Final Screening Assessment

Pseudomonas fluorescens ATCC 13525

Environment Canada
Health Canada
February 2015

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Table of Contents

Table of Tables


Pursuant to paragraph 74(b)of the Canadian Environmental Protection Act, 1999 (CEPA 1999), the Ministers of the Environment and of Health have conducted a Screening Assessment on Pseudomonas fluorescens ATCC 13525.

P. fluorescens is considered to be a ubiquitous bacterium and it has the ability to adapt to and thrive in soil and on plants and aqueous surfaces. Multiple potential uses of P. fluorescens in various industrial and commercial sectors exist. These include pulp and paper and textile processing, in municipal and industrial wastewater treatment, for waste degradation, particularly in petroleum refineries, bioremediation and biodegradation, as well as in commercial and household drain cleaners and degreasers, enzyme and chemical production, septic tank additives and general cleaning and odour-control products. Other uses include pest control, plant growth promotion and use as an anti-frost agent on plants.

There is no evidence from the scientific literature to suggest that P. fluorescens ATCC 13525 is likely to have a significant impact on animal and plant populations in the environment.

There have been no reported infections in humans attributed specifically to the strain P. fluorescens ATCC 13525. Information from the scientific literature indicates that strains of P. fluorescens are unlikely to infect the general Canadian population, but that they can infect humans with compromised immunity, and human outbreaks, associated with contaminated medical devices and fluids, have been reported. P. fluorescens can also grow at temperatures typical of refrigerated storage, a characteristic which has enabled it to proliferate in stored blood products and cause sepsis in transfused patients.

This assessment considered human and environmental exposure to P. fluorescens ATCC 13525 from its deliberate use in household or commercial products or in industrial processes in Canada. The government launched a mandatory information-gathering survey (Notice) under section 71 of CEPA 1999 as published in the Canada Gazette, Part I, on October 3rd, 2009. Information submitted in response to the notice indicates that 100 to 1000 kg of products containing P. fluorescens ATCC 13525 were imported into or manufactured in Canada in 2008.

Based on the information available, it is concluded that P. fluorescens ATCC 13525 does not meet the criteria under paragraph 64(a) or (b) of CEPA 1999 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. It is also concluded that P. fluorescens ATCC 13525 does not meet the criteria under paragraph 64(c) of CEPA 1999 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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Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA 1999), the Ministers of Environment and of Health are required to conduct screening assessments of those living organisms listed on the Domestic Substances List (DSL) which were in commerce between 1984 and 1986, to determine whether they present or may present a risk to the environment or human health (according to criteria as set out in section 64 of CEPA 1999)Footnote[1].

This screening assessment considers hazard information obtained from the public domain as well as from unpublished research data and comments from researchers in related fields. Exposure information was obtained from the public domain, as well as from a mandatory CEPA 1999 section 71 Notice published in the Canada Gazette Part 1 on October 3, 2009. Further details on the risk assessment methodology used are available in the Risk Assessment Framework document titled “Framework on the Science-Based Risk Assessment of Micro-organisms under the Canadian Environmental Protection Act, 1999”.

Data that are specific to Pseudomonas fluorescensstrain ATCC 13525 are identified as such and include results of laboratory analyses conducted at Health CanadaFootnote[2]. Where data concerning the particular strain were not available, surrogate information from the scientific literature of other P. fluorescens strains and the genus Pseudomonaswas used. Surrogate organisms are identified in each case to the taxonomic level provided by the source. Information identified as of March 2014 was considered for inclusion in this report. Literature searches were conducted using scientific literature databases (SCOPUS, Google Scholar, and CABI), web searches and key search terms for the identification of human health and environmental hazards of each of the DSL strains assessed in this report.

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1. Hazard Assessment

1.1 Characterization of Pseudomonas fluorescens

1.1.1 Identification, Taxonomy and Strain History

Pseudomonas fluorescens is a Gram-negative, obligate aerobic, motile, rod-shaped bacterium. It grows at neutral pH and has an optimal growth temperature of 25-30oC (Palleroni, 1984), with growth possible as low as 4oC. P. fluorescens does not form spores or other survival structures and does not grow under acidic conditions ( less than pH 4.5) (Holt, 1994). Nutritional demands of P. fluorescens are modest, and so it can survive and multiply for months in moist environments. Most strains are strictly aerobic chemo-organotrophs requiring both oxygen and organic carbon for growth (Holt, 1994). Descriptions of the colony morphology of P. fluorescens ATCC 13525 are outlined in Table 1-1.

Table 1-1 Colony morphology of P. fluorescens ATCC 13525
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Table 1-1[a]Nutrient agar/broth at 26oCFootnote Table 1-1 [b]
Size (mm) diameter4No data
TextureMoistSmooth or rough
UV fluorescenceYesYes
PigmentDiffusing yellow pigmentA yellow-green fluorescent pigment is produced on some media
Footnote Table 1-1 a

Data generated by Health Canada’s Environmental Health Science and Research Bureau

Return to footnote Table 1-1 a referrer

Footnote Table 1-1 b

ATCC description

Return to footnote Table 1-1 b referrer

Health Canada scientists independently characterized P. fluorescens ATCC 13525 using growth kinetics at different temperatures (Appendix 1), growth on different media at 28°C and 37°C (Appendix 2) and fatty acid methyl-ester (FAME) analysis (Appendix 3). Other phenotypic methods, such as the API biochemical tests, can be used for the rapid identification of P. fluorescens in medical and agroalimentary settings (Bodilis et al., 2004), but these techniques do not differentiate the DSL-listed strain from other P. fluorescensstrains.

The genus Pseudomonas is one of the most diverse bacterial genera, and its taxonomy has undergone many changes. Earlier studies resulted in the division of the P. fluorescens group into 5 biovars (I-V, synonym of biotypes A, B, C, F and G) based on phenotypic characteristics such as metabolic tests, fatty acid composition and protein profiles (Palleroni, 2005). P. fluorescens ATCC 13525 is the type strain of biovar I (Palleroni, 2005). Phenotypic characteristics central to the identification and differentiation of the various P. fluorescens biovars are presented in Table 1-2.

Table 1-2 General phenotypic characteristics of P. fluorescens biovarsFootnote Table 1-2[a]
Characteristics/Substrates Used for GrowthBiovar IBiovar IIBiovar IIIBiovar IVBiovar V
Denitrification-Footnote Table 1-2 [b]+Footnote Table 1-2 [c]++-
Levan Formation++-+-
L-Arabinose++dFootnote Table 1-2 [d]+d
Propylene glycol-+d-d
Footnote Table 1-2 a

Adapted from Bergey’s Manual of Systematic Bacteriology (Palleroni, 2005)

Return to footnote Table 1-2 a referrer

Footnote Table 1-2 b

-, negative in most strains

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Footnote Table 1-2 c

+, positive in most strains

Return to footnote Table 1-2 c referrer

Footnote Table 1-2 d

d, different strains give different reactions

Return to footnote Table 1-2 d referrer

The taxonomy of the P. fluorescens group is continually under review. While phenotypic characteristics and the biovar I-V designation are still valid for P. fluorescens identification, molecular characterization is used more reliably to demonstrate the phylogenetic relationships and variations between P. fluorescens strains and among closely related Pseudomonas species. These include full genomic sequence analyses (Paulsen et al., 2005; Silby et al., 2009) and 16S–23S rDNA intergenic spacer-restriction fragment length polymorphism (ITS-RFLP) analyses (Milyutina et al., 2004; Scarpellini et al., 2004). For instance, phylogenetic analyses of the Pseudomonas species performed by Mulet et al. (2010) using four housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) have shown that the P. fluorescens group consists of 9 subgroups, namely P. fluorescens, P. mandelii, P. corrugata, P. gessardii, P. fragi, P. jessenii, P. koreensis, P. chloroaphis and P. asplenii. The P. fluorescens subgroup is composed of 20 species, including the P. fluorescens type strain, ATCC 13525.

Pseudomonas aeruginosa, a known opportunistic human and animal pathogen, is the pathogen that is most closely related to, but is distinct from, P. fluorescens. Phylogenetic trees based on 16S rDNA sequences of Pseudomonas species show that, while the P. fluorescens group is not closely clustered with the P. aeruginosa group, the two are nevertheless related (Palleroni, 2005).

The genome size of P. fluorescens varies from approximately 6.4 Mb to 7.07 Mb (Paulsen et al., 2005; Silby et al., 2009). Comparative studies of three fully sequenced saprophytic P. fluorescens (Pf-5, SBW25 and Pf0-1) revealed an unexpectedly high degree of diversity (Silby et al., 2009), relative to other Pseudomonas species, which are more conserved. These three strains of P. fluorescensshare only 61.4% of their gene content compared with five sequenced genomes of P. aeruginosa which share 80% to 90% of their gene content (Mathee et al., 2008; Silby et al., 2009).

Health Canada scientists independently demonstrated 100% homology of the 16S rRNA gene sequence of the DSL P. fluorescens strain to P. fluorescens ATCC 13525 sequences in the proprietary MicroSeq® ID library and 99% homology to four other library entries (P. fluorescens ATCC 17572, P. veronii DSM 11331, P. marginalisATCC 10844, and P. tolaasii ATCC 33618). In consensus sequence comparisons with the NCBI Blast (National Center for Biotechnology Information Basic Local Alignment Search Tool), ATCC 13525 matched most closely with two P. fluorescens strains (SBW25 and WH6) and an unidentified Pseudomonassp. shotgun sequence. Subsequent matches featured P. syringae and P. savastanoi.

