressurized flow-through columns in which the solid phase was diatomaceous mudstone and sandstone, and the liquid phase was synthetic groundwater supplemented with sodium acetate. Cell numbers increased over the experimental period of 28 days (Harrison et al. 2011; Wragg et al. 2012). Therefore, Pseudomonas sp. ATCC 13867 would be expected to be able to survive and persist in most terrestrial and aquatic environments.

The following exposure scenarios are based on known uses of other strains and probable future uses as described in Section 2.1. Uses such as bioremediation and water and wastewater treatment are likely to introduce Pseudomonas sp. ATCC 13867 to terrestrial ecosystems. Terrestrial invertebrates living in the soils at the site of application or disposal and plants growing in treated soils are likely to be the most directly exposed. Vertebrates could ingest Pseudomonas sp. ATCC 13867 while feeding on plants or invertebrates growing in treated or contaminated soils.

Aquatic and marine species may come into contact with Pseudomonas sp. ATCC 13867 from runoff subsequent to terrestrial application and from the direct application of Pseudomonas sp. ATCC 13867 to water bodies for uses such as water treatment (fresh and salt water), wastewater treatment, or disposal of wastewater from applications such as recovery of oil and metals or the manufacture of biochemicals and biofuels.

Aquatic applications could also expose terrestrial species. For example, grazing animals could ingest Pseudomonas sp. ATCC 13867 subsequent to its use in water restoration, and plants and soil invertebrates could be exposed subsequent to the treatment of irrigation ponds.

In the event that consumer, commercial or industrial activities resume, the environmental exposure to Pseudomonas sp. ATCC 13867 will likely increase. The environmental compartments and species that will be exposed to the DSL strain will depend on the uses outlined in the exposure scenarios described above.

2.2.2 Human

Based on the absence of industrial, consumer or commercial activity in Canada according to the section 71 Notice, the overall human exposure estimation for Pseudomonas sp. ATCC 13867 is low. Nevertheless, given the range and scale of known and potential applications provided in Section 2.1, there is potential for an increase in human exposure to products containing Pseudomonas sp. ATCC 13867, and exposure scenarios arising from these products have been considered.

Should potential uses identified in Section 2.1 be realized in Canada, human exposure could be greatest through the use of consumer products intended for the treatment of aquariums and decorative ponds, degreasing of kitchen drains, cleaning and deodorizing of septic tanks, and composting. Handling and application of such products would be expected to result in direct exposure to the skin. Inhalation of aerosolized droplets or airborne dust containing Pseudomonas sp. ATCC 13867 generated during the application of such products could also occur.

Secondary to product application, residual Pseudomonassp. ATCC13867 on surfaces and in reservoirs such as treated drains could result in dermal exposure; oral exposure through inadvertent ingestion where the organism persists on food preparation surfaces; and exposure via inhalation where aerosols are generated (e.g., from kitchen garbage disposal units). Since Pseudomonassp. ATCC13867 is expected to persist following application, such exposures may be temporally distant from the time of application.

Should commercial products containing Pseudomonas sp. ATCC 13867 become available in Canada, the general population could be exposed as bystanders during commercial product application. The extent of bystander exposure will depend on the mode of application, the volume applied, and the proximity of bystanders to the site of application, but in general is expected to be moderate.

Human exposure to bodies of water and soil treated with Pseudomonas sp. ATCC 13867, (e.g., through recreational activities), could also result in exposure of the skin and eyes, as well as inadvertent ingestion.

Indirect exposure to Pseudomonas sp. ATCC 13867 in the environment subsequent to its use in oil recovery, water and wastewater treatment, soil bioremediation, or disposal of waste from its use in the production of enzymes is also likely to occur in the vicinity of application or disposal sites, but is expected to be no greater than direct exposure from the use of the organism in consumer products.

In the event that the organism enters municipal drinking water treatment systems through release from potential uses, the water treatment process, which utilizes one or more of the following methods: coagulation, flocculation, ozonation, filtration, ultraviolet radiation and chlorination, is expected to effectively eliminate these micro-organisms from drinking water.

In the event that the potential consumer, commercial or industrial uses of Pseudomonas sp. ATCC 13867 are realized, human exposure through the exposure scenarios described above can be expected and could include direct, possibly repeated, exposure to concentrated preparations of Pseudomonas sp. ATCC13867.

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3. Risk Characterization

In this assessment, risk is characterized according to a paradigm whereby a hazard and exposure to that hazard are both required for there to be a risk. The risk assessment conclusion is based on the hazard, and on what is known about exposure from current uses.

Hazard has been estimated for Pseudomonas sp. ATCC 13867 to be low for both the environment and human health. Based on the absence of industrial, consumer or commercial activity in Canada according to the section 71 Notice, environmental and human exposure to Pseudomonas sp. ATCC 13867 is not currently expected (low exposure), so the risk associated with current uses is estimated to be low for both the environment and human health.

The determination of risk from current uses is followed by consideration of the estimated hazard in relation to foreseeable future exposures (from new uses).

Pseudomonas sp. ATCC 13867 has properties that make it of interest for applications that could expose the environment and the general Canadian population to this strain in the future. The risk to the environment and human health from foreseeable future uses of Pseudomonas sp. ATCC 13867 is also low given that there is no evidence of adverse ecological effects or adverse effects to human health.

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4. Conclusion

Based on the information presented in this screening assessment, it is concluded that Pseudomonas sp. ATCC 13867 is not entering the environment in a quantity or concentration or under conditions that:

Therefore, it is concluded that Pseudomonas sp. ATCC 13867 does not meet any of the criteria set out in section 64 of CEPA.

