Assessment of Enteral Bacteria

Assessment of Enteral Bacteria 1

UNIT 21.3

1

Alessandro Menozzi and Maria Cristina Ossiprandi 1

University of Parma, Parma, Italy

ABSTRACT The disruption of intestinal barrier leads to the penetration of noxious luminal compounds into the gut wall, causing further damage. This unit describes the assessment of enteric bacteria translocation into the intestinal wall of rats, an established method for the evaluation of bowel damage to the mucosal epithelial barrier. The Basic Protocol provided in the present unit describes collection and preparation of small intestine sample, performing of sample serial dilutions for bacterial culture, performing of the culture of aerobic and anaerobic bacteria on petri dishes, incubation of the cultured plates, and counting of bacterial colonies. The Support Protocols describes the procedures for the preparation of petri dishes for the culture, using different employable media for aerobes or anaerobes. The Alternate Protocol describes the use of the “inclusion method,” suitable for the culture of anaerobic bacteria. Curr. Protoc. Toxicol. 44:21.3.1-21.3.11. C 2010 by John Wiley & Sons, Inc.  Keywords: intestinal damage r bacteria r permeability

INTRODUCTION Among the several functions of intestinal epithelium, the ability to work as an effective shield against lumen noxious constituents is remarkable, considering that this barrier is made of a single lining of cells. The protective layers, which concur to hinder the penetration of potentially harmful compounds, include an extrinsic barrier that is made of a water lining and a mucous film, also known as glycocalix, mostly made of glycolipids and glycoproteins, and an intrinsic barrier constituted by enterocytes and paracellular spaces between these cells (Madara, 1990). At the apex of the intercellular space are located the so-called tight junctions, the most important structure of the intestinal barrier, which hold epithelial cells close together and block the permeation of microorganisms and large compounds such as hydrophilic macromolecules (proteins, LPS, or peptidoglycan, for instance; Balzan et al., 2007). The efficacy of this sophisticated barrier is therefore dependent on the integrity of the enterocyte brush-border membranes and of the tight junctions, besides the ability to produce the right array of epithelial secretory products. Many toxic substances and intestinal diseases are able to disrupt the enteric epithelial barrier. Intestinal damage is often characterized by lesions ranging from microscopic erosions of the mucosa to large macroscopic ulcers or even perforations. When the anatomical structure of the intrinsic barrier is severely altered and the enterocyte monolayer becomes discontinuous, there is obviously no protection from the passage of macromolecules from the lumen to inner tissues. In several cases, however, intestinal damage can be more subtle and devious and, even if the wholeness of the epithelium seems to be preserved, tight junctions can become dysfunctional and large molecules are able to pass through and to trigger further damage. Increased gut permeability is thought to be relevant in the pathogenesis of many intestinal diseases like food allergies, inflammatory bowel disease (IBD), autoimmune disorders, celiac sprue, and the damage induced by radiations, toxins, and drugs (Bjarnason et al., 1995). There is evidence, as a matter of fact, that one of the crucial initiating events of Gastrointestinal Toxicology Current Protocols in Toxicology 21.3.1-21.3.11, May 2010 Published online May 2010 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471140856.tx2103s44 C 2010 John Wiley & Sons, Inc. Copyright 

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nonsteroidal anti-inflammatory drug (NSAID)–induced enteropathy is the uncoupling of oxidative phosphorylation in enterocyte mitochondria, leading to a fall in ATP production and to impaired epithelial tight junctions (Somasundaram et al., 1995; Mahmud et al., 1996). Additionally, any event or compound that is able to reduce the oxygenation of intestinal mucosa could cause a depletion of intracellular ATP and an increase of oxidative radicals, which in turn decrease tight junction impermeability (Bjarnason et al., 1986; Matthews et al., 1994). Among the potentially noxious luminal components, one of the most important is represented by enteric bacteria, because they represent one of the largest parts of intestinal contents (Simon and Gorbach, 1986). While some bacteria of enteric microflora are potential pathogens, the majority of them are mostly beneficial for the host, by contributing to digestive functions and to immune system development and by working as a shield from enteroinvasive pathogenic microorganisms (Berg, 1995; Steinberg, 2003). When intestinal permeability is abnormally increased, though, even saprophytic bacteria are able to translocate beyond the epithelial barrier and to become noxious by triggering host immune reaction. A suitable and commonly employed technique for the assessment of enhanced gut permeability as an index of bowel damage is, therefore, the count of bacteria that have translocated from lumen into the intestinal wall (Reuter et al., 1997). This unit describes basic methods regarding the collection of small intestine samples of rats, the preparation of tissues, and the measurement of the number of translocated enteral bacteria, as an indirect measure of intestinal mucosal injury (Basic Protocol). Additionally, there are protocols for preparing plates for bacterial culture (Support Protocols 1 and 2) and an alternate inclusion method for culturing anaerobes (Alternate Protocol). NOTE: All protocols using live animals must first be reviewed and approved by an Institution Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

BASIC PROTOCOL

ASSESSING ENTERIC BACTERIA NUMBER A segment of rat small intestine is surgically collected and homogenized to prepare the sample for assessment of bacterial growth. Dilutions of the sample are cultured under aerobic and anaerobic conditions to evaluate the bacterial load. NOTE: Maintain sterility of these cultures by working under a laminar vertical airflow hood.