The following are superseded names for P. fluorescens: “Bacillus fluorescens liquefaciens Flugge 1886, “Bacillus Fluorescens” Trevisan 1889, “Bacterium fluorescens” (Trevisan 1889) Lehmann and Neumann 1896 and “Liquidomonasfluorescens” (Trevisan 1889) Orla-Jensen 1909(Skerman et al., 1980). According to Hugh et al. (1964), ATCC 13525 was isolated in 1951 from water works pre-filter tanks in Reading, England. P. fluorescens ATCC 13525 is recognized by different accession numbers in other culture collections, for example it is referred to as DSM 50090 from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and as NCCB 76040 from the Netherlands Culture Collection of Bacteria.

1.1.2 Biological and Ecological Properties Biocontrol and plant growth promotion

P. fluorescens has been recognized as beneficial to plant growth (Kloepper et al., 1988; Weller and Cook, 1986). The majority of fluorescent pseudomonads produce complex peptidic siderophores called pyoverdines or pseudobactins, which are very efficient iron scavengers. P. fluorescens can enhance plant growth through production of siderophores which efficiently complex environmental iron, making it unavailable to other components of the soil microflora.

Certain strains of P. fluorescens produce molecules that make them ideal candidates for biocontrol against a wide spectrum of species including weeds (downy brome and annual bluegrass), aphids, termites, nematodes and zebra mussels, and various insects (see Appendix 4) as well as against a variety of pests in wheat, sugar beets, chickpea, tomato, cotton and cabbage (Kamilova et al., 2006; Khan et al., 2006; Schmidt et al., 2004; Someya et al., 2007; Srivastava et al., 2001; Weller, 2007). Some isolates have also been shown to suppress infections from Aeromonas hydrophila in fish (Das et al., 2006).

Below are some examples of strains, different from P. fluorescens ATCC 13525 on the DSL, in which biocontrol characteristics have been identified. There are no available data comparing the DSL strain, P. fluorescens ATCC 13525 with the biocontrol strains reported below. In addition, the DSL strain has not been reported in the literature as a candidate for biocontrol or able to produce the following toxins or metabolites.

Other metabolites:

Other mechanisms, separate from those that are mediated by specific toxins or metabolites, have been reported for biocontrol such as colonization of the host organism. For example, P. fluorescens MF0 shows virulence towards the fruit fly by its ability to evade its host immune system, growing rapidly within the insect (de Lima Pimenta et al., 2006). In the case of use as an antagonist of plant pathogens (bacteria, fungi or nematodes), the control of root diseases by some stains of P. fluorescens involves a combination of complementary mechanisms. The most prominent mechanisms are antibiosis towards plant pathogens, degradation of virulence factors produced by pathogens and induction of defence mechanisms in host plants. Efficient competition for colonization sites and micro- and macro-nutrients in the rhizosphere is an important prerequisite for effective biocontrol (Compant et al., 2005; Haas and Défago, 2005; Lugtenberg et al., 2001; Van Loon et al., 1998). P. fluorescens ATCC 31948 (referred to as A506) was assessed by the Pest Management Regulatory Agency (PMRA) in 2010 for use as biopesticide for the control of fire blight on apples and pears. It competes with the plant pathogen, Erwinia amylovora, for the same ecological niche (PMRA-HC, 2010). Biogeochemical cycling and metal resistance

Pseudomonads play a role in biogeochemical cycling as ecologically important micro-organisms in soil and water that assist in the degradation of many soluble compounds derived from the plant and animal materials (Palleroni, 1981).

P. fluorescens is also well known for its tolerance to metals (Appanna et al., 1996; Lemire et al., 2010). Pathogenic and Toxigenic Characteristics

No relevant reports were found in the publicly available literature, which investigated specifically the potential pathogenicity traits of P. fluorescens ATCC 13525 towards plants or animals. However, P. fluorescens, as a species, is known to produce a variety of enzymes, toxins, and other metabolites (Appendix 5).

Pathogenicity and toxicity testing studies on terrestrial organisms were performed by scientists at Environment Canada laboratories using the DSL-listed P. fluorescens ATCC 13525. Results of 21-day toxicity testing study using soil invertebrates, springtail (Folsomia candida) and earthworm (Eisenia andrei) exposed to P. fluorescens ATCC 13525 at 108 CFU/g dry soil and 106 CFU/g dry soil respectively, demonstrate no adverse effect on adult mortality or juvenile reproduction. Plant testing using red fescue grass (Festuca rubra) grown with P. fluorescens ATCC 13525 (1010 CFU/g dry soil) demonstrated no adverse effect on seedling emergence, shoot and root length, or shoot dry mass; however, a significant decrease in root dry mass was observed, (33% reduction) when exposed to ATCC 13525, relative to control growth (Princz, 2010). This result was not characterized further but it might be explained by the fact that P. fluorescens is capable of producing inhibitory metabolites that reduce root elongation, as is the case for strain A313 (Åström et al., 1993) .

In the absence of other published test data on the DSL-listed strain ATCC 13525, data from pathogenicity and toxicity testing on non-target species of other P. fluorescens strains, were reviewed as surrogate information. These data were provided to support the registration of these strains as biopesticides in Canada (PMRA-HC, 2010; PMRA-HC, 2012) and the United States (USEPA, 2009) (Appendix 6). Testing results of ATCC 55799 showed no evidence of mortality in the ciliate (Colpidium colpoda)or the cladoceran (Daphnia magna) and no mortality or adverse effects in terrestrial organisms. However, mortality was observed in four fish species in experiments with high concentrations, not recommended for the intended pesticidal use (Appendix 7).

An acute toxicity study was performed with another biocontrol strain, P. fluorescens ATCC 31948 (PMRA-HC, 2010). No signs of phytopathogenicity were observed when vascular plantsFootnote[3]were exposed to 106 or 108 CFU/mL of P. fluorescens ATCC 31948. However, the test protocol was not done according to guidelines as the test doses were below the maximum label rate (3.7 × 109 CFU/mL).

P. fluorescens ATCC 55799 and ATCC 31948 were observed to be not toxic to rats when exposed via oral or pulmonary route nor was it infective after intravenous injection. Mice were exposed to P. fluorescens ATCC 31948 via an intraperitoneal injection. Despite the study not meeting the recommended length of 21 days, general signs of toxicity were observed including scruffy coats, discharge from eyes, lethargy and diarrhea which may be attributed an immune reaction caused by the lipopolysaccharide.

There are no available data comparing P. fluorescens ATCC 13525 with the above two biocontrol strains (ATCC 31948 or ATCC 55799). In addition, the DSL strain has not been reported in the literature as a candidate for biocontrol or able to produce toxins and metabolites identified in other P. fluorescensstains. On this basis, we consider that P. fluorescens ATCC 13525 is unlikely to be toxic towards terrestrial or aquatic organisms.

Human Health

P. fluorescens is a psychrotrophic organism that is capable of proliferation at low temperatures typical of refrigerated storage (de Lima Pimenta et al., 2003; Gennari and Dragatto, 1992; Prescott et al., 2005). It is known as a spoilage organism, but not a pathogen, in refrigerated foods; however P. fluorescens can grow rapidly in refrigerated stored blood products. A seven to eight log increase in P. fluorescens cell number was observed after only one week of incubation at 4oC (Khabbaz et al., 1984). P. fluorescens contamination of blood products is strongly associated with post-transfusional sepsis (Gibaud et al., 1984; Gibb et al., 1995; Gibb, 2000; Khabbaz et al., 1984; Murray et al., 1987; Pappas et al., 2006; Scott et al., 1988), a rare, but often fatal systemic inflammatory response to bacteria in the bloodstream (Gottlieb, 1993; reviewed in Guinet et al., 2011; Riedemann et al., 2003). It is characterized by a rapid onset of symptoms, including fever, neutrophilia, rapid heart rate and loss of blood pressure, occurring during or immediately following transfusion of contaminated blood products (Brecher and Hay, 2005; Guinet et al., 2011; Murray et al., 1987).

Lipopolysaccharide (LPS) is a major structural component of the cell wall of all Gram negative bacteria, including P. fluorescens. In sepsis, LPS stimulates the innate immune response. In the tissues, this response acts effectively to contain infection, but in the bloodstream the widespread activation of phagocytes and the resulting cytokine cascade is catastrophic, inducing widespread endothelial cell activation, disseminated intravascular coagulation and organ dysfunction (reviewed in Guinet et al., 2011; Riedemann et al., 2003; Vincent, 2002). The structure of LPS affects its potency (reviewed in Netea et al., 2002), and in P. fluorescens LPS structure is known to be influenced by growth temperature (Picot et al., 2004); however it is unclear whether structural changes observed at low growth temperatures increase the potency of P. fluorescens LPS as an inflammatory mediator.

P. fluorescens has also been implicated as the causative agent of infection, mainly in patients with compromised immune function (de Lima Pimenta et al., 2003; Kanj et al., 1997; Nelson et al., 1991; Pappas et al., 2006). Unbroken skin and mucous membranes are the principle barriers against microbial invasion (reviewed in Ki and Rotstein, 2008). There is no evidence in the scientific literature that P. fluorescens has specific mechanisms for bypassing physical, chemical or lymphoid barriers to infection at epithelial or mucosal surfaces, and cases are usually associated with injury or medical interventions that breach normal physical barriers (Grice et al., 2008; Roth and James, 1988; Segre, 2006). Its abundance in the environment (Chapalain et al., 2008; Rebière-Huët et al., 2002) and its occurrence as a commensal member of the normal body flora (Chapalain et al., 2008; Madi et al., 2010; Stenhouse and Milner, 1992; Sutter et al., 1966; Wei et al., 2002) allow it to readily take advantage of such breaches.

