Anatomy

We are all connected to the external environment through our gastrointestinal (GI) tract (Figure 75-1). What we swallow enters the GI tract and passes through the esophagus into the stomach, through the small and large intestines, and finally to the anus. During passage, fluids and other components are added to this material as secretory products of individual cells and as enzymatic secretions of glands and organs, and they are removed from this material by absorption through the gut epithelium.

The major components of the tract are listed in Box 75-1. The nature of the epithelial cells lining the GI tract varies with each portion. The lining of the GI tract is called the mucosa. Because of the differing nature of the mucosal surfaces of various segments of the bowel, specific infectious disease processes tend to occur in each segment.

The wall of the small intestine has folds that have millions of tiny, hairlike projections called villi. Each villus contains an arteriole, venule, and lymph vessel (Figure 75-2). The function of villi is to absorb fluids and nutrients from the intestinal contents. Epithelial cells lining the surface of villi have a surface resembling a fine brush, referred to as a brush border. The brush border is formed by nearly 2000 microvilli per epithelial cell. Intestinal digestive enzymes are produced in brush border cells toward the top of the villi. Villi and microvilli help make the small intestine the primary site of digestion and absorption by significantly increasing the surface area; more than 90% of physiologic net fluid absorption occurs here. Mucus-secreting goblet cells are found in large numbers of villi and intestinal crypts.

Similar to the small intestine, the large intestine is composed of several segments (see Box 75-1). The wall of the large intestine consists of columnar epithelial cells, many of which are mucus-producing goblet cells. In contrast to the small intestine, there are no villous projections into the lumen. The remaining excess fluid within the GI tract is resorbed through the cells lining the large intestine before waste is finally discharged through the rectum.

In addition to the previously discussed components of the GI tract, numerous other organs and structures are either located in the main digestive organs or open into them. These accessory organs and structures include the salivary glands, tongue, teeth, liver, gallbladder, and pancreas. Except for the teeth and salivary glands, these organs are illustrated in Figure 75-1.

Resident Microbial Flora

The GI tract contains vast, diverse normal flora. Although the acidity of the stomach prevents any significant colonization in a normal host under most circumstances, many species can survive passage through the stomach to become resident within the lower intestinal tract. Normally, the upper small intestine contains only sparse flora (bacteria, primarily streptococci; lactobacilli; and yeasts; 10¹ to 10³/mL), but in the distal ileum, counts are about 10⁶ to 10⁷/mL, with Enterobacteriaceae and Bacteroides spp. predominantly present.

Infants usually are colonized by normal human epithelial flora, such as staphylococci, Corynebacterium spp., and other gram-positive organisms (bifidobacteria, clostridia, lactobacilli, streptococci), within a few hours of birth. Over time, the content of the intestinal flora changes. The normal flora of the adult large bowel (colon) is established relatively early in life and consists predominantly of anaerobic species, including Bacteroides, Clostridium, Peptostreptococcus, Bifidobacterium, and Eubacterium.

Aerobes, including Escherichia coli, other Enterobacteriaceae, enterococci, and streptococci, are outnumbered by anaerobes 1000 : 1. The number of bacteria per gram of stool within the bowel lumen increases steadily as material approaches the sigmoid colon (the last segment). Eighty percent of the dry weight of feces from a healthy human consists of bacteria, which can be present in numbers as high as 10¹¹ to 10¹² colony-forming units (CFU)/g of stool.

Gastroenteritis

Worldwide, diarrheal diseases are the second leading cause of death; about 48 million enteric infections occur each year. These infections cause significant morbidity and death, particularly in elderly people and children younger than 5 years of age. It has been estimated that 4 million to 6 million children die each year of diarrheal diseases, particularly in developing countries in Asia and Africa. Even in developed countries, significant morbidity occurs as a result of diarrheal illness. Although acute diarrheal syndromes are usually self-limited, some patients with infectious diarrhea require diagnostic studies and treatment.

Toxins

Enterotoxins.

Enterotoxins alter the metabolic activity of intestinal epithelial cells, resulting in an outpouring of electrolytes and fluid into the lumen. They act primarily in the jejunum and upper ileum, where most fluid transport takes place. The stool of patients with enterotoxic diarrheal disease involving the small bowel is profuse and watery, and blood or polymorphonuclear neutrophils are not prominent features.

