Final Screening Assessment

Escherichia hermannii ATCC 700368

Environment Canada
Health Canada
August 2015

(PDF Format - 942 KB)

Table of Contents

• Synopsis
• Introduction
• Decisions from Domestic and International Jurisdictions
• 1.  Hazard Assessment
  • 1.1 Characterization of Escherichia hermannii
  • 1.2 Effects
  • 1.3 Hazard Severity
• 2.  Exposure Assessment
  • 2.1 Sources of Exposure
  • 2.2 Exposure Characterization
• 3.  Risk Characterization
• 4.  Conclusion
• 5.  References
• A. Appendices
• Appendix A: Characterization of E. hermannii ATCC 700368
• Appendix B: Virulence and Pathogenicity Testing of E. hermannii ATCC 700368
• Appendix C: Potential Uses of E. hermannii

List of Tables

• Table 1-1: Morphological characteristics of E. hermannii
• Table 1-2: Physiological properties of E. hermannii
• Table 1-3: Molecular analyses of E. hermannii
• Table 1-4: Antimicrobial susceptibility of 32 E. hermannii strains (adapted from Brenner et al. 1982)
• Table 1-5: Minimal inhibitory concentration (MIC) and antibiotic susceptibility of E. hermannii ATCC 700368
• Table 1-6: Human case reports in which E. hermannii was isolated.
• Table 1-7: Biochemical characteristics of E. hermannii compared to other members of Enterobacteriaceae
• Table A-1: Fatty acid methyl ester (FAME) analysis of E. hermannii ATCC 700368 using MIDI environmental and clinical database
• Table A-2: Growth of E. hermannii ATCC 700368 in liquid media at various temperatures
• Table A-3: Growth of E. hermannii ATCC 700368 on solid media
• Table A-4: Biochemical characteristics of E. hermannii ATCC 700368
• Table A-5: In vitro cell culture: cytotoxicity
• Table A-6: Mouse model: endotracheal exposure data
• Table A-7: List of patents and potential uses of E' hermannii

List of Figures

• Figure 1-1: Persistence of E. hermannii strain ATCC 700368 in soil (reproduced from Xiang et al. 2010)
• Figure A-1: Fatty acid methyl ester (FAME) analysis of P. stutzeri ATCC 17587 using MIDI clinical and environmental databases
• Figure A-2: Multi-locus sequence analysis of E. hermannii ATCC 700368

Synopsis

Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA 1999), the Minister of the Environment and the Minister of Health have conducted a screening assessment of E. hermannii strain ATCC 700368.

E. hermannii strain ATCC 700368 is a non-spore-forming Gram-negative bacterium. Reports of isolation of E. hermannii are rare but include a range of sources, including humans, raw and processed food, animals and their food products, plants, and terrestrial, aquatic and marine environments. E. hermannii plays a role in the nitrogen and sulphur cycle, and can tolerate environments contaminated with toxic hydrocarbons and metals. These properties make it of possible commercial interest. Potential uses of E. hermannii strain ATCC 700368 reported in the public domain include bioremediation, biodegradation, industrial effluent treatment, municipal wastewater treatment (particularly oil and grease traps as well as sewage sludge), odour control, organic waste treatment, and composting.

There is no evidence in the scientific literature to suggest that E. hermannii strain ATCC 700368 is likely to have adverse effects on animal or plant populations in the environment. However, because E. hermannii has been only rarely isolated, there may not have been sufficient exposure of environmental species to E. hermannii strains in nature to observe or document an ability to cause disease in plants or animals. Therefore, there is some uncertainty about the pathogenic potential of E. hermannii strain ATCC 700368 and its effects on the environment.

Although E. hermannii has occasionally been described as an opportunistic human pathogen, and there is evidence in the literature to demonstrate that some strains contain determinants of pathogenicity, there are no reports linking E. hermannii to the production of known toxins. Infections involving E. hermannii as the putative primary pathogen are very rare, and occur in individuals predisposed to infection or involve a significant breach in normal barriers against infection; like most micro-organisms, E. hermannii can cause adverse effects if introduced into normally sterile body compartments. The majority of case reports involving E. hermannii were polymicrobial, and the other micro-organisms involved were considered to be the primary pathogens. E. hermannii has also been isolated from diarrheal stools, although rarely so; and has never been demonstrated to be the cause of disease.

This assessment considers the aforementioned characteristics of E. hermannii strain ATCC 700368 with respect to environmental and human health effects associated with product use and industrial processes subject to CEPA 1999, 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 1999, as published in the Canada Gazette, Part I, on October 3, 2009 (section 71 notice). Information submitted in response to the section 71 notice, as well as latest available information, indicates that E. hermannii strain ATCC 700368 is not imported into or manufactured in Canada.

Considering all available lines of evidence presented in the Screening Assessment, there is a low risk of harm to organisms and the broader integrity of the environment from E. hermannii strain ATCC 700368. It is concluded that E. hermannii strain ATCC 700368 does not meet the criteria under paragraphs 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.

Also, based on the information presented in the Screening Assessment, it is concluded that E. hermannii strain ATCC 700368 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.

Introduction

Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA 1999), the Minister of the Environment and the Minister of Health are required to conduct screening assessments of living organisms listed on the DSL that 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 strain was added to the DSL under subsection 25(1) of CEPA 1988 and the DSL under subsection 105(1) of CEPA 1999 because it was manufactured in or imported into Canada between January 1, 1984 and December 31, 1986.