Once entry into the host is achieved, adhesion to a host cell is the earliest step in the establishment of infection through colonisation of the host tissues (de Lima Pimenta et al., 2003; Hahn, 1997; Picot et al., 2001). Adhesion requires interaction between molecules present on the surfaces of the micro-organism and the host cell, and this interaction determines the tissue tropism of the ensuing infection (de Lima Pimenta et al., 2003). Like P. aeruginosa, P. fluorescens uses many different factors as adhesins including exopolysaccharides, lipopolysaccharides (LPS) and outer membrane proteins (reviewed in de Lima Pimenta et al., 2003; Dé et al., 1997; Picot et al., 2003), pili, flagellar proteins (reviewed in de Lima Pimenta et al., 2003; reviewed in Hahn, 1997), and porins (Rebière-Huët et al., 2002). Some of these are known to adhere to the human extracellular matrix protein fibronectin (de Lima Pimenta et al., 2003; Rebière-Huët et al., 2002). Fibronectin binding is an important mechanism in the pathogenicity of P. fluorescens, as fibronectin is involved in many cellular processes including tissue repair (To and Midwood, 2011), and is more available where tissue damage has occurred. As a consequence, breaches in the natural physical barrier, not only permit entry, but also favour colonization and infection.

The infectious potential of P. fluorescens in neurons was investigated because the closely-related opportunistic pathogen P. aeruginosa has been implicated in central nervous system infections with high rates of morbidity and mortality (Picot et al., 2001). The adherence of P. fluorescens to nerve and glial cells is mediated by LPS and generally causes apoptosis (Veron et al., 2008). In one study, adherence of P. fluorescens to cortical neurons and glial cells from rat neonate cell lines was comparable to that of P. aeruginosa (Picot et al., 2001). P. fluorescens has specific physiological characteristics, such as the expression of acetylcholinesterase (Rochu et al., 1998), γ-aminobutyric acid (GABA) aminotransderase, and high affinity GABA binding protein (Guthrie et al., 2000), which make it particularly harmful to nervous tissues (reviewed in Picot et al., 2004). It is able to exert cytotoxic effects that can induce cell death most likely through an apoptotic mechanism. P. fluorescens shows most features of an opportunistic pathogen in nerve cells and could therefore behave as a pathogen in certain situations (Picot et al., 2001). Nevertheless, P. fluorescens has been implicated as the cause of meningitis in only one report (Sarubbi et al., 1978).

Following adhesion, changes in enzyme production and protein secretion, including the expression of virulence factors, are regulated by quorum sensing. It is a cell-to-cell signalling system that functions through the secretion of diffusible autoinducer molecules that, when sensed by cell-surface receptors of other bacterial cells in the vicinity, regulates gene expression (Singh et al., 2010). Quorum sensing functions similarly in most Gram-negative bacteria, acting like a ‘switch’ to regulate expression of virulence factors to adapt to changes in environmental conditions (Singh et al., 2010). This mechanism enables bacteria to track changes in cell population density by monitoring the flux in autoinducer concentration, and associated coordination of gene expression (Swem et al., 2009). The two-component GacS-GacA regulatory system, which is conserved among Gram-negative bacteria like P. fluorescens, plays a role in quorum sensing and can determine virulence or biocontrol activities at the protein translation level (Blumer et al., 1999). In P. fluorescens, the GacS-GacA system controls the expression of extracelluar products such as antibiotics, exoenzymes, and hydrogen cyanide (Blumer et al., 1999).

P. fluorescens secretes an exotoxin related to the β-exotoxin of Bacillus thuringiensis (Picot et al., 2001); exoenzymes, including proteases (Koka and Weimer, 2000; Liao and McCallus, 1998; Sacherer et al., 1994); lipases (Dieckelmann et al., 1998) and a cholinesterase-like enzyme (Rochu et al., 1998); and other toxins and secondary metabolites reported in Appendix 5. Haemolysis has been observed in some, mainly clinical, strains of P. fluorescens (Sperandio et al., 2010). No haemolytic activity was observed when P. fluorescens ATCC 13525 was plated on blood sheep agar in tests conducted at Health Canada.

Quorum sensing is also important in biofilm formation.Biofilms have been extensively reported as a mechanism of pathogenicity in Pseudomonads through the adhesion to and colonization of biotic surfaces as well as abiotic surfaces (O'Toole and Kolter, 1998). P. fluorescens has been reported to form biofilms (Costerton et al., 1999; Heffernan et al., 2009). Once formed, biofilms may be more resistant to antimicrobial agents and could contribute to persistent and chronic bacterial infections (Costerton et al., 1999). P. fluorescens biofilms, which colonised catheter lumens, were implicated as a contributing factor in a series of delayed onset bloodstream infections as a result of exposure to contaminated heparin flush (CDC, 2006).

Some strains of P. fluorescens have been implicated in the pathogenesis of Crohn’s Disease (CD) and Inflammatory Bowel Disease (IBD) (Sandborn, 2007; Wei et al., 2002). Certain P. fluorescens strains carry the I2 antigen, which is a T-cell superantigen that induces the proliferative immune response and IL-10 secretion by CD4+ T-cells that is associated with CD and IBD (Cuffari, 2009; Dalwadi et al., 2001; Wei et al., 2002). P. fluorescens ATCC 13525 has not been associated with CD, and it is unknown whether it carries I2.

1.1.3 Effects Environment

P. fluorescens is a naturally occurring micro-organism that may occasionally be associated with some effects in plants and animals but, under normal circumstances, is unlikely to be a serious hazard.

No relevant reports, in the publicly available literature, specifically investigated the potential adverse effects of P. fluorescens ATCC 13525 towards plants or animals. However, the literature search performed with P. fluorescens at the species level, excluding the biocontrol context, revealed several cases of pathogenesis where P. fluorescens either caused a secondary infection or was found in the context of routine monitoring of diseases (see Appendix 8). Stress, compromised natural defenses, or comorbidity with primary viral or fungal infections are generally preconditions to the reported P. fluorescens infections.

In conclusion, while several studies have shown that different strains of P. fluorescens have toxigenic and pathogenic potential, it is important to note that adverse effects reported in Appendices 6 to 8 are mostly attributable to other strains and are not expected to occur to biota in the environment in Canada from P. fluorescens ATCC 13525. Human Health

Human infections have been mainly nosocomial in immune compromised or seriously ill patients, some of which resulted in serious and occasionally fatal disease. It is less virulent than other pseudomonads, such as P. aeruginosa (Foulon et al., 1981; Pappas et al., 2006).

P. fluorescens has also been reported as the causative agent in a diverse array of infections, many involving immune compromised patients, or those with debilitating co-morbidities, and with injury or medical interventions that breach normal physical barriers or introduce P. fluorescens-contaminated fluids into the body (Burgos et al., 1996; Carpenter and Dicks, 1982; Dalamaga et al., 2005; Essex et al., 2004; Foulon et al., 1981; Kitzmann et al., 2008; Manfredi et al., 2000; Nelson et al., 1991; Pappas et al., 2006; Rais-Bahrami et al., 1990; Rutenburg et al., 1958; Sarubbi et al., 1978). Pseudomonas infections, as a primary cause of sepsis in neonates, are increasingly recognized. Most are associated with P. aeruginosa, but one case of P. fluorescens sepsis resulting in the death of a neonate was reported (Rais-Bahrami et al., 1990). P. fluorescens-contaminated amphotericin B was implicated in one case of nosocomially-acquired meningitis and bacteraemia (Sarubbi et al., 1978). Pelviperitonitis caused by P. fluorescens was diagnosed in a patient wearing an intrauterine device (Foulon et al., 1981). A patient with positive HIV status was diagnosed with a P. fluorescens infection likely the result of the central venous catheter (Nelson et al., 1991). P. fluorescens endophthalmitis was reported in two patients: one secondary to trauma (Essex et al., 2004), the other associated with a corneal ulcer (Kitzmann et al., 2008). Urinary tract infections caused by P. fluorescens (Carpenter and Dicks, 1982; McLean and Nickel, 1991; Rutenburg et al., 1958) are often related to bladder catheterisation (Carpenter and Dicks, 1982). However, a cancer patient, with no history of catheterisation was also diagnosed with a urinary tract infection caused by P. fluorescens (Pappas et al., 2006). The death of a cystic fibrosis patient 18 months post-lung transplantation was attributed to pericarditis caused by P. fluorescens(Kanj et al., 1997). Other causes of P. fluorescensinfection include anabolic androgen steroid use (Kienbacher et al., 2007) and a dog bite which resulted in cutaneous abscess and recurrent bacteraemia (Dalamaga et al., 2005).

P. fluorescens was identified as the causative agent of infection in two outbreaks of P. fluorescens bacteremia associated with the presence of indwelling devices and contaminated fluids (e.g. heparin/saline flushes) (Gershman et al., 2008). One, reported in Taiwan (Hsueh et al., 1998), involved four patients with underlying malignancies and was associated with the use of Port-A-Cath implants. All recovered with treatment. The second, reported in the United States, originated from a single contaminated heparin/saline flush product distributed to several States (Gershman et al., 2008). Approximately 80 patients, being treated for various conditions, were infected. All recovered after antibiotic treatment.

