Experimental toxicity of Aeromonas spp. In mouse’s small intestine: Ultrastructural aspects

Aurora Longa-Briceño ¹, Zulma Peña-Contreras ², Delsy Dávila-Vera ³, Rosa Virginia Mendoza-Briceño ⁴ and Ernesto Palacios-Prü† ⁵

¹ Bioanalyst, Universidad de Los Andes (ULA), Venezuela. M.Sc. in Microbiology, ULA, Venezuela. Professor, ULA, Venezuela. e-mail: auroralon@ula.ve

² Biologist, ULA, Venezuela. M.Sc. in Neurobiology, ULA, Venezuela. Researcher and Professor, ULA, Venezuela. Address: Centro de Microscopía Electrónica "Dr. Ernesto Palacios Prü" Universidad de Los Andes. Calle 32. Entre Avenidas 4 y Febres Cordero. Mérida, Venezuela. e-mail: zulmap@ula.ve

³ Biologist, ULA, Venezuela. Researcher, ULA, Venezuela. e-mail: delyda@ula.ve

⁴ Surgeon, ULA, Venezuela. Specialist in Neurobiology, ULA, Venezuela. Researcher and Professor, ULA, Venezuela. e-mail: rovirmen@ula.ve

⁵ † Surgeon, ULA, Venezuela. Doctor in Medical Sciences, ULA, Venezuela.

SUMMARY

Ultrastructural aspects of mouse small intestinal tissue cultures infected with Aeromonas spp. strains are described. High resolution light and transmission electron microscopy were used to assess the bacterial pathogenic mechanism, the ultrastructural changes that take place during the colonization of the intestinal tract and the interaction of Aeromonas spp. with the host epithelium. After 24h of culture, chains of vesicles were seen on the outer surface of the Aeromonas’ membrane. The vesicles were also found on the enterocyte surface. After 48h of culture, lysis of the epithelial intestinal cells, mononuclear phagocytic cells, phagocytic eosinophils and phagocyted Aeromonas were observed.

KEYWORDS / Aeromonas spp. / Mouse / Pathogenic Mechanism / Small Intestine Tissue Culture /

Aspectos ultraestructurales de la toxicidad experimental producida por Aeromonas spp. En el intestino delgado de ratón

RESUMEN

Se describen aspectos ultraestructurales del tejido intestinal de ratón cultivado e infectado con Aeromonas spp. Se utilizó microscopía de luz de alta resolución y microscopía electrónica de transmisión para evaluar los mecanismos patogénicos bacterianos, los cambios ultraestructurales que ocurren durante la colonización del tracto intestinal por Aeromonas spp. y la interacción de éstas con el epitelio huésped. En los cultivos de 24h se observaron vesículas distribuidas en cadenas sobre la superficie de la membrana externa de las Aeromonas. Estas vesículas se observaron unidas a la superficie del enterocito. En los cultivos de 48h se observó lisis de la superficie epitelial del intestino, migración de células fagocíticas mononucleares y presencia en la cavidad intestinal de eosinófilos fagocíticos, algunos conteniendo Aeromonas en su interior.

Aspectos ultra-estruturais da toxicidade experimental produzida por Aeromonas spp. No intestino delgado de rato

RESUMO

Descrevem-se aspectos ultra-estruturais do tecido intestinal de rato cultivado e infectado com Aeromonas spp. Utilizaram-se microscopia de luz de alta resolução e microscopia eletrônica de transmissão para avaliar os mecanismos patogênicos bacterianos, As mudanças ultra-estruturais que ocorrem durante a colonização do tracto intestinal por Aeromonas spp. e a interação destas com o epitélio hospede. Nos cultivos de 24h se observaram vesículas distribuídas em cadeias sobre a superfície da membrana externa das Aeromonas. Estas vesículas se observaram unidas à superfície do enterócito. Nos cultivos de 48h se observou lise da superfície epitelial do intestino, migração de células fagocíticas mononucleares e presença na cavidade intestinal de eosinófilos fagocíticos, alguns contendo Aeromonas no seu interior.

Received: 09/11/2006. Modified: 04/18/2008. Accepted: 05/15/2008.

Introduction

Aeromonas species have acquired clinical relevance due to their changing phylogenetic relationships, evolving taxonomy and controversial role in gastrointestinal diseases, which render them troublesome to treat (Janda and Abbott, 1998). In recent years Aeromonas spp. have emerged as an important human pathogen, with increasing incidence among travelers (causative agent of traveler’s diarrhea), due to their presence in food as well as in treated water for human consumption. The Environmental Protection Agency has included these bacteria in the "Candidate to Contaminant List" and began monitoring their presence in the water supply in the USA since 2002 (Galindo et al., 2004).