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 CEPA 1999 section 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 Risk Assessment Framework 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, E. hermannii ATCC 700368, are identified as such. Strain-specific data are limited and originate from four sources: the Nominator, the American Type Culture Collection (ATCC), and unpublished data generated by Health Canada^Footnote[2] and Environment Canada^Footnote[3] research scientists. Where strain-specific data were not available, appropriate surrogate information from literature searches was used. When applicable, literature searches conducted on the organism included its synonyms, and common and superseded names. Surrogate organisms are identified in each case to the taxonomic level provided by the source. Literature searches were conducted using scientific literature databases (SCOPUS, Google Scholar, CAB Abstracts and NCBI Pubmed), web searches, and key search terms for the identification of potential human health and environmental hazards. Information identified as of November 2013 was considered for inclusion in this report.

Decisions from Domestic and International Jurisdictions

E. hermannii is a Risk Group 1 human and terrestrial animal pathogen according to the Public Health Agency of Canada (PHAC). It is not listed as a reportable or notifiable disease of aquatic animals under the Health of Animals Act, the Reportable Disease Regulations or the Health of Animals Regulations, nor is it listed by L'office international des epizooties (OIE 1997). It is not listed as a Regulated Plant Pest in Canada (personal communication, Canadian Food Inspection Agency, 2013) and is not a Regulated Pest of member countries of the International Plant Protection Convention (IPPC) or Invasive Species Database of the Global Invasive Species Programme (GISP).

The use of E. hermannii in a feed, in a fertilizer, or as a pest control product would be considered under other legislation in Canada.

There are no E. hermannii decisions or reports for pesticide registrations on file with the United States Environmental Protection Agency (U.S. EPA) or Canada's Pest Management Regulatory Agency (PMRA), no vaccine registrations with the United States Food and Drug Administration (U.S. FDA), and no veterinary biologic registrations on file with United States Department of Agriculture (USDA) or Canadian Food Inspection Agency (CFIA).

1. Hazard Assessment

1.1 Characterization of Escherichia hermannii

1.1.1 Taxonomic identification and strain history

Binomial name: Escherichia hermannii

Taxonomic designation:

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacteriales

Family: Enterobacteriaceae

Genus: Escherichia

Species: Escherichia hermannii (Brenner et al. 1982)

Type strain: ATCC 33650 (NBRC105704)

DSL strain: ATCC 700368

Common and superseded names: Escherichia hermannii; CDC^Footnote[4] Enteric Group 11 (atypical E. coli)

Strain history: E. hermannii strain ATCC 700368 was isolated from an unspecified lagoon by Sybron Chemical Inc. for its ability to oxidize sulphide. The strain was deposited to the ATCC by Sybron Chemical Inc. in 1997, and nominated to the DSL in March 1997.

1.1.1.1 Phylogeny of E. hermannii

E. hermannii has a complex and ambiguous taxonomy: it was originally classified as an atypical E. coli, referred to as CDC Enteric Group 11 (Brenner et al. 1982), and later reclassified as a new Escherichia species distinct from E. coli based upon genetic relatedness (DNA-DNA homology) and a combination of metabolic characteristics atypical of E. coli: production of yellow pigment, ability to grow on potassium cyanide (KCN) and utilization of cellobiose (Brenner et al. 1982). On the sole basis of DNA relatedness, E. hermannii could have been assigned to Enterobacter, Citrobacter (including C. freundii), Klebsiella, Escherichia or Salmonella (including S. enterica) E. hermannii is biochemically most similar to Citrobacter (Levinea) amalonaticus and E. coli, but fits neither biochemical profile perfectly, so Brenner proposed the creation of a new genus intermediate between the two genera (Brenner et al. 1982; Cilia et al. 1996; Christensen et al. 1998). The state of the science on the genus Escherichia now indicates that E. hermannii is not a valid member of the genus Escherichia (Walk et al. 2009; Clermont et al. 2011). E. hermannii has been dropped from the genus under the Clermont system for classifying Escherichia (Retchless and Lawrence 2010; Luo et al. 2011; Carlos et al. 2010; Clermont et al. 2011, 2013; Oh et al. 2012). This change is based on established criteria for classification (Stackebrandt et al. 2002,; Wertz et al. 2003; Tindall et al. 2010), and on 1) its minimal genetic similarity to Escherichia (39-46% compared with the established threshold of 70%), 2) the small number of isolates used to determine classification (genetic similarity was established for only eight strains), and 3) the requirement for a genus to be monophyletic, while phylogenetic analyses indicate that E. hermannii does not cluster with the genus Escherichia on the basis of 16S rRNA gene sequence analysis or on more sensitive indicator sequences from the genes gap, ompA, dnaJ, tuf and atpD (Lawrence et al. 1991; Hartl 1992; Christensen et al. 1998; Paradis et al. 2005; Iversen et al. 2007; Walk et al. 2009; Pham et al. 2007). Phylogenetic studies showed that E. hermannii is most closely related to Citrobacter freundii (Brenner et al. 1982), Salmonella enterica (Scheutz and Strockbine 2005; Lawrence et al. 1991; Christensen et al. 1998), Enterobacter sp. (E. cowanii [now reclassified to Kosakonia cowanii comb. nov.]), E. cloacae, E. dissolvens, E. sakazakii (many organisms now identified as Cronobacter sakazakii) and Klebsiella pneumoniae (Iversen 2007; Paradis et al. 2005).

