Tissue Culture to Assess Bacterial Enteropathogenicity

It is quite clear nowadays that the pathogenesis of infectious disease, although determined by pathogenic features of their causative agents, cannot be fully comprehended without structural (morphologic) analysis of immediate interaction between these agents and cells, tissues, and defense systems of the host. In diseases caused by agents adapted to certain cellular targets, the pathogenesis is, in addition, influenced by histophysiologic features of these targets wich are utilized or distorted or both by the pathogen.


Introduction
It is quite clear nowadays that the pathogenesis of infectious disease, although determined by pathogenic features of their causative agents, cannot be fully comprehended without structural (morphologic) analysis of immediate interaction between these agents and cells, tissues, and defense systems of the host. In diseases caused by agents adapted to certain cellular targets, the pathogenesis is, in addition, influenced by histophysiologic features of these targets wich are utilized or distorted or both by the pathogen. Acute diarrheal diseases caused by Gram-negative bacteria: Vibrio cholerae, diarrheagenic Escherichia coli, Shigella, Salmonella, Aeromonas and enteropathogenic Yersinia, are examples of this type of infection. All of these agents target the same cell type, the enterocyte, and produce potent exotoxinas. [1] The morphologic method either produced an impetus to bacteriologic studies or served as a tool in the evaluation of bacterial pathogenicity, including that of genetically altered microorganisms. In particular, morphologic studies have revealed that enteric bacteria either colonize enterocytes while remaining epicellular ( i.e., bound to the cell surface) with or whithout affecting cellular architecture, or they invade the cell with or whithout its destruction. On the other hand, biochemical studies have shown that prevalence of the secretory or destructive inflammatory disturbances in the gut is, for the most part, determined by bacterial exototoxins. Although exotoxins affect enterocytes by a variety of mechanisms, they can be combined into two groups that are referred to as cytotonic and cytotoxic or respectively, as enterotoxins and cytotoxins. It has also been established that each step in host-pathogen interaction is governed by multiple determinants encoded in bacterial plasmid and chromosome genes. [1,2,3] The genus Aeromonas comprises gram-negative bacteria that can be isolated from water and a diversity of foods. Some strains are important diarrhea producers, particularly in children under five years and in older adults [4,5].
The clinical manifestations of diarrhea vary from autolimited symptoms to severe cases with presence of mucus and blood in faeces, suggesting that, as in Escherichia coli pathogenic types, Aeromonas virulence is multifactorial [6,7]. 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 [8].
The aim of this chapter, is to show that with the development of an in vitro animal model, to explore the mechanisms related to the colonization of the digestive tract, as well as the determination of the mechanisms of interaction with the host epithelium, provide a valuable tool in the study of bacterial entropathogenicity that the genus Aeromonas

Interaction host/bacterial pathogen
The Aeromonas enteropathogenic capability is usually underrated [7,9,10]. Information about decisive diarrhea virulence factors is limited, due to lack of appropriate animal models for the study of Aeromonas genus. [7,10,11]). With the models developed up to now, it has obtained scarce information about the interaction between the host and Aeromonas spp. [10,11,12,13,14].
In this regard, the development of an in vitro model to explore the mechanisms related to the colonization of the digestive tract by Aeromonas spp. as well as the determination of the mechanisms of interaction with the host epithelium, provides a valuable tool in the study of Aeromonas pathogenicity. On the other hand, the critical steps in the pathogenesis of virulent strains involve the adhesion and colonization of the intestinal mucosa by Aeromonas.
The present focuses in the analysis of the mechanisms causing the pathogenicity of Aeromonas strains in co-cultures of isolated bacteria with intestinal cells. Ultrastructural aspects of mouse small intestinal tissue cultures infected with Aeromonas strains are described using a novel experimental procedure which allows to culture Aeromonas inside a previously sterilized short cylinder of mouse´s small intestine.