Virulence factors produced by Aeromonas spp. include proteases, lipases, pili or adhesin, "S-layer", hemolysin, cytotoxin, enterotoxin and endotoxin (Chopra et al., 2000; Sha et al., 2002; Sen and Lye, 2007). Endotoxins are considered responsible for most of the pathogenicity of gram-negative bacteria (Zhang et al., 1998). The endotoxin, or lipopolysaccharide or lipid "O", is an integral active component of the outer membrane of all gram-negative bacteria, where it plays an important role in the induction of sepsis. It is released during bacterial growth and has been associated with cellular lysis.

In this study, several changes observed in the enterocytic epithelial cells are described, using a novel experimental procedure which allows to culture Aeromonas inside a previously sterilized short cylinder of mouse's small intestine. Focus was placed on studying the ultrastructural changes occurring in the Aeromonas’ outer membrane, in the host tissue cytological organization during infection, and the possible relationship between both.

Materials and Methods

One strain of Aeromonas spp. was isolated as the unique enteropathogen from a patient with diarrhea. The strain was cultured in trypticase soy agar (Himedia Laboratories Ltd., India) and incubated for 24h at 37ºC. Subsequently, samples from this culture were incubated in basal medium Eagle (BME), adjusting the inoculum at a concentration of 1.50×10⁸ CFU/ml, and incubated for 24h at 37ºC.

Young adult NMRI mice obtained from the animal care facility of the Universidad de Los Andes, Mérida, Venezuela, were used. Segments of small intestine were removed for the elaboration of intestinal cylinders in which Aeromonas spp. were cultured. The intestinal cavity was prewashed with a 10% chlorine solution and immediately submerged in a 350mOsm BME solution, pH 7.2. Intestinal segments were tied at one end and filled with 1.5 CFU/ml of the Aeromonas spp. strain suspended in 1.0ml BME. After filling, the cylinders were tied close with surgical thread. The intestinal cylinders were incubated in culture media containing 98.9% BME with glucose, L-lysine and L-glutamine; 1% horse serum; and 0.1% penicillin/streptomycin (5000IU/5000µg), under constant rotation at 70rpm, at 37ºC, during 24h and 48h. The culture medium was oxygenated every 4h.

Cylinders of small intestinal segments were incubated for 24h and 48h using the same culture conditions but without filling them with Aeromonas, to be used as control cylinders.

After the programmed incubation time, intestinal cylinders were removed from THE culture medium and immediately submerged in a fixing solution of 3% glutaraldehyde and 3% formaldehyde prepared in 0.1M cacodylate buffer, pH 7.2 (Palacios-Prü and Mendoza-Briceño, 1972) during 6h at 4ºC. The cylinders were cut into small sections of ~3mm³, washed in cacodylate buffer and postfixed in 1% OsO₄ prepared in the same cacodylate buffer. Dehydration was achieved in ethanol series and propylene oxide, after which the samples were embedded in Eponate resin. Sections 1µm thick were stained with 1% toluidine blue for observation in a Reichert Polyvar light microscope. Sections 90nm thick were contrasted by using a modification (Palacios-Prü et al., 1981) of the classic method of uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). Thin sections were analyzed in a Hitachi H-7000 transmission electron microscope.

Results

Controls

The cylinders of small intestinal cultures incubated for 24h and 48h revealed good preservation (Figures 1a, b). The glycocalyx was clearly observed covering the microvilli (Figure 1b). Cells without signs of atrophia were seen and there were no visible epithelial protrusions or microulcerations of the intestinal wall. No vesicular chains were observed in or between the microvilli (Figure 1b), nor was any type of bacteria observed in the intestinal lumen.

Figure 1. a: Segments of enterocytes from a control sample with normal ultrastructural characteristics after 48h of culture. Bar: 1.5μm. b: detail of the normal ultrastructural features of an intestinal epithelial cell. Note the absence of vesicles between microvilli. Arrows, glycocalyx. Bar: 0.5μm

Incubation for 24h

After 24h of incubation, the Aeromonas spp. strain cultured with the intestinal tissue showed vesicular elements attached to the bacterial outer membrane (Figure 2a). These vesicles were seen alone, in pairs, or in groups of five or more vesicular units organized in chains emerging from the external membrane. Adjacent to these vesicles some complex membranous structures were observed (Figure 2b).

Figure 2. Cross sections of Aeromonas spp. cultured for 24h. Short chains of vesicles (in a) and large chains of vesicles (in b) can be seen emerging from the external bacterial membrane. Notice adjacent complex membranous structures (curved arrow) to these chains. Thick arrows: bacterial external membrane; short arrows: chains of vesicles. Bars: 0.17μm in a, 0.15μm in b.

The small intestine tissue cultivated with Aeromonas spp. and incubated for 24h did not present any alteration or modification in its cellular structure. The only outstanding observation was the presence of vesicular chains composed of four to ten vesicular units, which were aligned between microvilli spaces (Figure 3).

Figure 3. At a higher magnification, it is possible to identify the glycocalyx (asterisk) of the microvilli, and the particular vesicular chains (curved arrows) appearing between microvilli after 24h of culture with an Aeromonas spp. strain. Some microvilli fibrillary roots (straight arrow) are clearly seen. Bar: 0.35μm.