The DSL-listed E. hermannii strain ATCC 700368 was analyzed by fatty acid methyl ester (FAME) analysis and multi-locus sequence analysis (MLSA) of the recN, rpoA and thdF genes by Health Canada scientists. In the FAME analysis, E. hermannii ATCC 700368 clusters with Citrobacter amalatonicus, Citrobacter koseri, Kluyvera cryocresens, Pantoea agglomerans and Enterobacter (E. cloacae) (Appendix 1, Figure A-1). A phylogenetic tree of the top hits within Genbank of the recN gene shows that strain ATCC 700368 clusters closely with the E. hermannii type strain and another E. hermannii isolate (both clinical isolates). Additionally, E. hermannii ATCC 700368 clusters closely with proposed new Cronobacter species(C. helveticus, C. zuricensis, C. pulveris [basonyms (former nomenclature) Enterobacter helveticus, E. pulveris and E. turicensis] and C. mutjensisii, C. dublinensis and C. sakazakii), and Klebsiella pneumonia (Appendix 1, Figure A-2). Similar results were obtained from a Basic Local Alignment Search Tool (BLAST) search of the ATCC 700368 thdF gene. A BLAST search of the ATCC 700368 rpoA gene retrieved hits that were 97% identical to Leclercia adecarboxylata and 96% identical to Enterobacter cloacae. Although these phylogenetic analyses show that E. hermannii strain ATCC 700368 clusters closely with known opportunistic pathogens, the relevance of these phylogenetic associations is unclear in the absence of clinical data showing evidence of pathogenicity or virulence and given significant inter- and intra-species variability in virulence within the family Enterobacteriaceae.

1.1.1.2 Phenotypic and molecular characteristics

E. hermannii strain ATCC 700368 was initially identified with the Biolog system. The ATCC subsequently validated the organism’s identity based on morphology and phenotype as well as biochemical analyses (VITEK 2 and BioMerieux API). VITEK 2 is known to falsely identify Shigella sonnei as E. hermannii. However, certain biochemical and physiological characteristics (yellow pigment, motility, indole production and amygdaline fermentation) demonstrate that the strain is not Shigella sonnei. Other characteristics (yellow pigment, growth on KCN and cellobiose) also demonstrate that the strain is not Escherichia coli (Cedarlane, personal communication). E. hermannii has sometimes been confused with Citrobacter diversus (ATCC, personal communication), C. freundii (Fernandez et al. 2011), Shigella sonnei (Biomerieux) and E. coli (Tardio et al. 1988), indicating that identification of E. hermannii is not clear-cut.

Researchers at Health Canada were unable to reproduce the definitive characteristics of E. hermannii in the DSL strain. In independent testing at Health Canada, neither the E. hermannii type strain ATCC 33650, nor the DSL strain ATCC 700368, grew on KCN in the standard assay (0.75% KCN (w/v)); the type strain did grow at 0.012% KCN but not at 0.02% KCN, while the DSL strain ATCC 700368 did not grow at any of the tested KCN concentrations. The production of yellow pigment was observed for the type strain, but only pale yellow pigmentation was observed in the DSL strain, and then only when many colonies were scraped off the plate with a coverslip (Appendix 1, Table A-4). This observation, in conjunction with phylogenetic analysis from the same laboratory demonstrating that the DSL strain is closely related to other E. hermannii strains, may suggest that phylogenetic methods, in this case, are more reliable for accurate identification. Morphological characteristics (Table 1-1), physiological properties (Table 1-2) and molecular analyses (Table 1-3) of E. hermannii are shown below.

Rapid serological tests may be unsuitable for E. hermannii because of cross- reactivity with the O-antigens of other pathogens such as E. coli O157:H7 (Rice et al. 1992; Perry and Bundle 1990; Perry and Richards 1990), Brucella abortus and Brucella melitensis (Perry and Bundle 1990; Jacques and Dubray 1991; Beynon et al. 1990), Yersinia enterocolitica serotype O:9, Vibrio cholera O1 and Salmonella group N (O:30) (Godfroid et al.1998; Reeves and Wang 2002; Muñoz et al. 2005).

1.1.2 Biological and ecological properties

1.1.2.1 Natural habitats

E. hermannii has been identified in many countries, and is found in a diverse range of habitats, including host-associated, aquatic marine and terrestrial sites, as well as raw and processed food sources, as follows:

Living organisms:

• Chickens (egg shells, 1 isolate from feces) (Chang 2000; Praxedes et al. 2013)
• Marine birds (2 isolates) (Bogomolni et al. 2008)
• Swine (4 isolates) (Jackson et al. 1992)
• Mussels exposed to municipal effluent (Douville et al. 2010)
• Bullfrogs (Tee and Najiah 2011)
• Citrus plants, as an endophyte on the leaves (Liu et al. 2011)
• Plants grown at sandy beaches (rhizosphere) (Seo and Song 2013)
• Humans (isolates from wounds, sputum/lung, stools including diarrhea, blood, urine, cerebrospinal fluid, conjunctiva, peritoneal fluid, duodenal ulcers, periodonta) (see Table 1-6)

Aquatic, marine and terrestrial sites:

• Pebblestone (Fernandez 2011)
• Marine water (Palmer et al. 1993)
• Drinking water distribution systems (Rice et al. 1991)
• Sugar cane agro-ecosystem (De Lima et al. 1999)
• Sludge containing chlorobenzene from an industrial wastewater treatment plant (Kiernicka et al. 1999)
• Contaminated soil from an oil refinery (Hernández et al. 1998)

Raw and processed food sources:

• Milk, milk products and infant formula (Borczyk et al.1987; Estuningsih et al. 2006; Loaiza et al. 2011; Saad et al. 2012)
• Eggs (Loaiza et al. 2011; Shin et al. 2009; Chang 2000)
• Beer malt premature yeast flocculation (Zhao et al. 2011)
• Corn syrup (Robison 1984)
• Processed milk and formula, including powdered milk substitutes (Muytjens et al. 1988; Hwang et al. 2008; Estuningsih et al. 2006; Robison 1984)

In spite of the diversity of environments from which it has been isolated, reports of isolation of E. hermannii are rare (60-70 isolations reported since 1982), which could be a result of 1) lack of surveillance for E. hermannii, 2) lack of reporting, 3) lack of significance (it does not cause disease), 4) inability to culture it (viable but not culturable), 5) inaccurate identification, 6) lack of competitive fitness or 7) in fact being rare.

1.1.2.2 Growth parameters and metabolism

The ATCC recommends culturing the DSL strain in ATCC Medium 3 Nutrient Agar or Nutrient Broth, at 30°C under aerobic conditions. Health Canada data regarding the growth characteristics of E. hermannii ATCC 700368 (Appendix 1, Table A-2 and Table A-3) show that the strain can grow well in both liquid and on solid general purpose media (Trypticase Soy Broth/Agar). In trypticase soy broth (TSB), growth is best at 28-30ºC, but it can also grow at 37ºC and there is low-level growth at 42ºC. However, its growth was delayed in sheep plasma (SP), fetal bovine serum (FBS) and Dulbecco's modified eagle medium (DMEM) at 28ºC. At 37ºC there was growth only in FBS, and this growth was observable only after 15 hours. In these media (SP, FBS, DMEM), there was no growth at 42ºC. The strain also grew on specialized solid media, such as Maconkey agar and TSI agar (without fermentation). Lysine decarboxylase, starch, urea and catalase tests were positive, while citrate, mannitol and hemolysis tests were negative.

Like other members of the genus Escherichia, E. hermannii reduces nitrate to nitrite (Brenner et al. 1982), presumably (like E. coli) only under anaerobic conditions, and it thereby plays a part in the nitrogen cycle. E. hermannii strain ATCC 700368 also plays a role in the sulphur cycle, in that it was selected for its ability to oxidize sulphides and reduce sulphites.

E. hermannii has been isolated from contaminated environments where it can tolerate and metabolize toxic hydrocarbons such as chlorobenzene (Kiernicka et al. 1999) and metals such as nickel and vanadium (Hernández et al. 1998). These characteristics, in addition to its role in the nitrogen cycle, make it of interest for use in commercial products aimed at enhancing waste biodegradation and water treatment.

1.1.2.3 Survival, persistence and dispersal

There is very little survival and persistence or ecological information regarding strain ATCC 700368 or E. hermannii as a species. Given that E. hermannii has been isolated from pigs, birds, frogs, mussels, soil, freshwater and marine water, it can obviously survive in environmental media and certain organisms. Some E. hermannii strains are known to produce biofilms, which may increase their ability to survive and persist. Although E. hermannii does not form spores, it has been cultured from powdered milk formula, and thus does survive heat and desiccation. E. hermannii is also able to survive in environments contaminated with hydrocarbons and heavy metals.

Xiang et al. (2010) investigated the persistence of E. hermannii ATCC 700368 in clay loam microcosm soil (50.3% sand, 41.6% silt, 9.7% clay, 9.5% organic matter) and reported that populations of this strain dropped from 108 CFU/g in the inoculum added to dry soil to below the detection threshold of 6.41 × 10⁴ CFU/g of soil, after 21 days (Figure 1-1). The persistence observed may or may not be from viable cells, but the populations that were inoculated abruptly dropped below the detection level. The above information indicates that E. hermannii ATCC 700368 persistence is short, most likely due to its low capacity for colonization of the tested soil. Also, maintenance of high numbers beyond background levels is unlikely due to competition (Leung et al. 1995) and microbiostasis (Van Veen et al. 1997), which is an inhibitory effect of soil that results in the rapid decline of populations of introduced bacteria.

Figure 1-1: Persistence of E. hermannii strain ATCC 700368 in soil (reproduced from Xiang et al. 2010)

1.1.2.4 Antibiotic resistance

E. hermannii is resistant or marginally sensitive to beta-lactam antibiotics such as penicillin, ampicillin, carbenicillin, ticarcillin and amoxicillin, owing to its ability to produce beta-lactamase, which is thought to be chromosomally encoded (Beauchef-Havard et al. 2003). This author showed that E. hermannii produces a novel class A beta-lactamase HER1. It is susceptible to cephalosporins with the exception of cefoperazone and cefeprime (Stock and Wiedemann 1999). Compared to E. coli and Shigella, E. hermannii is less susceptible to nitrofurantoin and slightly more susceptible to several aminoglycosides (Table 1-4; Fitoussi et al. 1995; Stock and Wiedemann 1999; Beauchef-Havard et al. 2003).

