COMPARING POTENTIAL HEALTH EFFECTS OF INTRA-NASAL AND PERORAL EXPOSURE OF MICE TO PSEUDOMONAS AERUGINOSA AND ESCHERICHIA COLI

COMPARING POTENTIAL HEALTH EFFECTS OF INTRA-NASAL AND PERORAL EXPOSURE OF MICE TO PSEUDOMONAS AERUGINOSA AND ESCHERICHIA COLI

Gail M. Nelson^a, S. Elizabeth George^a, Michael J. Kohan^a, and Jørgen Schlundt^b

^aEnvironmental Carcinogenesis Division (MD-68), National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, (919)541-3095, fax (919)541-0694

^bNational Food Agency of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark

SUMMARY

Mouse models were evaluated to determine if health effects associated with intranasal and peroral exposure to microbial biotechnology agents could be detected. Pseudomonas aeruginosa AC869 resulted in mortality by either exposure route. Similar doses of E. coli BJ19 were not fatal. Both strains were recovered from lungs, small and large intestines, and cecum following intranasal or peroral treatment. Translocation to the mesenteric lymph nodes, liver, and spleen, and negative effects on tissue and body weights were observed with both exposure routes.

Key words: Intranasal exposure, Pseudomonas aeruginosa, Escherichia coli, biotechnology agents, peroral exposure

INTRODUCTION

Pseudomonas spp. have a multitude of environmental applications including biodegradation of environmental contaminants. Pseudomonads can metabolize a variety of chemicals such as polychlorinated biphenyls, oil, and the herbicides 2,4,5-trichlorophenoxyacetic acid, and atrazine. Escherichia coli has been used by industry for the production of specialty chemicals and does have some potential for environmental applications. Intranasal, peroral, and dermal exposure of humans to high numbers of microorganisms may occur during the production and application of microbial products.

P. aeruginosa, an opportunistic human pathogen, can produce several pathogenicity factors and can cause illness in immunosuppressed patients. Following inhalation of endotoxin, a gram-negative cell wall constituent found in both P. aeruginosa and E. coli, acute bronchoconstriction, fever, increased permeability, and macrophage migration can occur. Translocation of microorganisms from the intestinal tract to the mesenteric lymph nodes, spleen and liver in mice, also is a potential consequence of endotoxin exposure.

This study addresses the potential health effects of two biotechnology agents via an intranasal and a peroral exposure model: P. aeruginosa strain AC869, a 3,5-dichlorobenzoate degrader which has a pathogenic potential, and E. coli strain BJ19, an intestinal inhabitant originally isolated from the rat, which is avirulent in various animal species. Morbidity, mortality, clearance and translocation are compared in the two models.

MATERIALS AND METHODS

Two independent experiments were performed and repeated. Each experiment included the following variables: dose level (four levels including controls), time (termination at day 2 and 7), and exposure route (intranasal and peroral). For each combination of variables, 3 animals were used per experiment resulting in a total of 6 animals in each dose x time x exposure group. For peroral exposure, CD-1 mice were used, but because this mouse strain is very sensitive to endotoxin, C3H/HeJ mice were used for intranasal exposures. For comparative purposes, an extra group of CD-1 mice were used for intranasal exposure to E. coli.

Intranasal Exposure. Food was withheld 16 hours prior to treatment. The mice were anesthetized with methoxyfluorane. Each mouse was administered 50 µl by unilaterally placing the bacterial suspension or PBS at the base of the nostril with a feeding needle.

Peroral Exposure. Mice were dosed by gavage. Each animal received 0.1 ml of the bacterial suspension or PBS.

Enumeration of bacterial strains. At 2 and 7 days following treatment, mice were asphyxiated by CO₂and dissected aseptically. The lungs, liver, spleen, mesenteric lymph nodes, small intestine, cecum, and large intestine were removed and each placed into 5 ml of PBS. The tissues were weighed, homogenized, diluted and plated on Pseudomonas Isolation Agar + 50 µg/ml kanamycin (P. aeruginosa, 48 hr incubation at 30C) or MacConkey Agar + 40 µg/ml nalidixic acid (E. coli, 24 hr incubation at 37C). Following enumeration, representative colonies were isolated and antibiotic sensitivities were determined in order to confirm isolate origin.

RESULTS AND DISCUSSION

Intranasal challenge of mice with strain AC869 resulted in 100% (1.7 x 10⁸ CFU) and 50% (1.7 x 10⁷CFU) mortality. Peroral treatment with strain AC869 caused 33% and 8% mortality upon treatment with 2.2 x 10⁹ and 2.2 x 10⁸ CFU respectively. E. coli was not fatal by either intranasal (7.8 x 10⁷ CFU) or peroral (1.2 x 10⁹ CFU) exposure. Recovery of AC869 from lungs, small and large intestines, and cecum was observed following intranasal or peroral treatment with that strain. E. coli BJ19 was recovered from the same four organs after dosing by either exposure route. Intranasal challenge resulted in a higher concentration and a longer persistence of strain BJ19 in the intestinal system than direct peroral challenge. In addition, increases in lung weights were associated with strains BJ19 and AC869 treatment. Translocation from the intestinal tract to the mesenteric lymph nodes, liver, and spleen, and negative effects on tissue and body weights were detectable for both exposure routes and were dependent on the dosed strain. In summary, adverse health effects were detected when mice were dosed ndependently with microbial agents from different genera.

ACKNOWLEDGEMENTS

The authors would like to thank Ms. Bette Terrill and Ms.Peggy Mathis for their valuable technical assistance and Mr. Douglas Whitehouse for the graphics.

Although the research in this article has been supported in part by the U.S. Environmental Protection Agency, it has not been subject to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Part of the research has been supported by the Danish Centre for Microbial Ecology. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.