In vitro and in vivo tests were conducted by Health Canada’s scientists to evaluate the potential of P. fluorescensATCC 13525 to cause cytotoxicity and adverse immune effects. Results indicate no evidence of cytotoxic effects on human colonic cells (HT29) after 6, 12 and 24 hours of exposure. No haemolytic activity was observed on sheep blood agar. BALB/c mice exposed to 1 × 106 CFU/μL of ATCC 13525 by endotracheal instillation showed no changes in behavior and physical appearance. No significant increase in lung granulocytes, or lung and blood cytokines, or in serum amyloid A were observed over the one week sampling period. All bacteriawere cleared 96h and 168h after exposure from lungs, trachea and esophagus. In the absence of complete pathogenicity/toxicity test data on P. fluorescens ATCC 13525, data on other strains was considered as surrogate information. Health Canada’s Pest Management Regulatory Agency (PMRA) has registered two strains of P. fluorescens (ATCC 55799 and ATCC 31948) as microbial pest control agents under the strain designations CL45A and A506, respectively. Studies reviewed by PMRA as part of their decisions found that neither strain was toxic or pathogenic in standard acute pathogenicity/toxicity tests. P. fluorescens ATCC 55799 was observed to be not toxic to rats when exposed via oral (2.42 × 107 CFU/kg bw) or pulmonary (3.4 × 108 CFU/animal) route nor was it infective after intravenous injection (4.7 × 106 CFU/mL to 1.95 × 107 CFU/mL). No significant adverse effects were reported when a mixture (P. fluorescens ATCC 31928 and other P. fluorescens strains) or P. fluorescens AGS 3001.2 (a strain similar to P. fluorescens ATCC 31928) was administered orally to rats. P. fluorescens ATCC 31948 was observed to be not toxic to rats when exposed via oral (8.4 × 1010 CFU/animal) route. In mice exposed to P. fluorescens ATCC 31948 via intraperitoneal injection (2.0 × 108 CFU/animal) there were no mortalities, but general signs of toxicity were observed including scruffy coats, discharge from eyes, lethargy and diarrhea. The study did not meet the recommended 21 day observation period to assess infectivity. It is likely that the observed signs of toxicity can be attributed to an immune reaction to the lipopolysaccharide. These studies have been included in Appendix 7 (PMRA-HC, 2010; PMRA-HC, 2012).

The most serious effect of P. fluorescens on human health is associated with its ability to proliferate in blood products stored at refrigeration temperatures. Post-transfusional sepsis (Gibaud et al., 1984; Gibb et al., 1995; Gibb, 2000; Khabbaz et al., 1984; Murray et al., 1987; Pappas et al., 2006; Scott et al., 1988) occurs rarely, but is fatal in approximately 60% of reported cases, and affects people of all ages and states of health. Antibiotic Susceptibility

Antibiotics used successfully to treat the P. fluorescens infections described above include amikacin, aminoglycosides, ampicillin, ceftazimide, cefazolin, ciprofloxacin, chloramphenicol, gentamicin, methotrexate, moxifloxacin, prednisone, tetracycline, ticarcillin, ticarcillin/clavulanate, tobramycin, and vancomycin. Table 1-3 represents an antibiogram generated by Health Canada for the characterization of P. fluorescens ATCC 13525.

Table 1-3 Minimal Inhibitory Concentration (MIC) for P. fluorescens ATCC 13525Footnote Table 1-3[a]
AntibioticMIC (μg/mL)
Amoxycillingreater than 50
Amphotericingreater than 50
Aztreonamgreater than 50
Cephotaximegreater than 50
Doxycycline0.8 +/- 0.4
Erythromycingreater than 50
Gentamicin2.3 +/- 2.3
Nalidixic acid24
Trimethoprimgreater than 50
Vancomycingreater than 50
Footnote Table 1-3 a

Work conducted using TSB-MTT liquid assay method to characterize the P. fluorescens DSL strain (Seligy et al., 1997). The reported values are based on a minimum of three independent experiments. Values correspond to the minimal inhibitory concentration (μg/ml) for select P. fluorescens (20, 000 CFU/well) grown in the presence of antibiotic for 24 hrs. at 37°C.

Return to footnote Table 1-3 a referrer

MIC tests performed by Sader and Jones (2005) on P. fluorescens clinical isolates indicate that the most active compound was amikacin (MIC50, 2 mg/l; 88.5% susceptible), followed by cefepime (MIC50, 4 mg/l; 84.2% susceptible), which showed the lowest resistance rate (6.3%). Other compounds with reasonable activity against P. fluorescens include tobramycin, imipenem, polymyxin B and ceftazidime.

All micro-organisms contain components, such as lipopolysaccharides, antigens, toxins and enzymes, which may act as potential sensitizers. Though P. fluorescens possesses antigens which are known to cause hypersensitivity (Bernstein et al., 1995; Fishwick et al., 2005; Skorska et al., 2005; Yadav et al., 2003), there are no reported cases directly implicating allergenicity of P. fluorescens ATCC 13525 in healthy individuals.

1.2 Hazard Severity

P. fluorescens, as a species,is a well characterized micro-organism. A combination of morphological, biochemical and physiological traits allow it to be reliably discriminated from other Pseudomonas species, especially closely related pathogens such as P. aeruginosa. Despite the widespread presence of P. fluorescens in soil and water and the use of certain strains for biocontrol against specific pest organisms, only a few reports were found regarding the pathogenic and toxigenic potential of some strains of the species. Stress, compromised natural defenses or comorbidity with primary viral or fungal infections are generally preconditions to the reported P. fluorescens infections. No relevant reports, in the publicly available scientific literature, specifically investigated the potential adverse effect of P. fluorescens ATCC 13525 towards plants or animals. Furthermore, there have been no reports regarding the pathogenic or toxigenic potential of P. fluorescens ATCC 13525, nor has it been associated with toxins or metabolites that may lead to adverse effects.

Therefore, environmental hazard severityfor P. fluorescens ATCC 13525 is estimated to be low.

Despite adverse effects reported at the species level, there have been no reported infections attributed specifically to P. fluorescens ATCC 13525 in the scientific literature. Its inability to grow at normal human body temperature may limit its ability to invade and cause disease in immune competent individuals. The human health hazard for P. fluorescensATCC 13525 to the general population is estimated to be low. Nonetheless, there are reports of adverse effects including P. fluorescens nosocomialinfections and sepsis resulting from contaminated medical devices and blood products, suggesting a hazard to individuals undergoing medical treatment. The human health hazard associated with these individuals is estimated to be medium. The overall human hazard severityfor P. fluorescens ATCC 13525 is therefore estimated to be low-medium.

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2. Exposure Assessment

2.1 Sources of Exposure

In 2007, a voluntary questionnaire was sent to a subset of key biotechnology companies. These results combined with information obtained from other federal government regulatory and non-regulatory programs indicate that 10,000 to 100,000 kg of products potentially containing P. fluorescens ATCC 13525 (formulation and concentration unknown) were imported into or manufactured in Canada in 2006-2007.

In 2009, the government conducted a mandatory information-gathering Notice (survey) under section 71 of CEPA 1999 as published in the Canada Gazette Part 1 on October 3rd, 2009 (hereafter “s. 71 Notice”). The s. 71 Notice applied to any persons who, during the 2008 calendar year, manufactured or imported a DSL substance, whether alone, in a mixture, or in a product. Anyone meeting these reporting requirements was legally obligated to respond. Respondents were required to submit information on the industrial sector, uses and any trade names associated with products containing these strains, as well as the quantity and concentration of the strain imported or manufactured in the 2008 calendar year. A variety of environmental, industrial and household applications using P. fluorescens ATCC 13525 were reported in response to the s. 71 Notice. According to information submitted, 100 to1000 kg of P. fluorescens ATCC 13525 was imported into or manufactured in Canada in 2008.

The focus of this screening assessment is exposure to P. fluorescens ATCC 13525 from its deliberate use in consumer or commercial products and industrial processes.

The species P. fluorescens has properties that make it of commercial interest in a variety of industries. P. fluorescens has been shown to have the ability to degrade a wide variety of compounds, including; 3-chlorobenzoic acid (Fava et al., 1993); naphthalene, phenantrene, fluorine and fluoranthene (Weissenfels et al., 1990); chlorinated aliphatic hydrocarbons (Vandenbergh and Kunka, 1988); styrene (Baggi et al., 1983); and pure hydrocarbons and crude oil (Janiyani et al., 1993). P. fluorescens can also be used in biosensor applications. For example, the recombinant P. fluorescensstrain HK9, which lights up in the presence of contaminants such as PAHs (due to insertion of lux genes), allows easy detection of bioavailable fractions of pollutants in soils and sediments (King et al., 1990).

A search of the public domain (internet, patent databases) suggests multiple potential uses, including in pulp and paper and textile processing, municipal and industrial wastewater treatment, waste degradation, particularly in petroleum refineries, bioremediation and biodegradation as well as in commercial and household drain cleaners and degreasers, enzyme and chemical production, septic and RV tank additives and general cleaning and odour-control products. For agricultural applications, P. fluorescens has been used for pest control, for plant growth and disease suppression and as an anti-frost agent.

Due to expanding commercialization of products potentially containing P. fluorescens, there is a likelihood of an increase in the use and release of P. fluorescens ATCC 13525 in the environment through human activity (Chatzipavlidis et al, 2013).

2.2 Exposure Characterization

2.2.1 Environment

Based on reported uses identified through Section 71, the most likely routes of introduction of P. fluorescens ATCC 13525 into the environment could be directly and indirectly into water and soil.

P. fluorescens is a normal inhabitant of the soil rhizosphere and several studies were reported about the survival and persistence of different strains. P. fluorescens cells are able to withstand low temperatures, and could survive better at 4°C than at 15°C or 27°C following introduction into natural soil (George et al., 1999) and P. fluorescens R2f survived above 107 CFU/g dry soil for up to 84 days in loamy sand microcosms when encapsulated (Van Elsas et al., 1992), while free cells declined below 105 cells/g dry soil after 21 days. A study by Troxler et al. (1997) showed that under aerobic conditions, numbers of culturable P. fluorescens CHA0 cells declined in effluent water, from 107 to approximately 10>sup>2

, in the span of 115 days. The authors hypothesized that the decline was due to competition-antagonism with indigenous micro-organisms or grazing by protozoa.