Multi-drug resistance is associated with the expression of cryptic efflux pump systems, and with reduced expression of outer membrane proteins involved in the transport of antibiotics into bacterial cells. Similar types of pumps are involved in heavy metal resistance (Hernández et al. 1998; Nies 1999). Genes encoding efflux pumps involved in multi-drug and heavy metal resistance can be horizontally acquired and may be located closely together on the same plasmid, and are thus more likely to be transferred together in the environment during horizontal gene transfer (Spain and Alm 2003). Some strains of E. hermannii are known to be resistant to heavy metals. Growth of these strains of E. hermannii in the presence of vanadium induced multi-drug resistant phenotypes of E. hermannii, possibly involving up-regulation of the efflux pump system (Hernández et al. 1998). It is not known whether strain ATCC 700368 is resistant to heavy metals.

Antibiotic susceptibility of strain ATCC 700368 was confirmed by Health Canada (Table 1-5), and is similar to that reported for the species, with the exception of resistance to Trimethoprim. Trimethoprim resistance is encoded by a number of dihyrofolate reductase genes (dfr) and transmitted by HGT among both Gram-negative and Gram-positive bacteria (Kadlec and Schwarz 2009; Brolund et al. 2010).

Like many micro-organisms, E. hermannii contains or produces compounds, such as lipopolysaccharides and enzymes, that may be immunostimulatory or act as sensitizers. Hypersensitivity 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). No reported cases of hypersensitivity to E. hermannii were found in the literature.

1.1.2.5 Pathogenic and toxigenic characteristics

Based on an extensive literature review, there are no reports directly linking E. hermannii to the production of known toxins. Assays for verotoxin, heat-labile (LT) and heat-stable (ST) toxins in organisms initially thought to be E. coli but later confirmed to be E. hermannii were all negative (Robison 1984; Borczyk et al. 1987), and colony DNA hybridizations were negative for LT, STa and STb genes (Robison 1984). Shiga toxin-producing strains of E. hermannii have not been reported (Nataro and Kaper 1998).

There are few reports in the literature to demonstrate that some strains of E. hermannii contain determinants of pathogenicity. Chaudhury et al. (1999) demonstrated possible enteropathogenicity of E. hermannii in a Charles-Foster albino rat ileal loop model and hypothesized that this was mediated through enterotoxin(s). Yamanaka et al. (2010) demonstrated that another strain of E. hermannii (YS-11) produced persoamine, an O-chain epitope of lipopolysaccharide (LPS), within its biofilm, which mediated persistence, survival and tissue invasion as a mechanism of pathogenesis. It is not known if the DSL strain ATCC 700368 contains this biofilm constituent.

1.2 Effects

1.2.1 Environment

An in-depth scientific literature search yielded few reports of E. hermannii being isolated from the environment, but reported sites of isolation were diverse, including terrestrial, aquatic and marine, as well as host-associated, sites. There are no reported cases of infectivity or pathogenicity due to E. hermannii in non-human species under natural environmental conditions.

However, E. hermannii has been isolated from healthy swine (Jackson et al. 1992). In a case of bloody diarrhea in a human that was attributed to E. hermannii, there was a history of exposure to bloody diarrhea in pigs concurrent with the onset of disease (McCollum 1988). It was not determined whether the epidemic of bloody diarrhea in pigs was due to E. hermannii. Similarly, E. hermannii and many other species were isolated from the internal organs of bull frogs with external signs of ulcers, red leg, and torticollis, but the role of E. hermannii in producing these effects was not determined (Tee and Najiah 2011).

Pathogenicity studies in rodents showed that some strains of E. hermannii have the ability to invade tissue and cause abscesses (associated with the presence of persoamine contained within a biofilm), and to cause fluid accumulation in ileal loops, indicating possible determinants of enteropathogenicity (Chaudhury et al. 1999; Yamanaka et al. 2010). Another study showed no pathogenic effects in rodents when injected (see Section 1.2.2 for details) (Pien et al. 1985). E. hermannii may have other virulence factors related to its ability to adhere and colonize wounds and sterile sites. Health Canada in vitro data indicates that E. hermannii strain ATCC 700368 is cytotoxic to human HT 29 colonic epithelial cells after 6-24 hours exposure (without gentamicin). Administration of E. hermannii ATCC 700368 in a murine model at Health Canada showed no adverse effects following endotracheal exposure (see Section 1.2.2 for details).

Tests conducted at Environment Canada laboratories using standard methods (Environment Canada 2004, EPS 1/RM/44) evaluating the effects of E. hermannii ATCC 700368 on the soil invertebrate Folsomia candida (springtail) showed no significant effect on adult survival, but a significant reduction of the rate of juvenile reproduction of 35% and 42% at concentrations of 10⁹ and 10⁸ CFU/g, respectively, in clay loam dry soil (unpublished data). The test confirmed the potential for sub-lethal adverse effects of E. hermannii 700368 on F. candida reproduction. However, given the percent reduction that occurred, it is unlikely that a dilution test would yield an IC₅₀ (median inhibitory concentration) for juvenile production. Testing at a higher test concentration yields the risk of mould formation as a potential confounding effect on test results.

Other tests conducted by Environment Canada scientists using standard methods (Environment Canada 2004, EPS 1/RM/44) resulted in no significant adverse effects on the terrestrial plant Red Clover (Trifolium pretense) when the E. hermannii strain ATCC 700368 was applied at a concentration of 10¹⁰ CFU/g dry soil (10⁴ CFU/g of soil above the recommended maximum hazard concentration of 10⁶ CFU/g soil) (unpublished data).