Bacterial strains and growth conditions
Two strains of Aeromonas caviae were used, one (A) isolated from an asymptomatic patient and the other one (D) from a patient with diarrhea in which the isolated Aeromonas was the only enteropathogen. The strains were inoculated in trypticase soya agar (HIMEDIA laboratories Ltd., Bombay, India 400 086) at a concentration of 1.5 X 10 8 CFU/ml and incubated for 24h. After incubation the media were gently filtered and the corresponding Aeromonas strain resuspended in 1ml Basal Medium Eagle (BME).

Tissue culture procedure
Segments of the small intestine were removed from the abdominal cavity of young adult NMRI mice to prepare small intestine cylinders of ~3cm in length that were sterilized with a 10% chlorine solution. One end of each intestinal cylinder was tied with sterile surgical thread and the cavity was filled with 1.5 X 10 8 CFU/ml of a given one of the two Aeromonas strains suspended in BME. After filling, the cylinders were tied close with surgical thread. The whole preparations were co-cultured in 50ml of tissue culture media containing 90% BME with Earle´s salts and L-glutamine, 10% horse serum, 5000IU/ml penicillin and 5mg/ml streptomycin, 360mOsm, pH 7.2, at 37°C, under constant rotation at 70rpm, for 24h and 48h. During the incubation period, the media were oxygenated every 4h during daytime. Control small intestine cylinders were cultured for 48h under the same conditions but without Aeromonas inside. Once the programmed culture time was accomplished, the intestinal cylinders were removed from the flask and immediately immersed in a fixing solution containing 3% glutaraldehyde and 3% formaldehyde in 0.1M cacodylate buffer, pH 6.3 [15] during 6h at 4°C.
The intestinal cylinders were cut in small sections of approximately 3mm 3 , washed in 0.1M cacodylate buffer, pH 7.2, and postfixed for 24h in 1% osmium tetroxide prepared in the same buffer. The tissue was then dehydrated in ascending concentration ethanol solutions, followed by propylene oxide and finally embedded in Epon 812. Sections of 1μm were stained with 1% toluidin blue and observed under a high resolution light microscope. Sections of 90 nm were contrasted with uranyl acetate [16] and lead citrate [17] using a modification of this classic method [18] and were analyzed using a Hitachi-7000 transmission electron microscope. [19,20].

