Bull. Eur. Ass. Fish Pathol., 27(5) 2007, 200 Isolation of agglomerans from Brown Trout (Salmo trutta) from Gilchrist Creek, Michigan, USA

T.P. Loch* and M. Faisal

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.

Abstract Pantoea agglomerans is a gram-negative environmental epiphyte that is associated with plants, found in soil, and is emerging as a source of localized and systemic infections in humans. To date, there have been two reports of P. agglomerans infecting fish, none of which have occurred within the Great Lakes basin. Herein, we report on the isolation of P. agglomerans from the kid- ney of an infected feral brown trout (Salmo trutta) caught from the Gilchrist Creek, Lake Huron watershed, Michigan, USA.

Introduction (Oncorhyncus tshawytscha) from the Abernathy Pantoea agglomerans (Gavini et al., 1989), Salmon Culture Technology Center in formerly known as Erwinia herbicola or Longview, Washington, USA (Sauter et al., agglomerans, is a ubiquitous 1987). With the exception of poor egg quality epiphytic gram-negative bacterium belonging from one P. agglomerans-infected chinook to the family Enterobacteriaceae (Gavini et al., salmon, no lesions or adverse health effects 1989). Pantoea agglomerans is found primarily were reported. In humans, this epiphyte is one on the surfaces of plants (Monier and Lindow, of the most common organisms transmitted 2005; Dye 1969); however, it has also been through plant thorn injuries (Flatauer & Khan, isolated from the gut of a number of insects 1978; De Champs et al., 2000; Kratz et al., (Hogg & Lehane, 2001; DeMaio et al., 1996; 2003). Most recently, a number of studies Pidiyar et al., 2004) and the cephalothorax of connected P. agglomerans to serious localized the mangrove crab, Ucides cordatus (Vieira et and systemic infections in humans, such as al., 2004). In fish, P. agglomerans has been neonatal pneumonia (Van Rostenberghe et al., recovered from a mortality event in hatchery- 2006), peritonitis (Lim et al., 2006), arthritis raised dolphin fish (Coryphaena hippurus) in (Ulloa-Gutierrez et al., 2004), bacteremia (De Bermuda (Hansen et al., 1990). It is unknown, Baere et al., 2004), sepsis (Cicchetti et al., however, if P. agglomerans was the etiologic 2006), as well as , urinary tract agent behind the mortality event. In another infections, and abscesses (Cruz et al., 2007). instance, P. agglomerans was isolated from the The prevalence or potential pathogenicity of heart blood and surface-disinfected non- Pantoea agglomerans to other animal species is fertilized eggs of spawning chinook salmon currently lacking.

* Corresponding author’s E-mail: [email protected] Bull. Eur. Ass. Fish Pathol., 27(5) 2007, 201