There are no reported studies regarding effects for the DSL-listed strain ATCC 700368 or any other E. hermannii strain on aquatic species.

1.2.2 Human health

E. hermannii has occasionally been described as an opportunistic human pathogen; however, an extensive literature review has identified only three reports in which E. hermannii was isolated from the stools of patients with diarrhea, and only occasional reports of E. hermannii in association with human infection in extra-intestinal sites (Table 1-6). Most reported infections were polymicrobial and the other bacteria and fungi involved were considered to be the primary pathogens. Infections involving E. hermannii as the putative primary pathogen were in individuals predisposed to infection because of compromised immunity, debilitating disease or extremes of age, or involved a significant breach in normal barriers against infection; like most micro-organisms, E. hermannii can cause adverse effects if introduced into normally sterile body compartments. Deaths involving E. hermannii as a possible causative agent (sepsis) occurred only in neonates, and involved co-infection with known pathogens; all of the other cases for which outcomes were reported were successfully treated with antibiotics.

E. hermannii is phylogenetically closely related to other Entrobacteriaceae, including species of the genera Enterobacter, Cronobacter, Klebsiella and Citrobacter, some species of which have been implicated in human disease. E. hermannii has sometimes been confused with Citrobacter diversus (ATCC 55236), C. freundii (Fernandez et al. 2011), Shigella sonnei (Biomerieux), E. coli (Tardio et al. 1988) and Cronobacter sakazakii (Choudhury and Seet 2013), but standard biochemical methods can generally differentiate E. hermannii from its close phylogenetic relatives, as summarized in Table 1-7. Leclercia adecarboxylata is biochemically very similar to E. hermannii, but is genetically distinct (Tamura et al. 1986). Clinical diagnostic laboratories relying solely on biochemical methods may not be able to differentiate infections caused by L. adecarboxylata from those caused by E. hermannii.

In a mouse model, 12 E. hermannii isolates from polymicrobial soft tissue infections were injected into four-week-old ICR-strain mice in subcutaneous, intramuscular and intradermal sites. Apart from a few instances of non-recurring swelling at an injection site, none caused persistent wound infection in the mice (Pien et al. 1985).

In cytotoxicity testing at Health Canada, E. hermannii ATCC 700368 was toxic toward HT29 human colonic epithelial cell cultures if applied at high concentrations (1 × 10⁶ CFU/mL), and was capable of inducing the expression of IL-8 in HT29 cells. Cytotoxicity could not be evaluated in J774A.1 murine macrophages, because phagocytosis of bacteria interfered with the assay. J774A.1 cells produced IL-6 and TNF-alpha upon exposure to high concentrations of E. hermannii ATCC 700368 (Appendix 2, Table A-5).

In in vivo experiments conducted by Health Canada scientists, there was no evidence of pathogenicity or toxicity in mice dosed with 106 CFU E. hermannii ATCC 700368 in a 25-μL volume using an endotracheal nebulizer method for pulmonary exposure (Appendix A, Table A-6). The mice did not show any signs of abnormal behaviour and rapidly cleared the bacteria from their lungs within two days. There was a small, transient local inflammation that resolved simultaneously with clearance.

1.3 Hazard Severity

1.3.1 Environment

There is a recognized ambiguity in the literature about the classification of E. hermannii. However, correct taxonomic placement based on phylogenetic analysis alone would not give sufficient information regarding its pathogenic potential. There are no reports in the literature of toxicity, infectivity or pathogenicity due to E. hermannii in non-human species under natural environmental conditions in general, and none on the DSL strain ATCC 700368.

Only three studies have reported on determinants of pathogenicity for other strains of E. hermannii. Although some adverse effects following experimental challenge with high concentrations of the DSL-listed strain ATCC 700368 in a soil invertebrate were observed, the percentage reduction in juvenile production shown is not significant enough to allow for determination of an IC₅₀ value. No effects were shown in tested plants.

There are sources of uncertainty in the assessment of hazard related to the lack of familiarity with this micro-organism and difficulty identifying a suitable surrogate to alleviate this uncertainty. Also, if the rarity of its isolation reflects rare occurrence (Section 1.1.2.1), there may have been insufficient exposure to E. hermannii for potential effects to be manifested in non-human species. This challenges an accurate determination of its behaviour and effect in the environment, and increases the uncertainty level.

Thus, based on the limited available scientific information on fate and effect of E. hermannii ATCC 700368in the environment, which indicates that E. hermannii ATCC 700368 is not hazardous, and on the uncertainty derived from lack of familiarity, the environmental hazard severity for E. hermannii ATCC 700368 is estimated to be low.

1.3.2 Human

Although there is potential for misidentification, a combination of morphological, biochemical and physiological traits can be used to differentiate E. hermannii from related pathogenic organisms, including E. coli, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumonia, Salmonella spp., Shigella sonnei and Shigella dysenteriae (Table 1-7).

Since its recognition as a new species in 1982, a small number of E. hermannii wound infections have been reported in the literature (Table 1-6). These were mostly polymicrobial, and in cases where it was isolated with other micro-organisms, E. hermannii was rarely considered the primary pathogen; most likely, E. hermannii is a rare opportunistic pathogen. More recent reports identified E. hermannii as the sole pathogen responsible in cases of conjunctivitis, periodontitis, sepsis and septicemia; however, there were contributing factors in each of these cases, including breaches of normal physical and chemical barriers to infection, contamination of medical devices such as catheters, a history of antibiotic therapy, and debilitating disease or compromised immunity. Most infections were treated successfully with the administration of antibiotics. In a mouse model, there were no adverse effects following injection of human wound isolates into subcutaneous, intramuscular or intradermal sites.

