Enteropathogenic bacteria

Enteric Infections are caused by a variety of microorganisms, including bacterial pathogens. Among these infections, diarrheal diseases are a major cause of mortality in the children of Third World countries, due to malnutrition, poor personal hygiene, and insufficient environmental sanitation. In industrialized countries, diarrhea may result from foodborne outbreaks and is common in day care centers, hospitals, and chronic care institutions, among homosexual men and immunocompromised patients. Diarrhea can result from inflammatory infections in the colon and/or small intestine caused by pathogens, such as Shigella, Salmonella, and Campylobacter, or noninflammatory infections in the small intestine by pathogens such as Vibrio and enterotoxigenic Escherichia coli. Diarrhea can also result from ingestion of preformed toxins, produced by bacteria such as Clostridium perfringens and Bacillus cereus. In addition, infections by enteropathogenic bacteria may cause systemic syndromes, such as typhoid fever caused by Salmonella typhi. Another form of enteric infection, gastritis, results exclusively from infections with Helicobacter pylori. We will briefly review the epidemiology, pathogenesis, and clinical features of the most important bacterial enteric pathogens of humans.  

I. BACTERIAL AGENTS OF INFLAMMATORY DIARRHEA 

In the majority of cases, inflammatory diarrhea in the distal small bowel and colon occurs in response to bacterial invasion of the intestinal tissues (Table 34.1). However, in some infections, enterocolitis can be an outcome of bacterial toxicity without invasion. Causative agents of these infections include the following. A. Shigella spp. Shigella, the major etiologic agent of bacillary dysentery, is traditionally divided into four species based on biochemical and serological characteristics. These species include S. dysenteriae, S. flexneri, S. sonnei, and S. boydii. However, techniques such as multilocus enzyme electrophoresis have revealed that all Shigella strains are actually encompassed within the species E. coli. Clinical syndromes of shigellosis include a mild watery diarrhea, which is often followed by severe dysentery with blood, mucus, and inflammatory cells in feces. The incubation period ranges from 6 h to 5 days. Epidemiological studies show that Shigella is transmitted by the fecal–oral route or by contaminated food and water. As few as 100 organisms can cause infection in an adult. Shigella invades the colonic mucosa and this invasion involves entry and intercellular dissemination. Bacteria enter the cells by a micropinocytic process, which requires polymerization of actin at the site of entry. Shortly after entry, bacteria lyse the phagocytic vacuole and move into the cytoplasm where they multiply. Within the cytoplasm, Shigella recruits actin microfilaments at one pole of the bacterium, which leads to the formation of a polymerized actin tail behind the bacterium and, consequently, movement of the bacteria (Fig. 34.1). 

The movement of the bacteria leads to the formation of cell membrane protrusions that extend from one cell into the adjacent cell and allow dissemination of bacteria without their release into the extracellular environment. The ability of Shigella to spread from cell to cell is measured in vitro by the formation of plaques on a confluent cell monolayer (plaque assay) and in vivo by formation of keratoconjunctivitis in guinea pigs (Sereny test). All of the genes required for Shigella invasion are carried on a 200-kb virulence plasmid. A 30-kb fragment contains the genes that encode secreted proteins called IpaA, B, C, and D, the chaperones for these proteins called Ipgs, and a specialized type III secretion system for these proteins called Mxi–Spa. Several other proteins that might be important in the entry process are also secreted by the Mxi–Spa machinery. This machinery becomes activated upon contact of bacteria with the host epithelial cells. The icsA (virG) gene, which confers the ability of Shigella to spread from cell to cell, is located approximately 40 kb away from the ipa–ipg–mxi–spa region. Following secretion, IpaB and IpaC form a complex, which triggers recruitment of actin at the site of bacterial entry by a mechanism that involves the small host G protein, Rho. IpaA contributes to the entry process by interacting with other cytoskeletal proteins, such as vinculin and _-actinin. Contact with macrophages results in delivery of IpaB into the cytoplasm of macrophages where IpaB induces programmed cell death and release of interleukin-1. Release of this cytokine triggers a cascade of proinflammatory responses, which opens intercellular junctions and destabilizes the epithelia, thus facilitating bacterial invasion and ulceration of the colon. Infections due to S. dysenteriae are more severe and more likely to lead to complications than are infections with other Shigella species. 