Persistence data was specifically performed by Environment Canada on P. fluorescens ATCC 13525 in agricultural soil. After inoculation of soil with live cells, it was found that the DNA from this strain persisted for up to 28 days (Xiang et al., 2010). The persistence observed may or may not be from viable cells. However, given the ubiquity of the species, one could assume that this strain is also able to survive for considerable lengths of time in soil and other media even if there is no evidence of proliferation.

The above information indicates that in most exposure scenarios, P. fluorescens ATCC 13525 is theoretically able to survive and persist in the environment but maintenance of high numbers beyond those of background are unlikely, due to competition (Leung et al., 1995) and microbiotasis (Van Veen et al., 1997), which is an inhibitory effect of soil that results in the rapid decline of populations of introduced bacteria. Competition and competitive niche exclusion are likely to limit the growth of introduced pseudomonad inoculants. Competitors are likely to include closely related pseudomonads and other bacteria able to compete for the same ecological niches with similar nutritional requirements (Lindow, 1992).

No relevant reports concerning persistence in the environment of toxins produced by P. fluorescens have been found.

Thus, theoverall environmental exposure to P. fluorescens strain ATCC 13525 is estimated to be medium.

2.2.2 Humans

Hazards related to micro-organisms used in the workplace should be classified accordingly under the Workplace Hazardous Materials Information System (WHMIS)Footnote[4].

Human exposure is expected to be greatest through the use of consumer or commercial products containing P. fluorescens ATCC 13525. For consumer products, the routes and extent of user and bystander exposure will depend on the application method, the concentration of P. fluorescens ATCC 13525 in the product, and the amount of product applied. During product application, dermal exposure is likely for products applied by hand, and inhalation of aerosols or dust containing P. fluorescens ATCC 13525 is expected for products with spray or powder formulations. Secondary to product application, residual P. fluorescens ATCC13525 on surfaces and in reservoirs such as treated drains could result in dermal exposure, as well as inadvertent ingestion where the organism persists on food preparation surfaces, and inhalation, where aerosols are generated (e.g., kitchen garbage disposal units). Since P. fluorescens ATCC 13525 is expected to persist following application, such exposures may be temporally distant from the time of application.

For commercial products, the general population could be exposed as bystanders during product application. The route and extent of exposure will depend on the application method, the concentration of P. fluorescens ATCC 13525 in the product, the amount of product applied, and proximity to the site application. The general population could also come into contact with residual P. fluorescens ATCC 13525 on treated surfaces.

Use of P. fluorescens ATCC 13525 in waste and wastewater treatment or in industrial processes, may introduce the organism into bodies of water and soil. Nevertheless, human exposure to the strain through the environment is expected to be low. While large inputs of DSL-listed P. fluorescens ATCC 13525 into the environment could result in concentrations greater than background levels of P. fluorescens, high numbers of vegetative cells are unlikely to be maintained in water and in soil due to competition (Leung et al. 1995) and microbiostasis (van Veen et al. 1997), as previously mentioned. Moreover, drinking water treatment processes are expected to effectively eliminate these micro-organisms and so limit their ingestion in drinking water.

The overall human exposure estimation for P. fluorescens ATCC 13525 is medium. Given the range and scale of intended and potential uses, there is reason to expect that the quantity of P. fluorescens ATCC 13525 released into the Canadian environment will be greater than that reported in response to the section 71 Notice. In addition, specific exposure scenarios, such as the use of consumer cleaning products containing P. fluorescens, could result in direct, possibly repeated, exposure to larger quantities of the micro-organism.

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3. Decisions from other Jurisdictions

In Canada, the Pest Management Regulatory Agency (PMRA), under the authority of the Pest Control Products Act (PCPA) and Regulations, as well as the United States Environmental Protection Agency (USEPA) granted full registration for the sale and use of P. fluorescens strain (ATCC 31948, biovar 1) microbial pest control agents (MPCA) against a fireblight pathogen. Another strain of P. fluorescens (ATCC 55799, biovar 1, inactivated) was approved for use as a MPCA against zebra and quagga mussels by both the PMRA and the USEPA. An evaluation by PMRA of available scientific information found that, under the approved conditions of use, the products do not present an unacceptable risk to human health or the environment. Infectivity and toxicity testing for the latter strain is included in Appendix 7 (PMRA-HC, 2010; PMRA-HC, 2012). P. fluorescens was considered a plant pest of quarantine importance in the Commonwealth of Dominica in 2005 (Regulated Pests of member countries of the International Plant Protection Convention).

P. fluorescens is considered a Risk Group 1 animal and human pathogenic by the Public Health Agency of Canada. It is not considered to be an animal pathogen, nor does the Canadian Food Inspection Agency require a plant protection permit to import this organism into Canada (CFIA, Import Permit Office - Plant Health Program). The Public Health Agency of Canada considers P. fluorescens to be a Risk Group 1 bacterium, based on the fact that it is an opportunistic human pathogen, capable of causing human infection, but unlikely to do so in healthy individuals.

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4. Risk Characterization

In this assessment, risk is characterized according to a paradigm embedded in section 64 of CEPA 1999 that 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 P. fluorescens ATCC 13525 to be low and exposure, as assessed through the s. 71 Notice for the 2008 calendar year, from its deliberate use in industrial processes or consumer or commercial products in Canada isexpected to be medium for environmental species.

Based on the considerations outlined above, the risk to the environment from current and foreseeable future uses of Pseudomonas fluorescens ATCC13525 is expected to be low.

Based on the low level of human health hazard of P. fluorescens ATCC 13525 to the general population and the medium potential for exposure as assessed through the s. 71 Notice for the 2008 calendar year, the risk is estimated to be low with respect to the general population. Although individuals undergoing medical treatment could be at greater risk than the general population, current use patterns do not suggest a risk that medical devices or blood products could become contaminated from deliberate uses of P. fluorescens ATCC 13525.

It is therefore proposed to conclude that P. fluorescens ATCC 13525 does not meet any of the criteria set out in section 64 of CEPA 1999.

The determination of risk from current uses is followed by consideration of the estimated hazard in relation to foreseeable future exposures (from new uses). If a risk may be associated with new uses or activities, the Government can take action to require assessment of these new activities before they begin.

The risk to the environment from foreseeable future uses is expected to be low. Aquatic and terrestrial species may be exposed to the DSL strain when used in activities at higher concentrationscompared to whatwould be expected in a naturally-occurring microbial community. Nevertheless, no evidence of adverse effects have been reported at the population or ecosystem levels in Canada that can be specifically attributed to P. fluorescens ATCC 13525 and the dilution factor of products containing theDSL strain is expected to be significant, so the concentrations required to see any potential adverse effects are not anticipated to be reached.

The risk to human health from foreseeable future uses is expected to be low, but could increase to medium for individuals undergoing medical treatment, if they occur in healthcare settings.

P. fluorescens ATCC 13525 has properties that make it suitable for use in a range of products, and there is reason to expect new uses of P. fluorescens ATCC 13525 in health care settings could emerge. In particular, there is growth in the market for “greener” microbial-based cleaning products, (Spök and Klade, 2009). As these products have potential uses in health care settings, there is some potential for harm.

Therefore, although effects in the general population are not expected, it is possible that new activities not considered in this assessment could increase the risk of nosocomial infections or sepsis resulting from contamination of medical devices or blood products.

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5. Conclusion

Based on responses to the 2009 s. 71 Notice, it is concluded that P. fluorescens ATCC 13525 is not entering the environment in a quantity or concentration or under conditions that:

Therefore, it is concluded that those substances do not meet the criteria as set

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Appendix 1: Characteristics of P. fluorescens ATCC 13525 – Growth Kinetics

Growth kinetics was 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). Measurements were taken at OD 500nm every 15min over a 24h period.

Table A1-1 Growth kinetics of P. fluorescens ATCC 13525Footnote Appendix A Table A1-1 [a]
Trypticase Soy Broth+Footnote Appendix A Table A1-1[b]~Footnote Appendix A Table A1-1[c]-Footnote Appendix A Table A1-1[d]-
Sheep Plasma(+)Footnote Appendix A Table A1-1[e](+)--
Fetal Bovine Serum~---
Dulbecco’s Modified Eagles Medium (mammalian cell culture)(+)---
Footnote Appendix A Table A1-1 a

Data generated by Health Canada’s Healthy Environments and Consumer Safety Branch

Return to footnote Appendix A Table A1-1 a referrer

Footnote Appendix A Table A1-1 b

+, growth

Return to footnote Appendix A Table A1-1 b referrer

Footnote Appendix A Table A1-1 c

~, low level growth

Return to footnote Appendix A Table A1-1 c referrer

Footnote Appendix A Table A1-1 d

-, no growth

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Footnote Appendix A Table A1-1 e

(+), delayed growth (after 15h)

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Appendix 2: Characteristics of P. fluorescens ATCC 13525 - Growth on Different Media at 28°C and 37°C (48 hours)