It is unclear whether E. hermannii can cause diarrhea. Although it has been isolated from stools during episodes of diarrhea, and although its culture supernatant induced fluid accumulation in a mouse ileal loop model, its role as a causative agent has never been definitively shown.

The DSL strain ATCC 700368 has no history of pathogenicity in humans and it did not induce adverse effects upon endotracheal administration in a mouse model. Based on these findings, the human hazard severity for E. hermannii ATCC 700368 is low for the general population, but it may be low to medium in individuals who are susceptible because of compromised immunity, debilitating disease or extremes of age.

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

2. Exposure Assessment

2.1 Sources of Exposure

This assessment considers exposure to E. hermannii ATCC 700368 resulting from its addition to consumer or commercial products and its use in industrial processes in Canada.

E. hermannii ATCC 700368 was nominated to the DSL in 1997, and although it was nominated for use in a variety of products for the treatment of water and wastewater as well as for biodegradation and bioremediation, it is not currently used by the proprietary owner, who has declared that there are no plans to use it in the future. A second company that imported the strain, for research purposes only, also declared that there is no intention to use it in commercial 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 E. hermannii ATCC 700368 was not in commercial use in 2006.

The Government conducted a mandatory information-gathering survey under section 71 of CEPA 1999, 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 E. hermannii ATCC 700368, whether alone, in a mixture or in a product. No industrial, commercial or consumer activities using E. hermannii ATCC 700368 were reported in response to the Notice.

Although it appears to be longer in commercial use, E. hermannii ATCC 700368 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 application of other strains of E. hermannii. These represent possible uses of the DSL strain, as strain ATCC 700368 is likely to share characteristics (modes of action) with other commercialized E. hermannii strains (See Appendix 3, Table A-7 for details.):

• Biodegradation, bioremediation, waste and wastewater treatment for the removal of oil and grease, sewage sludge; and heavy metals,
• Aquarium and aquaculture treatments,
• Live attenuated subunit vaccine,
• Fertilizer,
• Antifouling additive in paint,
• Feed additive for biocontrol of Campylobacter jejuni in chickens,
• Pest control products such as mosquito repellant.

The reported uses are largely industrial, but include possible applications in certain consumer products, including septic tank treatments, drain cleaners, degreasers, odour control products, compost starters and aquarium treatments.

2.2 Exposure Characterization

2.2.1 Environment

Based on the absence of consumer or commercial activity in Canada according to the section 71 Notice, the overall environmental exposure estimation for E. hermannii ATCC 700368 is low. Nevertheless, given the range and scale of known and potential applications of the species E. hermannii listed in Section 2.1, there is potential for an increase in environmental exposure to E. hermannii ATCC 700368 and exposure scenarios arising from potential uses have been considered.

Should potential uses of E. hermannii ATCC 700368 identified in Section 2.1 be realized in Canada the most likely routes of introduction of E. hermannii ATCC 700368 into the environment would be into aquatic ecosystems through wastewater treatment, through runoff from direct application to soil of products containing E. hermannii or of land-applied treated sewage sludge, or waste effluent from commercial or industrial activities. Additionally, it may enter terrestrial ecosystems through direct and frequent application to soils during bioremediation, biodegradation and composting of organic and sludge waste. Aquatic applications could also expose terrestrial species through irrigation systems.

Naturally occurring strains of E. hermannii have been isolated from animals and their food products, and from terrestrial, aquatic and marine environments. Environmental isolations are rare; no data on background levels of E. hermannii in the environment were identified; standardized routine testing for fecal coliforms to determine water quality does not screen for E. hermannii (Rice et al. 1991). E. hermannii ATCC 700368 does not persist in soil with low organic content, but it may survive and persist in environments rich in organic carbon such as industrial effluent, sewage, organic rich soils, and sediment. The ecology and life cycle of E. hermannii is not fully known.

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. Commercialization of certain of these uses could result in large amounts of the organism being spread on fertile lands or being released into organic, rich waters. This exposure could result in large amounts of the organism being released, which could result in persistence and survival where there is a sufficient supply of organic carbon to sustain growth. Nevertheless, it is generally recognized that micro-organisms introduced into soils decline to a concentration that is in equilibrium with other microbial competitors (Leung et al. 1995; Van Veen et al. 1997).

2.2.2 Human

Based on the absence of consumer or commercial activity in Canada, according to the section 71 Notice, the overall human exposure estimation for E. hermannii ATCC 700368 is low. Nevertheless, given the range and scale of known and potential applications of the species E. hermannii listed in Section 2.1, there is potential for an increase in human exposure to E. hermannii ATCC 700368, and exposure scenarios arising from potential uses have been considered.

Should potential uses of E. hermannii ATCC 700368 identified in Section 2.1 be realized in Canada, human exposure could be greatest through the handling and application of consumer products intended for wastewater treatment (e.g. septic tank additives), degreasing (e.g. drain cleaners) or for the treatment of aquaria and ornamental ponds. These uses could result in direct exposure of the skin, and inhalation of aerosolized droplets or dusts containing E. hermannii. Secondary to product application, residual E. hermannii ATCC 700368 on surfaces or 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 E. hermannii may persist in sites that are rich in organic carbon, e.g., in drains, such exposures could be temporally distant from the time of application.