Herein, we report the isolation of P. agglo- from the following carbohydrates was merans from a brown trout (Salmo trutta) examined in phenol red broth base at a final collected from Gilchrist Creek, Lake Huron concentration of 1%: arabinose, galactose, watershed, Michigan, USA. inositol, lactose, malonate, maltose, mannitol, mannose, rhamnose, salicin, sucrose, Materials and methods trehalose, and xylose. All biochemical tests A total of sixty brown trout were collected via were incubated at 22°C. Unless otherwise electro-shocker from two different counties described, all ingredients were purchased bordering Gilchrist Creek (GPS coordinates from Remel Inc. Lenexa, KS. Results were N 44.8404; W 84.00155) and transported to the followed for seven days, with the following Aquatic Animal health Laboratory (AAHL) at exceptions: methyl red, Voges-Proskauer, Michigan State University. Immediately upon indole production, phenylalanine deaminase, arrival, the fish were necropsied and samples and TSI reactions were read at 2 days. aseptically obtained from the kidneys. Tissues sampled were streaked onto trypticase soy Results/Discussion agar (TSA, Remel Inc., Lenexa, KS) and Primary bacterial isolation from one brown incubated at 22 °C for 72 hours. Subcultures trout yielded a pure culture of yellow- of individually picked colonies were pigmented, undulate, translucent colonies incubated for 18-24 hours at 22°C before that exhibited umbonate surfaces with testing for morphological, cultural, and slightly concave centers. The colonies biochemical characteristics. measured 5 mm in diameter, and a colony count estimated the presence of 2.2 × 103 Gram reaction, motility testing (at 22°C and colony forming units/g kidney tissues. The 37°C), and biochemical reactions were bacterium was a gram-negative, straight rod performed according to standard bacterio- (0.5 × 1.25 mm) arranged in palisade-like logical methodologies (MacFaddin, 2000). structures and was motile at 22°C, but not at Biochemical reactions tested included: 37°C. The isolate partially hemolysed sheep cytochrome oxidase, catalase reaction (3% red blood cells (α-hemolytic). Other H O ), indole production, hydrogen sulfide 2 2 biochemical reactions are listed in Table 1. production, oxidation/fermentation reactions (O/F Basal medium; BD, Sparks, MD), mixed The phenotypic characteristics observed acid production (methyl red test), 2,3- strongly agree with those characteristic of P. butanediol production from glucose (Voges- agglomerans described in the Bergey’s Manual Proskauer test), nitrate reduction, citrate of Determinative Bacteriology (Holt et al., utilization, triple sugar iron reaction, ONPG 2000; Table 1) and reported by Gavini et al. (β-galactosidase), lysine decarboxylase, (1989), Iimura and Hosono (1996), Stiles and ornithine decarboxylase, arginine dihy- Lai-King (1981), and MacFaddin (2000). The drolase, hemolysis reaction on sheep blood production of phenylalanine deaminase, the agar (SBA), phenylalanine deaminase (BD), hydrolysis of esculin, the reduction of nitrate, and esculin hydrolysis. Production of acid acid production from salicin, and the Bull. Eur. Ass. Fish Pathol., 27(5) 2007, 202

Comparison of the biochemical characteristics of the brown trout isolate to those previously reported P. agglomerans E. agglomerans isolate Characteristics of isolate of this study reported P. agglomerans as reported by Hansen et al. (1990) by Holt et al. (2000) Gram Stain - - - Production of H2S - - - Simmons Citrate + + + Production of Indole - - - TSI Reaction K/A/-/- NR NR Esculin Hydrolysis + NR + Oxidative-Fermentative F F F (Glucose) Methyl Red - + d Voges-Proskauer + + + Nitrate Reduction + + + Lysine Decarboxylase - - - Ornithine Decarboxylase - - d Arginine Dihydrolase - - - Catalase + + + Oxidase - - - ONPG + + + Phenylalanine Deaminase + NR + Urease - - - Gas Production from - - - Glucose Utilization of Sodium + NR + Malonate Acid Production From: Arabinose + NR + Galactose + NR NR Inositol - NR - Lactose - NR - Maltose + NR + Mannitol + NR + Mannose + NR + Rhamnose + NR + Salicin + NR + Sucrose + NR + Trehalose + NR + Xylose + NR + Table 1: Phenotypic characteristics of Pantoea agglomerans isolated from brown trout. Results performed on isolate in this study were recorded at 7 days of incubation at 22°C unless otherwise noted. +, positive reaction; -, negative reaction; d, 11-89% positive; F, fermentative; NR, no results; TSI results, K/A/-/-, alkaline slant over acid butt without any production of H2S or gas. utilization of malonate allowed for the allowed for the elimination of other possible differentiation between P. agglomerans and bacterial species lacking pigment production. P. dispersa (Holt et al., 2000). In addition, the In the same context, other yellow-pigment production of yellow pigment by our isolate producing members of the family Bull. Eur. Ass. Fish Pathol., 27(5) 2007, 203

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