Table A2-1 Growth of P. fluorescens ATCC 13525 on different mediaFootnote Appendix A Table A2-1[a]
Nutrient+Footnote Appendix A Table A2-1[b]-Footnote Appendix A Table A2-1[c]
TSBFootnote Appendix A Table A2-1[d]+-
StarchFootnote Appendix A Table A2-1[e]-GrowthNTFootnote Appendix A Table A2-1[f]-
Maconkey AgarFootnote Appendix A Table A2-1[g]+-
Lysine IronFootnote Appendix A Table A2-1[h]+-
Triple Sugar Iron - w phenol redFootnote Appendix A Table A2-1 [i]+-
Mannitol Egg Yolk Polymyxin supplementsFootnote Appendix A Table A2-1 [j]+-
MannitolFootnote Appendix A Table A2-1[k]--
CitrateFootnote Appendix A Table A2-1[l]+-
UreaFootnote Appendix A Table A2-1[m]+-
Footnote Appendix A Table A2-1 a

Data generated by Heath Canada’s Environmental Health Science and Research Bureau

Return to footnote Appendix A Table A2-1 a referrer

Footnote Appendix A Table A2-1 b

+, positive growth

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Footnote Appendix A Table A2-1 c

-, negative growth

Return to footnote Appendix A Table A2-1 c referrer

Footnote Appendix A Table A2-1 d

Trypticase soy broth, an all-purpose medium

Return to footnote Appendix A Table A2-1 d referrer

Footnote Appendix A Table A2-1 e

Differential medium that tests the ability of an organism to produce extracellular enzymes that hydrolyze starch

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Footnote Appendix A Table A2-1 f

NT, not tested

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Footnote Appendix A Table A2-1 g

Detection of coliform organisms in milk and water; tests for ability of organism to ferment lactose

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Footnote Appendix A Table A2-1 h

Simultaneous detection of lysine decarboxylase and formation of hydrogen sulfide in the identification of Enterobacteriaceae, in particular Salmonella and Arizona according to Edwards and Fife

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Footnote Appendix A Table A2-1 i

Gram-negative enteric bacilli based on glucose, lactose, and sucrose fermentation and hydrogen sulfide production

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Footnote Appendix A Table A2-1 j

B. cereus selective agar

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Footnote Appendix A Table A2-1 k

Isolation and differentiation of Staphylococci

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Footnote Appendix A Table A2-1 l

Citrate utilization test, ability to use citrate as the sole carbon source

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Footnote Appendix A Table A2-1 m

Screening of enteric pathogens from stool specimens - urea metabolism

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Appendix 3: Characteristics of P. fluorescens ATCC 13525 – Fatty Acid Methyl Ester (FAME) Analysis

Data presented shows the best match between the sample and the environmental MIDI databases, along with the number of matches (fraction of total number of tests) and the fatty acid profile similarity index (in parentheses; average of all matches). MIDI is a commercial identification system that is based on the gas chromatographic analysis of cellular fatty acid methyl esters

Table A3-1 Environmental MIDI database results for P. fluorescens ATCC 13525Footnote Appendix A Table A3-1 [a]
Species MatchesFatty Acid Profile Similarity Index (average of all matches)
P. fluorescens biotype A6/22 (0.888)
P. fluorescens biotype B6/22 (0.800)
P. putida biotype A1/22 (0.805)
P. putida biotype B4/22 (0.229)
Corynebacterium diphtheriae-gravis & mitis1/22 (0.729)
Pseudoalteromonas nigrifaciens1/22 (0.117)
No Match3/22
Footnote Appendix A Table A3-1 a

Data generated by Health Canada’s Healthy Environments and Consumer Safety Branch.

Return to footnote Appendix A Table A3-1 a referrer

Appendix 4: LD50 Values for Toxins Produced by Some Strains of P. fluorescens

Table A4-1 LD50 values for toxins produced by some strains of P. fluorescens
SubstanceOrganismLD50 or LC50Strain
LPSFootnote Appendix A Table A4-1[a]10- to 12- week-old BALB/c mice(sensitized to D-galactosamine)LD50 7500 ng/mouseATCC 13525 (IMV 4125)
P. fluorescens (Fit Toxin producing strains)Footnote Appendix A Table A4-1 [b]Galleria mellonella
Manduca sexta
LD50 1.8 × 102cells
LD50 9.8 × 102 cells
LD50 4 × 103 cells
CHA0 and Pf-5
UnidentifiedFootnote Appendix A Table A4-1 [c]44 kDa protein (VCRC B426)Footnote Appendix A Table A4-1[d]Anopheles stephensi (larvae)LC50 70.4 µg protein ml-1P. fluorescens Migula
Unidentified[c] 44 kDa protein (VCRC B426)[d]Culexquinquefasciatus (larvae)LC50511.5 µg protein ml-1P. fluorescens Migula
Unidentified[c] 44 kDa protein (VCRC B426)[d]Aedes aegypti (larvae)LC50 757.3 µg protein ml-1P. fluorescens Migula
Unidentified[c] 44 kDa protein (VCRC B426)[d]Anopheles stephensi (pupae)LC50 2.0 µg protein ml-1P. fluorescens Migula
Unidentified[c] 44 kDa protein (VCRC B426)[d]Culexquinquefasciatus (pupae)LC50 9.4 µg protein ml-1P. fluorescens Migula
Unidentified[c] 44 kDa protein (VCRC B426)[d]Aedes aegypti (pupae)LC5019.2 µg protein ml-1P. fluorescens Migula
Unidentified[c] 44 kDa protein (VCRC B426)Footnote Appendix A Table A4-1[e]Musca domestica (net mortality of larvae and pulpae all together)LC50 8.25 µg protein ml-1P. fluorescens Migula
Footnote Appendix A Table A4-1 a

(Veremeichenko et al., 2005)

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Footnote Appendix A Table A4-1 b

(Péchy-Tarr et al., 2008)

Return to footnote Appendix A Table A4-1 b referrer

Footnote Appendix A Table A4-1 c

Proprietary information; toxin name undisclosed

Return to footnote Appendix A Table A4-1 c referrer

Footnote Appendix A Table A4-1 d

(Prabakaran et al., 2003)

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Footnote Appendix A Table A4-1 e

(Padmanabhan et al., 2005)

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Appendix 5: Toxin and Secondary Metabolite Production

Table A5-1 List of toxins and secondary metabolites produced by P. fluorescens
Lipopolysaccharide (LPS), endotoxin
  • LPS plays a leading role in the infectious process
  • LPS is a complex amphiphilic molecule essential for outer membrane functions, particularly during host-pathogen interactions
  • Major virulence factor responsible for membrane depolarisation in cerebellar granule neurons. Causes the reduction of two of the major voltage-dependant potassium currents
  • P. fluorescens can bind to glial cells and its LPS will modulate potassium channels in target cells. LPS induces the expression of a nitrite oxide synthase (NOS) associated to apoptosis. Cells invasion and cytotoxicity are not mutually exclusive events
  • In sepsis, the lipid A component stimulates the innate immune response by binding to the phagocyte LPS receptor. This activates the release of the inflammatory cytokines TNF, IL-1, IL-6, IL-8 and IL-12, which in the bloodstream can cause septic shock
(Mezghani-Abdelmoula et al., 2004; Mezghani-Abdelmoula et al., 2003; Picot et al., 2003; Picot et al., 2004; Veremeichenko et al., 2005; Veremeichenko and Zdorovenko, 2008)
  • Virulence factor that contributes to bacterial infection
  • Heat resistant extracellular alkaline metalloprotease of the serralysin family
  • Involved in nutrient utilization; ability to degrade proteins in the environment
  • Possesses two conserved binding domains (Zn2+ and Ca2+)
  • Associated with strain linked to spoilage of milk and dairy products
(Dufour et al., 2008; Marchand et al., 2009; Zhang et al., 2009)
AprA (alkaline protease)
  • Extracellular protease (EDTA-sensitive)
  • Anti-nematode factor
  • Serralysin-type metalloprotease
  • apra genes are regulated by quorum sensing
(Lenz et al., 2008; Maunsell et al., 2006; Siddiqui et al., 2005)
Phospholipase C
  • Substrate specificity different from phospholipase C from either Cl. welchii or B. cereus
  • Phosphatidyl ethanolamine is hydrolyzed more easily than other phospholipids
(Doi and Nojima, 1971)
Unidentified 44 kDa protein (VCRC B426)
  • A bacterial metabolite in simulated field conditions; mode of action unknown
  • Approximate molecular mass of 44 kDa with temperature stability at 120 ºC for 20 min
  • Nontoxic to mammals
  • Caused significant mortality of Culex quinquefasciatuspupae and suppression of adult emergence
  • VCRC B426 in a 0.09% emulsifiable concentration showed reduction of 80% in pupal density for Culex quinquefasciatus
(Murty et al., 1994; Padmanabhan et al., 2005; Prabakaran et al., 2003; Prabakaran et al., 2009; Sadanandane et al., 2003)
Hydrogen cyanide
  • Inhibitor of plants roots and a broad-spectrum of compounds
  • Produced by clinical isolates of P. aeruginosa from cystic fibrosis patients at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase
  • A potent inhibitor of cellular respiration that is produced under microaerophilic growth conditions at high cell densities
  • Cyanide levels are associated with impaired lung function
(Blumer and Haas, 2000; Castric, 1983; Flores-Vargas and O'Hara, 2006; Pessi and Haas, 2000; Ryall et al., 2008)
  • High-affinity strain specific, yellow-green fluorescent siderophore
  • In iron-limiting conditions, pyoverdine enables the acquisition of iron from the environment by chelating with iron when secreted in the extracellular environment and resulting in a ferri-pyoverdine complex that will be transported back into the bacteria by a cell surface receptor protein
  • Virulence factor in P. aeroginosa
(Gross and Loper, 2009; Moon et al., 2008)
  • Iron-scavenging metabolites with a very different scaffold than the pyoverdines
  • P. fluorescens produced a stereoisomer of pyochelin, named enantio-pyochelin
  • Enantio-pyochelin enables the bacteria to sequestered iron in an available form for them but not for competing bacteria
  • Virulence factor in P. aeroginosa
(Gross and Loper, 2009; Takase et al., 2000; Youard et al., 2007)
  • Secondary siderophorebutfunction like a siderophore
  • Consist of salicylic acid and two heterocyclic amino acids
(Reviewed in Gross and Loper, 2009)
  • Pyocins are antibacterial agents (active against closely related species or strains) usually associated with P. aeruginosa which exist in the three type (R, F and S)
  • Putative F- and R- pyocins appear to be ubiquitously distributed among strains of P. fluorescens
  • R- and F- type pyocins resemble tails of bacteriophage. The R- type has a non-flexible and contractile rod-like structure and the F- type has a flexible and non-contractile rod-like structure
  • R-type pyocin arrests the synthesis of macro molecules and releases intracellular material, which is followed by cell death caused by depolarisation of the cytoplasmic membrane
  • Production starts when adverse conditions provoke DNA damage and at optimal temperatures (37°C)
(Iwalokun et al., 2006; Mavrodi et al., 2009; Michel-Briand and Baysse, 2002)
Fit toxin (P. fluorescens insecticidal toxin)
  • Toxin produced only by the strain CHA0 and Pf-5 which have an anti-insect activity related to the insecticidal toxin Mcf (causes caterpillars to become floppy) from Photorhabdus luminescens
(Péchy-Tarr et al., 2008)
Rhizoxin analogs (such as (WF-1360 F, 22Z-WF-1360 F, WF1360 C, WF-1360 B and Rhizoxin D)
  • 16-member macrolide that exhibits phytotoxic, antifungal and antitumor activities (reported to be produced by strain Pf-5)
  • Activity related to the binding of those molecules to β-tubilin which will interfere with microtubule dynamics during mitosis
(Gross and Loper, 2009; Loper et al., 2008)
Cyclic lipopeptides (viscosin, massetolide, orfamide ,etc.)
  • Class of compounds with diverse structure containing a fatty acyl residue ranging from C5-C16 in length and chains of 7-25 amino acids of which 4-14 form a lactone ring
  • Divided in 6 groups: viscosin, syringomycin, amphisin, putisolvin, tolaasin and syringopeptin
  • Lower surface tension and altering cellular membranes integrity by interaction due to their amphiphilic properties
  • Increase the bioavailability of water-insoluble substrates, promote cellular swarming and enhance virulence or antagonism against other microorganism
(Reviewed in Gross and Loper, 2009)
  • Strong antifungal activity, compound use as a topical antimycotic in human
  • Inhibitor of fungal respiratory chain
(Reviewed in Gross and Loper, 2009)
  • Over 50 compounds in this large family of colorful nitrogen-containing tricyclic molecules
  • Antibiotic, antitumor and antiparasitic activity
  • Activity due to interaction with polynucleotides, topoisomerase inhibition and the generation of free radical
  • Intracellular signals have an influence on transcriptional regulation and broad effect on bacterial physiology and fitness
(Reviewed in Gross and Loper, 2009)
  • Biologic controls against plants disease produced by a subset of P. fluorescens.
  • Toxic effect on a range of plant pathogenic fungi
  • Antibacterial, anti-helmintic and phytotoxic properties at high concentration
  • DAPG triggers systemic resistance of plants against disease
(Gross and Loper, 2009)
  • Substance composed of a bichlorinated pyrrole linked to a resorcinol moiety
  • Shows antifungal properties
  • Moderate plant disease cause by fungal pathogen such as Oomycetes fungi
(Hammer et al., 1997; Nowak-Thompson et al., 1999)
Cyclo(-Pro-Val-) and cyclo(-Pro-Tyr-)
  • Phytotoxic activity
(Guo et al., 2007)