Effects of Aeromonas caviae co-cultured in mouse small intestine
The experimental desing 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, this experimental model employed using co-cultures of mouse intestinal mucosa with Aeromonas caviae revealed important information on the pathogenicity of this species in gastrointestinal infections. The strain A, isolated from asymptomatic patient, produce minor to mild alterations of the intestinal wall, while more pronounced alterations were found at both periods of incubations when strain D, from the patient with diarrhea, was used to prepare the co-cultures.
The damage produced was demonstrated by the alterations of the integrity of intestinal microvilli, disruption of the epithelium and presence of mucosal microulcerations, which were brought about by the fact that this strain is an high cytotoxin producer and may possess other virulence factors [4,21] These strains belong to the A. caviae species and were considered of lesser virulence than other strains of this species [22].
Light microscopical observations revealed a varying degree of histological alterations of the intestinal wall, according to the severity of the damage. The major tissue damage is shown in Figure 1, where a large bacterial cluster can be seen occupying the crypts between intestinal folds. In most cases, the intestinal wall showed a high degree of generalized cellular atrophy with tissue lysis when the diarrhea producing strain (D) was used. These images were seen at 24h as well as at the 48h samples of incubation. In cases of moderate damage, the basic cytoarchitecture of the intestinal mucosa was preserved and it was possible to recognize enterocytes, mucous cells and germinative or mother cells. The villi as well as the majority of the crypts were seen, althought some of them were shorter and thicker (Figures 2 and 3).
These mild damages were caused when the strain A from the asymptomatic species was employed for the co-culture. Microvilli were seen forming part of the apical surface of the enterocyte as well as cytoplasmatic protrusion that come from the apical portion of the enterocytes (Figure 4). Some segments of the intestinal cylinder co-cultured with Aeromonas strains showing a higher degree of alterations contains clusters of spheroidal cells associated to bacterial elements (Figures 3 and 6).  Transmission electron microscopy revealed minor alterations of the intestinal mucosa when the strain from asymptomatic patients (A) was co-cultured. Most enterocytes were seen with seen with typical ultrastructural characteristics, however, they showed numerous apical protrusion detachments (Figures 2 and 4). In more damaged regions, there was a progressive atrophy of the epithelial cells showing loss of microvilli and large cellular vacuoles loaded with cellular detritus ( Figure 5). In the intestinal regions having intermediate epithelial alterations, globular cells identified as blood and lymphatic cells were observed in the gut lumen associated to Aeromonas and cellular debris (Figures 3 and 6). Eosinophils were seen with multilobular nuclei and their characteristic lysosomes with crystal-like structures (Figures 7 and 8). In Figure 8 phagocytated Aeromonas within the eosinophilic cytoplasm are clearly visible.
Cells identified as lymphocytes ( Figure 7) and others as plasma cells (Figure 9) were also observed as part of the clusters of globular cells found in the small intestine cylinder lumen after two days of culture.    In Figure 10 an image is shown of a cylinder of small intestinal segment incubated for 48h using the same culture conditions but without Aeromonas in its lumen, with the purpose to compare the control cylinders to the ones co-cultured with the bacteriae. No atrophic cells are seen and there are no visible epithelial protrusions, extraepithelial cells nor microulcerations of the intestinal wall. The villi and crypts show minor changes due to the culture conditions.  The cylinders of small intestinal cultures incubated for 24h and 48h revealed good preservation. The glycocalyx was clearly observed covering the microvilli (Figure 11). No vesicular chains were observed in or between the microvilli, nor was any type of bacteria observed in the intestinal lumen. Figure 11. a: Segments of enterocytes from a control sample with normal ultrstructural characteristics alter 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: 0.5 μm After 24h de incubation, the Aeromonas spp. strain cultured with the intestinal tissue showed vesicular elements attached to the bacterial outer membrane (Figure 12). 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 12).
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 13). 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 14 and 15). Numerous defensive cells such as lymphocytes were also observed (Figure 15), as well as abundant eosinophils showing their typical crystal-like structures inside lysosomes (Figure 16), and phagocytic mononuclear cells (Figure 17). Inside the cytoplasm of both of these cell types, Aeromonas spp. could be observed (Figures 16 and 17) Figure 13. 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: 0.35&#956;m    The pathogenicity of Aeromonas has been confirmed with this procedure and its virulence has been corroborated as strain-specific and not species-specific.
When the intestinal epithelium lesions were observed no bacteriae adhered to the cells were seen, indicating that direct bacteria-epithelial cell contact was nor required for tissue alterations: on the contrary, it seems that Aeromonas triggers a chemotactic response which activates migratory actions to the lymphatic cells from the adjacent lymphatic plaques.
The lymphatic submucous plaques and the autonomous defensive structures are able to act in culture conditions even in the absence of the circulatory blood elements, bone marrow cells or other lymphatic organs. Among the defense elements that migrate in answer to the chemotactic stimulus are the eosinophilic granulocytes, which have been reported to participate only in parasitic diseases. In this investigation their capability to defend intestinal cells against bacterial aggressions [23,24] is ultrastructurally documented.
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 13), which were not distinguished from those vesicular elements seen attached to the bacterial outer membrane (Figure 12) in the 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 14 y 15).
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 suggest 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 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 suggest that this vesicular system is a part of the pathogenic mechanism of action that induces the migration of eosinophils and macrophages.
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, inmunologicals material or any other natural element responsible for the virulence and pathogenicity of Aeromonas spp., similar to what has been described by [25].
The strain isolated from the patient with diarrhea produced important alterations in the intestinal mucosa, indicating its enteropathogenic potential. This strain is a cytotoxin producer and, as has been pointed out for Shigella, a correlation exists between the severity of the lesions of the mucosal membrane and the toxigenicity of a particular strain [1].

Conclusion
In conclusion the genus Aeromonas has great enteropathogenic potential, although additional studies are necessary to understand more about the pathogenicity of other Aeromonas strains, this procedure also may useful to determinate currently unknown interactions between a variety of other enteropathogenic bacteria and, the results obtained in these studies give a valuable contribution for the future application of this procedure in the bacterial patogenicity research.