The classic example of an enterotoxin is that of Vibrio cholerae (Figure 75-3). This toxin consists of two subunits, A and B. The A subunit is composed of one molecule of A₁, the toxic moiety, and one molecule of A₂, which binds an A₁ subunit to five B subunits. The B subunits bind the toxin to a receptor (a ganglioside, an acidic glycolipid) on the intestinal cell membrane. Once bound, the toxin acts on adenylate cyclase enzyme, which catalyzes the transformation of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). Increased levels of cAMP stimulate the cell to actively secrete ions into the intestinal lumen. To maintain osmotic stabilization, the cells then secrete fluid into the lumen. The fluid is drawn from the intravascular fluid store of the body. Patients therefore can become dehydrated and hypotensive rapidly. V. cholerae inhabits sea and stagnant water and is spread in contaminated water. The organisms have been isolated from coastal waters of several states, and sporadic cases of cholera occur in the United States. Additional information about V. cholerae is provided in Chapter 26.

Other organisms also produce a cholera-like enterotoxin. A group of vibrios similar to V. cholerae but serologically different, known as the noncholera vibrios, produce disease clinically identical to cholera, effected by a very similar toxin. The heat-labile toxin (LT) elaborated by certain strains of E. coli, called enterotoxigenic E. coli (ETEC), is similar to cholera toxin, sharing cross-reactive antigenic determinants. The enterotoxins of some Salmonella spp. (including S. enterica subsp. arizonae), Vibrio parahaemolyticus, the Campylobacter jejuni group, Clostridium perfringens, Clostridium difficile, Bacillus cereus, Aeromonas, Shigella dysenteriae, and many other Enterobacteriaceae also cause positive reactions in at least one of the tests for enterotoxin (discussed later). The exact contribution of these enterotoxins to the pathogenicity of most stool pathogens remains to be elucidated.

Certain strains of E. coli, in addition to producing a heat-labile toxin (LT) similar to cholera toxin, also produce a heat-stable toxin (ST) with other properties. Although ST also promotes fluid secretion into the intestinal lumen, its effect is mediated by activation of guanylate cyclase, resulting in increased levels of cyclic guanylate monophosphate (GMP), which yields the same net effect as increased cAMP. Tests for ST include enzyme-linked immunosorbent assay (ELISA), immunodiffusion and cell culture. Molecular techniques, including the use of DNA probes as well as several amplification assays, have been used to identify ETEC directly in clinical samples or isolated bacterial colonies.

Several tests are available for the detection of enterotoxin. Immunodiffusion, ELISA, and latex agglutination tests are all available to identify specific toxins. Molecular probes and amplification assays for toxin detection are also available, primarily for research use.

Attachment.

An organism’s ability to cause disease can also depend on its ability to colonize and adhere to the bowel. To illustrate, ETEC must be able to adhere to and colonize the small intestine, as well as produce an enterotoxin. These organisms produce an adherence antigen, called colonization factor antigen (CFA). Certain strains of E. coli referred to as the enteropathogenic E. coli (EPEC) attach and then adhere to the intestinal brush border. This localized adherence is mediated by the production of pili. Subsequent to attaching, EPEC disrupts normal cell function by effacing the brush epithelium, thereby causing diarrheal disease. This complete process is referred to as attachment and effacement. Genes responsible for the initial adherence of ETEC, EHEC, and EPEC to intestinal epithelial cells reside on a transmissible plasmid. EHEC has the same ability to attach to intestinal epithelial cells and cause effacement. In addition, EHEC produces a Shiga toxin that spreads to the bloodstream, causing systemic damage to vascular endothelial cells of various organs, including kidney, colon, small intestine, and lung. EHEC is believed to have arisen as a result of an EPEC strain having become infected with a bacteriophage carrying the Shiga toxin gene (Figure 75-4).

Giardia lamblia, a parasite, has increasingly become more common as an etiologic agent of GI disease in the United States. Excreted into fresh water by natural animal hosts such as the beaver, the organism can be acquired by drinking stream water or even city water in some localities, particularly in the Rocky Mountain states. The organism, a flagellated protozoan, adheres to the intestinal mucosa of the small bowel, by means of a ventral sucker, destroying the mucosal cells’ ability to participate in normal secretion and absorption. No evidence indicates invasion or toxin production.

Cryptosporidia and Isospora spp., parasitic etiologic agents of diarrhea in animals and poultry and more recently recognized as causing human disease, probably also act by adhering to intestinal mucosa and disrupting function. Cryptosporidia are often seen in the diarrhea of patients with acquired immunodeficiency syndrome (AIDS), as well as in travelers’ diarrhea, day care epidemics, and diarrhea in people with animal exposure. Cryptosporidia and Isospora spp. may cause severe, protracted diarrhea in AIDS patients. Other coccidian parasites, such as microsporidia, produce diarrhea by destroying intestinal cell function.

Invasion.

Following initial and essential adherence to GI mucosal cells, some enteric pathogens are able to gain access to the intracellular environment. Invasion allows the organism to reach deeper tissues, access nutrients for growth, and possibly avoid the host immune system.