The general population could be exposed to E. hermannii ATCC 700368, as bystanders, during the application of commercial products. The route and extent of bystander exposure would depend on the nature of the product, 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 to low.

Indirect exposure to E. hermannii ATCC 700368 released into the environment subsequent to its use in water and wastewater treatment or soil bioremediation is also likely to occur in the vicinity of treated sites, but is expected to be no greater than direct exposure from the use of the organism in consumer products. Human exposure to bodies of water and soils treated with E. hermannii ATCC 700368 (e.g. through recreational activities) could result in exposure of the skin and eyes, as well as inadvertent ingestion, but exposure levels are likely to be low relative to household application scenarios.

Although E. hermannii has been isolated from drinking water distribution systems (Rice et al. 1991), possibly as a resident bacterium, the municipal drinking water treatment process, which includes coagulation, flocculation, ozonation, filtration and chlorination, would be expected to effectively remove E. hermannii from the source water entering the system.

In the event that potential consumer, commercial or industrial uses of E. hermannii ATCC 700368 are realized, human exposure to this micro-organism is expected to change based on the exposure scenarios described above. Such uses could result in direct and possibly repeated exposure to larger quantities of E. hermannii ATCC 700368.

3. 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 E. hermannii ATCC 700368 to be low for the environment and human health (low for the general population, and low-medium for individuals made susceptible by compromised immunity, debilitating disease or breaches in normal barriers to infection). Environmental and human exposure to E. hermannii ATCC 700368 from its deliberate use in industrial processes or consumer or commercial products in Canada 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).

There is no evidence in the scientific literature to suggest that E. hermannii ATCC 700368 will cause adverse ecological effects at the population level for vertebrates, invertebrates and plants under foreseeable use scenarios. Aquatic and terrestrial species may be exposed to the DSL-listed strain when used for bioremediation and wastewater, but, considering all the available lines of evidence in this report and the status of the science for this micro-organism, it is unlikely that E. hermannii ATCC 700368 poses a risk to the environment at population and ecosystem levels. Thus, environmental risk from its foreseeable future uses in industrial processes is estimated to be low.

Human exposure to E. hermannii ATCC 700368 could increaseif potential (new) uses are realized. E. hermannii has only rarely been associated with human infection in spite of its isolation from a range of habitats in several countries, since its recognition as a new species in 1982. On a few occasions, it has been implicated as the etiologic agent of disease, but these cases involved predisposing factors such as compromised immunity and breaches of barriers to infection. In the unlikely event of infection with the DSL strain ATCC 700368, it is susceptible to a number of clinically relevant antibiotics. Given these findings, and notwithstanding the potential for increased exposure to E. hermannii ATCC 700368, if consumer or commercial products containing this strain become available in Canada, the risk to human health from E. hermannii ATCC 700368 from its foreseeable future uses in industrial processes or consumer or commercial products in Canada is low.

4. Conclusion

Based on the information presented in this Screening Assessment, it is concluded that E. hermannii ATCC 700368 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 in the environment or its biological diversity;
• constitute or may constitute a danger to the environment on which life depends; or
• constitute or may constitute a danger in Canada to human life or health.

Therefore, it is concluded that this substance does not meet the criteria as set out in section 64 of CEPA 1999.

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A. Appendices

Appendix 1: Characterization of E. hermannii ATCC 700368^{Appendix 1 Footnote [a]}

MIDI is a commercial identification system based on the gas chromatographic analysis of cellular fatty acid methyl esters. Data presented show the best match between the sample and different MIDI databases (clinical and environmental), 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).

Figure A-1: Fatty acid methyl ester (FAME) analysis of P. stutzeri ATCC 17587 using MIDI clinical and environmental databases

Figure A-2: Multi-locus sequence analysis of E. hermannii ATCC 700368

Appendix 2: Virulence and Pathogenicity Testing of E. hermannii ATCC 700368

The MTT Assay was used to determine the cytotoxic potential of E. hermannii ATCC 700368 toward HT29 (colonic epithelial cells) and J774A.1 (macrophage cells). MTT is a yellow, soluble bromide salt that is reduced to a purple, insoluble formazan crystal by dehydrogenase enzymes of living cells (indicating mitochondrial activity). In the crystal state after reduction, it is trapped inside the cell. DMSO or another solvent such as isopropanol or mineral oil can be used to solubilize the formazan, which can then exit the cell, turning the solvent a purple colour that is detectable with a spectrophotometer. This assay is suitable for animal cells that are adherent. Metabolically active bacterial cells can also reduce MTT. Given that most bacterial cells are not adherent, bacteria and their formazan contribution can be rinsed away with PBS prior to solubilization. HT29 and J774A.1 were incubated at 37°C in the presence of 5% carbon dioxide. Mammalian cells were dosed with 10⁶ CFU/well of bacteria for 2, 4 and 24 hours. Dosed cells were washed twice with PBS before adding MTT. Loss in bioreduction activity was measured to determine the cytotoxic potential of E. hermannii ATCC 700368 group strains. Cytotoxicity is related to increased losses in bioreduction activity of the cell lines.

Appendix 3: Potential uses of E. hermannii

Date modified:

2015-07-31