S. dysenteriae produces a lethal cytotoxin, called Shiga toxin. This toxin is composed of an enzymatically active Asubunit and five B subunits that mediate binding of the toxin to target cell receptors. The toxin functions by cleaving 28S rRNA of eukaryotic cells and inhibiting protein synthesis. Shiga toxin may play a role in manifestations of hemolytic uremic syndrome, an occasional consequence of shigellosis. B. Enteroinvasive E. coli E. coli is commonly regarded as a harmless commensal of the intestines of humans. However, at least six varieties have been identified that possess specific pathogenic mechanisms allowing them to cause diarrhea. These pathogens include enteroinvasive E. coli (EIEC). EIEC is closely related to Shigella spp. in biochemical and serological characteristics, pathogenic mechanisms, and virulence determinants (see preceding) (Tables 34.2 and 34.3). EIEC is identified as E. coli based on its biochemical profile but is distinguished from other strains of E. coli based on genotypic and phenotypic characteristics of Shigella spp. Therefore, the Sereny test and plaque assay are appropriate tests for identification of EIEC. Like Shigella spp., EIEC is transmitted by contaminated food and water. 

The infectious dose, however, is 2–3 logs higher than that for Shigella infection. Therefore, transmission from person to person is likely less than is the case for Shigella. Infection with EIEC leads to watery diarrhea, with dysentery syndrome in only some of the patients. C. Nontyphoidal Salmonella Salmonella spp. other than Salmonella typhi are the cause of salmonellosis in humans. There are over 2000 serotypes of Salmonella that infect a wide range of hosts, from humans to domestic animals, birds, reptiles, and insects. Most of the Salmonella serotypes associated with human infections belong to subgroup 1 of the six subgroups of Salmonella enterica. Many serotypes are species-specific; thus, a particular serotype may be nonpathogenic in one host species and cause severe infection in another. Gastroenteritis is the most common manifestation of Salmonella infections. Diarrhea begins 8–48 h after ingestion of contaminated food and lasts for 3–7 days. Infection is often food-borne and associated with consumption of foods of animal origin, such as chicken, raw milk, and undercooked eggs. The infectious dose ranges from 105 to 1010 organisms and depends on serotype, source of infection, and host factors. Foodborne salmonellosis seems to be predominantly a disease of industrialized countries. Infections can also be transmitted by the fecal–oral route, particularly among homosexual men. Immunocompromised hosts, such as HIV-infected individuals, are more prone to Salmonella infections. In these patients, nontyphoidal Salmonella usually causes bacteremia. Salmonella spp. invade mucosal cells of the small intestine by a bacterial-mediated endocytosis process similar to Shigella entry. Bacteria stimulate signal transduction pathways in epithelial cells that lead to cytoskeletal rearrangements and membrane ruffling, similar to those induced by growth factors on mammalian cells. 

Membrane ruffling results in uptake of bacteria into cells. Unlike Shigella, Salmonella remains in membrane-bound vacuoles, modifies the pH of the phagolysosome, and multiplies. The organisms can also be taken up by macrophages, where they multiply and penetrate into deeper tissues (see the section following on S. typhi). Most of the research on the molecular genetic basis of Salmonella invasion has been done on S. typhimurium. The genes that confer the ability of Salmonella to invade are located on a 40-kb pathogenicity island, called SPI-1, at the 63 min of S. typhimurium chromosome. SPI-1 encodes the components of a type III secretion pathway, called Inv–Spa, and the Sip proteins secreted via this machinery. Sip proteins have functions similar to Shigella Ipa proteins in the process of entry and induction of proinflammatory response and tissue destruction (see preceding section on Shigella). Lipopolysaccharide (LPS) is another factor involved in invasiveness of Salmonella. Rough mutants with short O-side chains are less virulent. Salmonella also produces several adhesins, which might facilitate attachment to epithelial cells prior to penetration. In addition, nontyphi Salmonella strains produce an enterotoxin that has cytoskeleton-altering activity. Salmonella strains induce secretion of cytokines such as IL-8 from epithelial cells. IL-8 is a chemoattractant for polymorphonuclear leukocytes (PMNs) and stimulates transmigration of PMNs through epithelial cell tight junctions into the intestinal lumen. The passage of PMNs through cellular junctions can lead to fluid leakage and consequent diarrhea. D. Campylobacter spp. Campylobacters are slender, spirally curved gramnegative rods, carrying a relatively small (1.6_106 bp) AT-rich genome that has been already sequenced. These organisms are microaerophilic, thermophilic, and require complex media for growth. Campylobacters are one of the most commonly reported causes of diarrhea worldwide. Infections with these organisms usually result in inflammatory dysentery-like diarrhea in adults of industrialized nations and watery diarrhea in children of developing countries. Campylobacters can also cause systemic disease. C. jejuni and C. fetus are the prototypes for diarrheal and systemic infections, respectively. 