In the case of diarrhea caused by Shigella, the primary mechanism of disease production consists of (1) the triggering and directing by Shigella entry into colonic epithelial cells by genes located on a plasmid, and once internalized, (2) the rapid multiplication of Shigella in the submucosa and lamina propria and its intracellular and extracellular spread to other adjacent colonic epithelial cells. Once in the host cell cytoplasm, Shigella spp. cause apoptosis and release of the cytokines interleukin (IL)-1 and IL-8. The inflammatory response to these cytokines damages the colonic mucosa and exacerbates (aggravates) the infection. The genes for invasiveness are located on a large invasion plasmid. These activities lead to extensive superficial tissue destruction. If these two steps do not occur, one does not get the clinical presentation of classic dysentery (Table 75-3). The entry process is illustrated in Figure 75-5.

Miscellaneous Virulence Factors.

Other virulence traits appear to be involved in the development of GI infections and include characteristics such as motility, chemotaxis, and mucinase production. Also, the possession of certain antigens, such as the Vi antigen of Salmonella typhi and certain cell wall components, are also associated with virulence.

Clinical Manifestations

The clinical symptoms experienced by a patient are largely dependent on how the enteric pathogen causes disease. To illustrate, patients infected with an enteric pathogen that upsets fluid and electrolyte balance have no fecal leukocytes present in the stool and complain of watery diarrhea; fever is usually absent or mild. Although nausea, vomiting, and abdominal pain may also be present, the dominant feature is intestinal fluid loss. In contrast, patients infected with an enteric pathogen that causes significant cell destruction and inflammation have fecal leukocytes present in the stool (Figure 75-6). Their diarrhea is often characterized by the presence of mucus and blood; in many of these patients, fever is a prominent component of their disease, as well as abdominal pain, cramps, and tenesmus. Finally, patients who become infected with a pathogen capable of penetrating the intestinal mucosa of the small intestine without producing enterocolitis and then subsequently spreading and multiplying at other sites will present with signs and symptoms of a systemic illness such as headache, sore throat, malaise, and fever; diarrhea in these patients is not a prominent feature and is absent or mild in many cases. Features of these three types of enteric infections are summarized in Table 75-3.

Epidemiology

Gastrointestinal infections occur in numerous epidemiologic settings. Awareness of these different settings is important because knowledge of a particular epidemiologic setting can help provide a basis for the diagnosis and clues to possible etiologies. When this knowledge is combined with clinical findings, the etiology of the infection can often be narrowed to three or four organisms.

Etiologic Agents

Many microorganisms are able to cause enteric infections. A discussion of each organism is beyond the scope of this chapter. Rather, these organisms are addressed in Parts III through VI of the textbook. Table 75-4 summarizes the general characteristics of the more common agents of enteric infections.

Other Infections of the Gastrointestinal Tract

Besides causing disease in the small and large intestine, microorganisms can also infect other sites of the GI tract, as well as the GI tract’s accessory organs.

If enteric pathogens are to be detected by the laboratory, adherence to appropriate guidelines for specimen collection and transport is imperative (see Table 5-1 for a quick guide to specimen collection, transport, and processing). If an etiologic agent is not isolated with the first culture or visual examination, two additional specimens should be submitted to the laboratory over the next few days. Because organisms may be shed intermittently, collection of specimens at different times over several days enhances recovery. Certain infectious agents, such as Giardia, may be difficult to detect, requiring the processing of multiple specimens over weeks, duodenal aspirates (in the case of Giardia), or additional alternative methods.

Culture of Fecal Material for Isolation of Etiologic Agents

Bacteria

Fecal specimens for culture should be inoculated to several media for maximal yield, including solid agar and broth. The choice of media is arbitrary and based on the particular requirements of the clinician and the laboratory. Recommendations for selection of media are included in this section.

Organisms for Routine Culture.

Stools received for routine culture in most clinical laboratories in the United States should be examined for the presence of Campylobacter, Salmonella, and Shigella spp. under all circumstances. Detection of Aeromonas and Plesiomonas spp. should be incorporated into routine stool culture procedures. The cost of doing a stool examination on every patient for all potential enteric pathogens is prohibitive. The decision as to what other bacteria are routinely cultured should take into account the incidence of GI tract infections caused by particular etiologic agents in the area served by the laboratory. For example, if the incidence of Yersinia enterocolitica gastroenteritis is high enough in the area served by the laboratory, then this agent should also be sought routinely. Similarly, because of the increasing prevalence of disease caused by Vibrio spp. in individuals living in high-risk areas of the United States (sea coast), laboratories in these localities may routinely look for these organisms. Conversely, unless a patient has a significant travel history, a laboratory located in the Midwestern United States should not routinely look for these organisms except by special request. Protocols for culture of enterohemorrhagic E. coli (e.g., E. coli O157:H7) vary greatly; based on incidence of disease, laboratories routinely culture for this organism when cases of severe diarrhea are implicated. Selective or screening media to detect E. coli O157:H7 also vary greatly, including using a 1% sorbitol-containing medium (most O157:H7 E. coli are sorbitol-negative), a specific trypticase blood agar (Unipath GmbH, Wesel, Germany), RambaCHROM (Gibson Laboratories, LLC, Lexington, Ky), CHROMagar (BD Diagnostics, Franklin Lakes, N.J.) or Rainbow Agar O157 (Bio-log, Inc., Hayward, California).