Materials Diethyl ether (Merck) Rats (Wistar, males, 220 to 240 g body weight) 40 mg/ml chlorhexidine gluconate (Hibiscrub) 0.9% (w/v) NaCl solution, sterile Sterile Ringer solution (Oxoid) TSA culture plates (Support Protocol 1) Blood agar base culture plates (Support Protocol 2)

Assessment of Enteral Bacteria

Laminar vertical air flow hood (Bio-Air) Foam board with pins Hair clipper (Oster) Sterile gauzes Sterile surgical gloves Sterile surgical towels

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Current Protocols in Toxicology

Sterile surgical instruments including: Blunt-end scissors Sharp-end scissors Surgical forceps 100-ml beakers 90-mm petri dishes, sterile Stomacher bags, sterile (Laboindustria) Stomacher (PBI) 15-ml polypropylene round-bottom tubes (Becton Dickinson), sterile 0.1-, 1-, and 10-ml pipets (Sarstedt), sterile Pipet aid (Drummond) Micropipettors (Gilson) Pipet tips (Sarstedt) Analytical scale (Acculab) Vortex mixer (Falc Instrument) Sterile spreaders (Laboindustria) Incubator 37 ± 1◦ C (Instruments) Anaerobic jars (Oxoid) Anaerobic sachets (Oxoid) Colony counter (PBI) Collect the small intestine sample 1. Anesthetize the rat with diethyl ether and sacrifice it by cervical dislocation. 2. Place it on its back on a foam board and fix front and rear paws to the table with pins. 3. Trim the hair of the animal on the abdomen and lower chest and thoroughly disinfect the area with 4% chlorhexidine solution, wiping with a soaked gauze. All the following procedures must be performed as sterile as possible.

4. Wearing surgical gloves, place a sterile towel on the table aside the body of the rat. Using blunt-end scissors, cut a U-shaped area of the abdominal wall from xiphoid process to the pubic region. 5. Carefully exteriorize the small intestine and cut it with sharp-end scissors 5 to 6 mm distal to the pylorus and 2 to 3 mm proximal to the ileo-caecal valve. Collect the excised intestinal tube, gently separating it from peritoneal ligaments with the scissors. 6. Place the excised intestine on the sterile towel and cut away a segment of ∼10 cm. The choice of which 10-cm sample is to be collected may depend on the kind of intestinal lesion you are dealing with. You can choose a segment from the proximal region if intestinal inflammation involves mostly the jejunum or a distal segment if an ileitis is present. In any case, it is important that you always collect the sample in the same position (e.g., 30 cm from proximal end of whole removed intestine).

7. Carefully remove the peritoneal fat on the mesenteric side of the intestinal segment with the aid of surgical forceps. 8. Open the intestinal segment along the antimesenteric side with sharp-end scissors and gently remove fecal contents. 9. Rinse the intestinal sample several times in a 100-ml beaker filled with sterile 0.9% NaCl solution, in order to remove all traces of fecal material from the mucosal surface.

Prepare small intestine samples 10. Weigh the intestinal sample, place it into an empty sterile petri dish, and mince it with scissors.

Gastrointestinal Toxicology

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11. Using the forceps, put all obtained gut pieces in a sterile Stomacher bag. Add 1 ml of sterile Ringer solution per 100 mg wet tissue into the bag and homogenize the sample by Stomacher action for 3 min. 12. Transfer 10 ml of the homogenized sample, using a sterile pipet, into a 15-ml round-bottom sterile tube.

Prepare sample dilutions 13. Using separate sterile pipets, prepare serial decimal dilutions until 10-8 , by transferring 1 ml from previous dilution into a sterile tube filled with 9 ml of Ringer solution each time, starting from the tissue homogenate. Carefully shake each tube using a vortex immediately before taking the sample for the next dilution. Perform the cultures All petri dishes to be employed must be prepared at this point (see Support Protocol 1). 14. After shaking the tube with the vortex, pipet 0.1 ml of each dilution into separate, duplicate, appropriately marked petri dishes. Repeat for each medium employed [usually Tryptone Soya Agar (TSA) for aerobic bacteria and Blood Agar Base for anaerobics]. 15. Spread the sample on the petri dish surface, until it is evenly distributed, by using a sterile spreader to reach a complete covering.

Incubate the cultures 16a. For aerobic growth: Put the TSA petri dishes upside down (to prevent condensation from dripping down onto the agar surface) into the incubator at 37◦ C and leave 24 hr.

Assessment of Enteral Bacteria

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Figure 21.3.1 Assessing colony numbers by means of Colony Counter. The plate is put upsidedown on the illuminated glass and the colonies are tallied directly on the back of the petri dish using a marker pen. The counter records the number of pressures and shows it on the display. Current Protocols in Toxicology

16b. For anaerobic growth: Put the blood agar base petri dishes upside-down into the anaerobic jar, together with an opened specific anaerobic sachet (when the sachet is removed from the outer wrapper, the sachet becomes activated by exposure to air), and immediately close the jar lid. Inspect it carefully to be sure that the jar is hermetically sealed, and then incubate 48 hr at 37◦ C.

Count the colonies 17. Select the plates for the bacteria colony count by the following criteria: any plate which has >200 colonies is designated as too many to count (TMTC); plates with 200 colonies or