Appendix 6: Strains of P. fluorescens used as biocontrol agents against plants and invertebrates

Table A6-1 P. fluorescens strains used as a biocontrol agent against plants
Downy brome (Bromus tectorum) seedsP. fluorescens strain D7(Johnson et al., 1993)
Annual bluegrass (Poa annua L.)Different P. fluorescens isolates(Banowetz et al., 2008)
Table A6-2 P. fluorescens strains used as a biocontrol agent against invertebrates
Aphid (Aphis gossypii)
Leaf hopper (Amrasca biguttula), whitefly (Bemisia abaci) -nymphs and adults
No strain designation provided(Gandhi and Gunasekaran, 2008)
Argentine stem weevil (Listronotus bonariensis) - adults collected from field populationsNo strain designation specified(Jackson and McNeill, 1998)
Fruit Fly (Drosophila melanogaster)P. fluorescens MF0(de Lima Pimenta et al., 2006)
Hazelnut beetle (Balaninus nucum, Curculio nucum)No strain designation specified(Sezen and Demirbag, 1999)
Lady beetle (Hippodamia convergens)No strain designation specified(James and Lighthart, 1992)
Leafhopper (Amrasca devastans)(Distant)Pf- 1®(Murugesan and Kavitha, 2009)
Mosquitoes (Culex quinquefasciatus, Anopheles stephensi, Aedes aegypti) and other species (late second instar stage larvae)P. fluorescens strain MSS-1, isolated from diseased mosquito larvae(Murty et al., 1994)
Subterranean termite (Odontotermes obesus)P. fluorescens CHA0(Devi and Kothamasi, 2009)
Southern pine beetle (Dendroctonus frontalis)No strain designation(Moore, 1972)
Burrowing nematode (Radopholus similis)Females and juveniles, root-knot nematode (Meloidogyne spp.) 2nd stage juveniles3 P. fluorescens strains(Aalten et al., 1998)
Zebra mussel (Dreissena polymorpha, D. Bugensis)P. fluorescens strain ATCC 55799

(Molloy, 2004)

See also (Mayer, 2009)

Daphnid (Daphnia magna)P. fluorescens ATCC 55799 (dead cells)(Mayer, 2009)
Giant freshwater prawn (Macrobrachium rosenbergii)No strain designation provided(Baruah and Prasad, 2001)
Field slug (Deroceras reticulatum)
  • No strain designation specified; isolated from a cadaver of D. reticulatum following death by an infection of Phasmarhabditis hermaphrodita
  • Cultures of P. fluorescens were grown in nutrient broth for 24 h at 22°C
(Wilson et al., 1994)

Appendix 7: Pathogenicity and Toxicity Studies of other Strains of P. fluorescens

Table A7-1 Toxicity and infectivity studies of P. fluorescens ATCC 55799 (CL145A)
TestTest OrganismTest Substance or ConcentrationLD50, LC50, EC50
Acute oral toxicity Footnote Appendix A Table A7-1[a]Mallard ducks2000 mg/kg bw (inactive)Footnote Appendix A Table A7-1 [b]LD50 greater than 2000 mg/kg bw/ kg bw
Toxicity studies (4-day static renewal) [a]Rainbow trout (Oncorhynchus mykiss)25, 50, 100, 200 or 400 mg a.i/L (inactive)59.09 mg a.i./LFootnote Appendix A Table A7-1 [c]
Toxicity studies (4-day static renewal) [a]Fathead minnow (Pimephales promelas)100, 200, 400 or 600 mg a.i/L (inactive)LD50: 569.9 mg a.i./L
Toxicity studies (30-day static renewal) [a]Fathead minnow (Pimephales promelas)3.8 × 109 CFU/mL (live)LC50: 6.9 × 106CFU/mL
Toxicity studies (30-day static renewal) [a]Fathead minnow (Pimephales promelas)3.8 × 103 CFU/mL (inactive)LC50 could not be calculated due to relatively low mortality rates
Toxicity studies (4-day static renewal) [a]Chinook salmon (Oncorhynchus tshawytscha)100, 200, 400 or 800 mg a.i./L (inactive)LC50: 183.5 mg a.i./L
Toxicity studies (4-day static renewal) [a]Sacramento split tail (Pogonichthys macrolepidotus)50, 100, 200, 400, 800 and 1600 mg a.i./L (inactive)LC50: 137.6 mg a.i./L
Toxicity study (48-hour static) [a]Shrimp (Hyalella azteca)25, 50, 100 or 200 ppm (inactive with active biotoxin)LC50 greater than 200 ppm
Toxicity study (48-hour static) [a]Shrimp (Hyalella azteca)25, 50, 100 or 200 ppm (inactive and heat-treated with inactive biotoxin)LC50 greater than 200 ppm
Toxicity study (10-day static [a])Daphnia magna200 ppm (killed, irradiated)LC50 greater than 200 ppm (48 hours)
Toxicity study (2-day static) [a]Daphnia magna15.625, 31.25, 62.5, 125, and 250 mg a.i./L (inactive)EC50: 143.59 mg a.i./L
Acute oral toxicity studySprague-Dawley rats2.42 × 107 CFU/kg bwLD50 greater than 2.42 × 107 CFU/kg bw
Acute pulmonary infectivity and toxicitySprague-Dawley rats3.4 × 108 CFU/animal (0.1 mL dose)LD50 greater than 3.4× 108 CFU/animal
Acute inhalation studySprague-Dawley ratsAerosolised dose of 2.25 mg/L (CFU/animal could not be determined)LC50 greater than 2.25 mg/L
Acute intravenous infectivity studySprague-Dawley ratsDoses ranged from 4.7 × 106CFU/mL to 1.95 × 107 CFU/mLNon-infective to rats
Footnote Appendix A Table A7-1 a

Adapted from (PMRA-HC, 2012)

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Footnote Appendix A Table A7-1 b

Inactive/killed (use in end-use product)

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Footnote Appendix A Table A7-1 c

Active ingredient (a.i.)