Routine Culture Methods.

An in-depth discussion regarding culture of all enteric pathogens is beyond the scope of this chapter. Because U.S. laboratories should routinely examine stools for the presence of salmonella, Shigella, and Campylobacter spp., culture of these organisms is addressed. Culture conditions for all other pathogens, including viruses, are covered in Parts III, IV, and VI. Specimens received for detection of the most frequently isolated Enterobacteriaceae and Salmonella and Shigella spp. should be plated to a supportive medium, a slightly selective and differential medium, and a moderately selective medium.

Blood agar (tryptic soy agar with 5% sheep blood) is an excellent general supportive medium. Blood agar medium allows growth of yeast species, staphylococci, and enterococci, in addition to gram-negative bacilli. The absence of normal gram-negative fecal flora or the presence of significant quantities of organisms such as Staphylococcus aureus, yeasts, and Pseudomonas aeruginosa can be evaluated. The use of blood agar also provides colonies for oxidase testing. Several colonies that do not resemble Pseudomonas from the third or fourth quadrant should be routinely screened for production of cytochrome oxidase. If numerous colonies are present, Aeromonas, Vibrio, or Plesiomonas spp. should be suspected.

The slightly selective agar should support growth of most Enterobacteriaceae, vibrios, and other possible pathogens; MacConkey agar works well. Some laboratories use eosin-methylene blue (EMB), which is slightly more inhibitory. All lactose-negative colonies should be tested further, ensuring adequate detection of most vibrios and most pathogenic Enterobacteriaceae. Lactose-positive vibrios (V. vulnificus), pathogenic E. coli, some Aeromonas spp., and Plesiomonas spp. may not be distinctive on MacConkey agar.

Salmonella/Shigella.

The specimen should also be inoculated to a moderately selective agar such as Hektoen enteric (HE) or xylose-lysine desoxycholate (XLD) media. These media inhibit growth of most Enterobacteriaceae, allowing Salmonella and Shigella spp. to be detected. Colony morphologies of lactose-negative, lactose-positive, and H₂S-producing organisms are illustrated in Figure 75-7. Other highly selective enteric media, such as salmonella-shigella, bismuth sulfite, deoxycholate, or brilliant green, may inhibit some strains of Salmonella or Shigella. All these media are incubated at 35° to 37° C in ambient air and examined at 24 and 48 hours for suspicious colonies.

Campylobacter.

Cultures for isolation of Campylobacter jejuni and Campylobacter coli should be inoculated to a selective agar containing antimicrobial agents that suppress the growth of normal flora. The introduction of a blood-free, charcoal-containing medium containing selective antibiotic components has improved recovery of most enteropathogenic Campylobacter spp. Brucella broth base has yielded less satisfactory recovery of Campylobacter spp. Commercially produced agar plates for isolation of campylobacters are available from several manufacturers. These plates are incubated in a microaerophilic atmosphere at 42° C and examined at 24 and 48 hours for suspicious colonies. Culture methods for other campylobacters associated with GI disease, such as C. hyointestinalis and C. fetus subsp. fetus, are provided in Chapter 34.

Enrichment Broths.

Enrichment broths are sometimes used for enhanced recovery of Salmonella, Shigella, Campylobacter, and Y. enterocolitica, although Shigella usually does not survive enrichment. Gram-negative broth (Hajna GN) or selenite F broth yields good recovery. Enrichment broths for Enterobacteriaceae should be incubated in air at 35° C for 6 to 8 hours and then several drops should be subcultured to at least two selective media. A commercial system that allows broth to be tested for antigen of Salmonella or Shigella directly has been described; however, the reported sensitivity is lower than desired. Stool would be inoculated to broth initially; those broths that test negative could be discarded without subculturing. Campy-thioglycollate enrichment broth increases the yields of positive cultures for Campylobacter spp., although it is not necessary for routine use. Enrichment broth for Campylobacter is refrigerated overnight or for a minimum of 8 hours before a few drops are plated to Campylobacter agar and incubated at 42° C in a microaerophilic atmosphere.