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Appendices

A. Phylogenetic tree

Figure A-1: Phylogenetic tree using 16S rRNA gene sequences with species representing major groups within the Pseudomonas genus indicated

Figure A-1 (See long description below)

Long description for figure A-1

Figure A-1: Phylogenetic tree generated by the Environmental Health Science and Research Bureau using 16S rRNA gene sequences of the Pseudomonas genus (available via the Ribosomal Database Project http://rdp.cme.msu.edu/). The alignment was generated by Muscle and a Maximum Likelihood Tree was constructed with the Gamma distributed with invariant sites method and 500 bootstrap replicates, using the MEGA version 5.2 platform (Tamura et al., 2011). Pseudomonas sp. ATCC 18367, Pseudomonas sp. SP2, P. denitrificans ATCC 19244 and P. aeruginosa have been highlighted. In the phylogenetic tree the DSL strain appears as ‘Pseudomonas denitrificans ATCC 13867’ and Pseudomonas denitrificans ATCC 19244 appears as ‘Pseudomonas denitrificans IAM 12023’.

Figure A-2: Partial phylogenetic tree using 16S rRNA gene sequences of Pseudomonas species

Figure A-2 (See long description below)

Long description for figure A-2

Figure A-2: Partial phylogenetic tree generated by the Environmental Health Science and Research Bureau using 16S rRNA gene sequences of the Pseudomonas genus (available via the Ribosomal Database Project http://rdp.cme.msu.edu/). The alignment was generated by Muscle and a Maximum Likelihood Tree was constructed with the Gamma distributed with invariant sites method and 500 bootstrap replicates, using the MEGA version 5.2 platform (Tamura et al., 2011). Pseudomonas sp. ATCC 18367, Pseudomonas sp. SP2, P. denitrificans ATCC 19244 and P. aeruginosa have been highlighted. In the phylogenetic tree the DSL strain appears as ‘Pseudomonas denitrificans ATCC 13867’ and Pseudomonas denitrificans ATCC 19244 appears as ‘Pseudomonas denitrificans IAM 12023’.

Phylogenetic trees were generated by the Environmental Health Science and Research Bureau using 16S rRNA gene sequences of the Pseudomonas genus (available via the Ribosomal Database Project). The alignment was generated by Muscle and a Maximum Likelihood Tree was constructed with the Gamma distributed with invariant sites method and 500 bootstrap replicates, using the MEGA version 5.2 platform (Tamura et al. 2011). Pseudomonas sp. ATCC 18367, Pseudomonas sp. SP2, P. denitrificansATCC 19244 and P. aeruginosa have been highlighted. In the phylogenetic tree the DSL strain appears as "Pseudomonas denitrificans ATCC 13867" and Pseudomonas denitrificans ATCC 19244 appears as "Pseudomonas denitrificans IAM 12023".

B. Fatty Acid Methyl Ester (FAME) AnalysisFatty Acid Methyl Ester (FAME) Analysis

Unpublished data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Data presented based on clinical and environmental MIDI databases. MIDI is a commercial identification system that is based on the gas chromatographic analysis of cellular fatty acid methyl esters. The environmental and clinical databases showed the closest relationship of Pseudomonas sp. to Pseudomonas aeruginosa.

Table A-1: Fatty Acid Methyl Ester (FAME) Analysis of Pseudomonas sp. ATCC 13867
ContextEnvironmental databaseClinical database
Frequency6/65/5
Similarity index0.4850.847
First choicePseudomonas-aeruginosa-GC subgroup APseudomonas-aeruginosa-mucoid strains

Figure A-3: Environmental database nearest neighbor analysis and select strains

Figure A-3 (See long description below)

Long description for figure A-3

Figure A-3: Analysis group to simple links in the environmental database and selected strains. Dendrogram demonstrating that Pseudomonas sp. ATCC 13867 is closely related to Pseudomonas aeruginosa based on fatty acid methyl ester analysis.

Figure A-4: Clinical database nearest neighbor analysis

Figure A-4 (See long description below)

Long description for figure A-4

Figure A-4: Group analysis to simple links in the clinical database. Dendrogram demonstrating that Pseudomonas sp. ATCC 13867 is closely related to Pseudomonas aeruginosa based on fatty acid methyl ester analysis.

C. Growth Kinetics

Growth kinetics were investigated using Dulbecco's Modified Eagle Medium, Trypticase Soy Broth and Fetal Bovine Serum at various temperatures. Each table entry shows whether growth (increase in absorbance at 500nm) occurs at different temperatures (28, 32, 37, 42°C).

Table A-2: Growth Kinetics of Pseudomonas sp. ATCC 13867 in liquid media for 24 hours
Medium28°C32°C37°C42°C
Trypticase Soy Broth+++Footnote Table A-2a++++Footnote Table A-2b-Footnote Table A-2c
10% Fetal Bovine Serum (FBS)~Footnote Table A-2d~~-
100% Fetal Bovine Serum+++Footnote Table A-2e--
Dulbecco's Modified Eagles Medium with FBS and glutamine----

D. Growth in different media

Table A-3: Growth of Pseudomonas sp. ATCC 13867 at 28°C (48 hours) in different media
MediumFootnote Table A-3aResults
Trypticase soy brothaPositive
Cetrimide agarFootnote Table A-3bPositive
StarchFootnote Table A-3c: GrowthPositive
Starchc: HydrolysisWeakly positive
Maconkey AgarFootnote Table A-3dGrowth, non-pink colonies
Mannitol Egg Yolk Polymyxin supplementsFootnote Table A-3eNegative
Mannitol Salt AgarFootnote Table A-3fPositive but alkaline reaction
Citrate utilizationFootnote Table A-3gNegativeFootnote Table A-3h
Urea hydrolysisFootnote Table A-3iNegative

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