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Table A7-2 Toxicity and infectivity studies of P. fluorescens ATCC 31948 (A506)
TestTest OrganismTest Substance or ConcentrationLD50, LC50, EC50
Acute oral toxicity Footnote Appendix A Table A7-2[a]Sprague-Dawley rats5.0 g/kg bwFootnote Appendix A Table A7-2 [b]LD50 greater than 5.0 mg/kg bw
Acute oral toxicity [a]Sprague-Dawley rats8.4 × 1010 CFU/animalFootnote Appendix A Table A7-2 [c]LD50 greater than 8.5 × 1010 CFU/animal (no mortality, no significant toxicity)
Acute pulmonary toxicity [a]Sprague-Dawley rats5.3 mg/LFootnote Appendix A Table A7-2 [d]LD50 greater than 5.3 mg/kg bw
Acute infectivity (intraperitoneal injection) [a]Swiss Webster mice2.0 × 108CFU/animal[c]No mortalities, general signs of toxicity (scruffy coats, discharge from eyes, lethargy, diarrhea)
Contact toxicity [a]Italian honeybees (Apis mellifera)5 μL at 1.03 × 105 CFU/bee, 2.06 × 105 CFU/bee, 4.12 × 105 CFU/bee, 8.25 × 105 CFU/bee, or 1.65 × 106CFU/bee[c]LC50 greater than 1.65 × 106 CFU/beeFootnote Appendix A Table A7-2 [e]
Acute toxicity [a],Footnote Appendix A Table A7-2 [f]Vascular plantsFootnote Appendix A Table A7-2 [g]106 or 108CFU/mL[b], or bothNo signs of phyto-pathogenicity (doses below the max. label rate of 3.7 × 109 CFU/mL)
Footnote Appendix A Table A7-2 a

Adapted from (PMRA-HC, 2010)

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Footnote Appendix A Table A7-2 b

MPCA, microbial pest control agent

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Footnote Appendix A Table A7-2 c

Testing was conducted with Frostbans A, a mixture containing the MPCA, as well as other Pseudomonasspecies which are known to be more pathogenic than the MPCA (e.g., Pseudomonas syringae), rather than with Blightban A506 (ATCC 31948) itself

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Footnote Appendix A Table A7-2 d

Testing was conducted with Pseudomonas fluorescens strain AGS3001.2, a strain considered equivalent to the MPCA (Pseudomonas fluorescens ATCC 31948) rather than with the MPCA itself

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Footnote Appendix A Table A7-2 e

Cause of death not established, increase in mortality coincided with a decline in food supply. Infectivity was not assessed.

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Footnote Appendix A Table A7-2 f

Inoculation methods: puncture wound, leaf swab, hole punch, and leaf infiltration, depending on host.

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Footnote Appendix A Table A7-2 g

Tested plants: barley, maize, oats, sorghum, wheat, broccoli, cabbage, cauliflower, cucumber, snapbean and citrus, peach, pear, tomato and tobacco.

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Appendix 8: Adverse effects associated with other strains of P. fluorescensFootnote[5]

Table A8-1 Adverse effects reported in plants
OrganismStrainAdverse effects/disease symptomsReference
Sainfoin (Onobrychis viciaefolia)P. fluorescens strains SA-2-1, SB-1-1, SC-4-1, SD-2-4 and SF-2-1Crown and root rot(Hwang et al., 1989)
CactiAtypical strain of P. fluorescens(biotype II of Buchan & Gibbons, 1974)Orange soft rot(Anson, 1982)
Willow (Salix viminalis)No strain designation specifiedNecrotic tissue damage (discolouration, necrosis of the bark or glassy appearance of the woody tissue)(Nejad et al., 2004)
Black Pine (Pinus thunbergii)P. fluorescens biotype I and biotype IIWilting or browningFootnote Appendix A Table A8-1 [a](Han et al., 2003), see also (Guo et al., 2007)
Footnote Appendix A Table A8-1 a

Pine wood nematodes were also implicated in the disease progression

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Table A8-2 Adverse effects reported in vertebrates
OrganismIsolation, challenge methodFootnote Appendix A Table A8-2 [a] and strainAdverse effects/disease symptomsReference
Atlantic salmon (Salmo salar)P. fluorescens strain 92/3556 isolated from fish that demonstrated languid swimming behaviour, congestion at the base of the fins, tail erosion, and with patches of haemorrhagic petechiation on the flank
  • P. fluorescens strain 92/3556 did not cause re-infection in healthy fish
  • P. fluorescens strain 92/3556 may be considered to be an opportunisitic pathogen of cold-stessed fish with depressed immune function
(Carson and Schmidtke, 1993)
Elephantsnout fish (Mormyrus kannume)P. fluorescens (no strain designation) was isolated from diseased fishInvasion of fish with P. fluorescens may have enhanced saprolegnia infection(Ahmed, 1992)
European eel (Anguilla anguilla) and rainbow trout (Oncorhynchus mykiss)Footnote Appendix A Table A8-2 [b]P. fluorescens (no strain designation) isolated from diseased eel and fish
  • Cutaneous petechiation (ventral) and ulceration
  • Recovered from kidneys and liver of dead (challenged) fish
(Esteve et al., 1993)Footnote Appendix A Table A8-2 [c]
Rainbow trout (Oncorhynchus mykiss), Tilapia spp.Footnote Appendix A Table A8-2 [d]P. fluorescens (no strain designation) isolated from kidneys of diseased fish
  • Hemorrhage at the base of fin and anal regions
  • Petechia of organs (especially the intestine)
  • Mortality was reported

(Sakai et al., 1989)

See also (Barker et al., 1991; Ostland et al., 1999)

Scottish rainbow trout (Salmo gairdneri)P. fluorescens (no strain designation) isolated from diseased (chronic infectious pancreatic necrosis) and dead fish
  • P. fluorescens did not cause re-infection in healthy fish; likely an opportunistic pathogen in this case
(Roberts and Horne, 1978)
Tilapia spp. (Clarias lazera, Heterobranchus bidorsalis)P. fluorescens (no strain designation) isolated by tissue swab
  • P. fluorescens was associated with fin rot, dropsy, pop-eye and haemorrhagic septicaemia of freshwater fish
  • May contribute to mortality, especially in stress conditions
  • Did not cause re-infection in healthy fish
  • Likely an opportunistic pathogen
(Okaeme, 1989)
Carp (Cyprinus carpio L.)Intraperitoneal injection of P. fluorescens (no strain designation)Significant reduction in hematocrit and erythrocyte counts; elevation in leukocrit and total leukocyte numbers; decrease in plasma protein and albumin; and lowering of plasma electrolytes (Na+, K+, Ca2+ & P)

(Yildiz, 1998)

See also (Csaba et al., 1984) (silver carp and bighead carp in farms).

ChickenP. fluorescens (no strain designation) isolated from clinical sick or dead chickens and inoculated by intraperitoneal injection in health chickensMortalities as a result of bacterial pneumonia, sinusitis, and/or septicemia were reported(Lin et al., 1993)
Marine turtles (Chelonia mydas and Eretmochelys imbricate)P. fluorescens (no strain designation) isolated from diseased turtles
  • Associated with  ulcerative dermatitis, stomatitis, blepharitis and shell disease; rhinitis; broncho-pneumonia; kerato-conjunctivitis; adenitis; peritonitis; septicaemia-toxaemia; and osteomyelitis
  • Re-infection not performed to determine pathogenicity in healthy turtles
(Glazebrook and Campbell, 1990)
HorseP. fluorescens (no strain designation) isolated from clinically diseased horses during an outbreak of equine influenza virus E2/F Influenza A/Equi-2/Re-infection not performed to determine pathogenicity in healthy horses(Sarasola et al., 1992)
Mice (Mus musculus)A superficial burn lesion was made at the base of the mouse’s tail and subsequently inoculated with P. fluorescens strain 8/3
  • P. fluorescens was isolated from the lesion but was not isolated from the organs
  • Study demonstrated that P. fluorescens did not readily cause infection in the mice
(Liu, 1964)
Footnote Appendix A Table A8-2 a

Challenge method provided if applicable

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Footnote Appendix A Table A8-2 b

Fish were challenged

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Footnote Appendix A Table A8-2 c

Authors indicate that P. fluorescensis likely a secondary invader that took advantage of damaged tissues and suggest that it acts as an opportunistic pathogen

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Footnote Appendix A Table A8-2 d

Diseased fish (not challenged)

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Footnote 1

A determination of whether one or more of the criteria of section 64 of CEPA 1999 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposure in the general environment. For humans, this includes, but is not limited to, exposure from air, water and the use of products containing the substances. A conclusion under CEPA 1999, on Pseudomonas fluorescensstrain ATCC 13525, is not relevant to, nor does it preclude, an assessment against the hazard criteria for Workplace Hazardous Materials Information System (WHMIS) that are specified in the Controlled Products Regulations for products intended for workplace use.

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Footnote 2

Testing conducted by Health Canada’s Environmental Health Science and Research Bureau

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Footnote 3

Vascular plants tested include barley, maize, oats, sorghum, wheat, broccoli, cabbage, cauliflower, cucumber, snapbean, citrus, peach, pear, tomato and tobacco.

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Footnote 4

A determination of whether one or more of the criteria of section 64 of CEPA 1999 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposure in the general environment. For humans, this includes, but is not limited to, exposure from air, water and the use of products containing the substances. A conclusion under CEPA 1999, on these substances, is not relevant to, nor does it preclude, an assessment against the hazard criteria for WHMIS that are specified in the Controlled Products Regulations for products intended for workplace use.

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Footnote 5

Adverse effects excluding those reported in a biocontrol context.

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