Incubation for 48h

When the segments of the small intestine were cultivated with Aeromonas spp., and then incubated for 48h, severe damage of the enterocytic epithelial surface was seen, with regions of tissular lysis on the surface of the intestinal microvilli (Figures 4 and 5). Numerous defensive cells such as lymphocytes were also observed (Figure 5), as well as abundant eosinophils showing their typical crystal-like structures inside lysosomes (Figure 6), and phagocytic mononuclear cells (Figure 7). Inside the cytoplasm of both of these cell types, Aeromonas spp. could be observed (Figures 6 and 7).

Figure 4. Light microscopic image of a segment of a small intestinal cylinder cultivated with Aeromonas spp. for 48h. The enterocytic epithelial surface shows regions with tissular lysis (arrow). Bar: 7.0μm.

Figure 5. Notice the lysis (straight arrow) of the surface of the small intestine and the presence of defensive cells or lymphocytes (curved arrows) after 48h culture with Aeromonas from the symptomatic patient. Bar: 4.4μm.

Figure 6. Eosinophils containing numerous phagocyted Aeromonas spp. (straight arrows) from symptomatic strain, are a common feature of the 48h intestinal Aeromonas cultures. These eosinophils show lysosome crystal-like structures (curved arrows). N: nucleus, *: cellular debris. Bar: 0.95μm.

Figure 7. After 48h culture of Aeromonas spp. with small intestine tissue it is possible to see, within cellular clusters adjacent to the enterocytes, numerous phagocyte mononuclear cells. Inside the cytoplasm of these cells, phagocyted Aeromonas (arrows) can be identified. Bar: 0.65μm.

Discussion

The experimental design herein presented offers appropriate conditions for the physiopathologic study of the interrelationship between the intestinal wall and bacteria under in vitro conditions very similar to in situ conditions. On the other hand, the results provide information about the pathogenicity diarrhea induced by Aeromonas spp.

Under the conditions of the experiment, no alteration of the intestinal epithelium was observed in the cultures from the small intestine with Aeromonas spp. incubated during 24h, except for vesicular chains aligned between microvilli spaces (Figure 3), which were not distinguished from those vesicular elements seen attached to the bacterial outer membrane (Figure 2) in these same cultures.

On the other hand, in cultures with 48h incubation, Aeromonas initially induced important tissue damage, including necrosis and lysis of the intestinal microvilli (Figures 4 and 5). In these cultures, the presence of phagocyting eosinophils within the intestinal cylinder lumen was observed (Figure 6), indicating the possible eosinophilic migration from the submucosa lymphatic organs, as well as the transformation of the eosinophilic cells into authentic macrophages. Aeromonas spp. also seem to induce the migration of mononuclear macrophages from submucosal origin, like the one shown in Figure 7, with abundant bacteria in their cytoplasm.

The vesicles produced on the surface of the bacterial outer membrane are also constituted by a double membrane unit. The production of these vesicles was observed in all the cultures analyzed. However, the more important vesicular formation was detected in the cultures incubated for 24h, whereas in the samples incubated for 48h, the vesicles were found where the intestinal damage was more severe.

The ultrastructural analysis suggests that Aeromonas spp. produce vesicles that form chains and can be seen between the microvilli of the intestinal epithelium. The contact between them could be one of the mechanisms that trigger virulence factors produced by Aeromonas spp., contained in the vesicles, and that could subsequently initiate mechanisms that attract eosinophils and mononuclear cells into the intestinal lumen.

The vesicles do not appear to be intestinal exocytic vesicles because they are formed on the surface of the bacterial outer membrane. Moreover, no clathrin-like outer skeleton is seen, which suggests that this vesicular system is a part of the pathogenic mechanism of action that induces the migration of eosinophils and macrophages. The phagocytic action of eosinophils is not a normal function of these cells, and we found only one report of a bactericide action of eosinophils (Persson et al., 2001).

The fundamental aim of the present study was the ultrastructural analysis of Aeromonas spp. present in the mouse's intestine epithelium, to determine their relation when in contact. In the cultures with a 24h incubation period, the vesicular chains were found adhered to the bacterial outer membrane as well as occupying the free spaces between microvilli. In cultures incubated for 48h, the vesicles were absent, whereas some regions of the enterocytic epithelial surface lost their organization, leading to tissue lysis. The vesicles could constitute a carrier tool that facilitates the approximation and the interchange of enterotoxins, immunological material or any other natural element responsible for the virulence and pathogenicity of Aeromonas spp., similar to what has been described by Sen and Lye (2007). However, a certain time is needed for the tissular response to take place in the presence of the enteropathogen bacteria. Further studies implementing immunohistochemical techniques would enrich the present study.

ACKNOWLEDGEMENTS

The authors acknowledge the technical and photographic assistance of Nancy Pacheco, Emilitza Labarca-Villasmil and José Benigno-Ramírez. This study was partly supported by the University of Los Andes CDCHT-ULA, grant M-727-01-03-B.

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