Campylobacter spp. are found in the gastrointestinal tracts of most domesticated mammals and fowls. Transmission occurs by contaminated food or water or by oral contact with feces of infected animals or humans. The organism cannot tolerate drying or freezing, thus being limited in transmission. Infections occur all year long, with a sharp peak in summer. The infectious dose can be as small as 500 or as high as 109 organisms, depending on the source of infection. The incubation period ranges from one to seven days and the duration of the illness is usually one week, with occasional relapses in untreated patients. Campylobacter jejuni causes inflammation in both the colon and the small intestine. The resulting nonspecific colitis can be mistaken for acute ulcerative colitis. The inflammatory process can be extended to the appendix, mesenteric lymph nodes, and gall bladder. Bacteremia can also occur in some cases. Infections with C. jejuni may be followed by noninfectious complications, such as Guillain–Barre syndrome (GBS), an acute disease of peripheral nerves, and reactive arthritis. Inflammation and bacteremia caused by C. jejuni suggest tissue invasion by this organism. Invasion seems to be linked to the presence of flagellae. Flagellae enable the organisms to move along the viscous environments and penetrate the intestinal mucosa. Binding of C. jejuni to host epithelial cells leads to bacterial uptake by a complex mechanism that remains controversial. Bacteria also penetrate the underlying lymphoid tissue and survive within the macrophages. Campylobacter jejuni strains also produce several toxins. These toxins include a heat-labile cholera-like enterotoxin (CLT), which correlates with watery diarrhea, a cytolethal distending cytotoxin (CLDT), which alters the host cytoskeleton, and a hemolysin(s). Campylobacter fetus causes febrile systemic illness more often than diarrheal infections. This organism has a tropism for vascular sites, thus causing bacteremia. The organism uses lipopolysaccharide (LPS) and a surface (S) layer protein, which functions as a capsule, to resist phagocytosis and serum-killing. C. fetus can disseminate to cause meningoencephalitis, lung abscess, septic arthritis, and urinary tract infections. E. Clostridium difficile Clostridium difficile is a gram-positive anaerobic sporeforming bacillus. This organism is widespread in the environment and is found in the intestines of several mammals, including humans. C. difficile is the most common recognized cause of diarrhea in hospitals and chronic care facilities in developed countries. 

Antibiotic therapy disrupts the normal flora of the colon and allows colonization or proliferation of C. difficile. Infection occurs by ingestion of spores from the environment. The spores resist the gastric acid and germinate into the vegetative form in the colon. The symptoms of disease range from mild diarrhea to severe pseudomembranous colitis. Infection with C. difficile is more common in the elderly, whereas neonates are resistant to colonization by this organism. Pathogenic strains of C. difficile produce two very large toxins: toxin Aand toxin B. Toxin Ais a cytotoxin with a molecular weight of 308 kDa. This toxin stimulates cytokine production by macrophages and infiltration of PMNs, which results in the inflammation seen in pseudomembranous colitis. Toxin B is a protein of 207 kDa that also possesses cytotoxic activity. This toxin has glucosyltransferase activity that catalyzes transfer of glucose to the small GTP-binding protein, Rho. Modification of Rho leads to actin cytoskeletal disruption, which results in rounding of the cells. C. difficile also produces hydrolytic enzymes, such as hyaluronidase, gelatinase, and collagenase, which might contribute to destruction of connective tissue and subsequent fluid accumulation. 

II. BACTERIAL AGENTS OF NONINFLAMMATORY DIARRHEA 

The noninflammatory watery diarrhea caused by bacteria is usually associated with the production of an enterotoxin after bacterial colonization of the small intestine or with the presence of preformed enterotoxins in food. Occasionally, bacteria can cause a drastic effect on intestinal epithelial cells in the absence of an enterotoxin. This category of diarrhea is caused by the following. A. Vibrio cholerae Vibrio cholerae belongs to the family Vibrionaceae and has been the cause of seven cholera pandemics since 1817. It is transmitted by contaminated food and water. Food-borne transmission often occurs by ingestion of raw or undercooked shellfish. Since the acidsensitive bacteria must pass through the stomach to colonize the small intestine, a high inoculum of 109 organisms is required to cause disease. The diarrhea can be extremely severe, with characteristic “rice water” stools, which can lead to rapid dehydration, circulatory collapse, and death. Cholera is caused by toxigenic strains of V. cholerae O1 and O139 Bengal. The O1 strains can be divided into E1 Tor and classical biotypes that are epidemiologically distinct. V. cholerae O139 is a new strain that caused a major epidemic in 1992 in India in a population which was already immune to V. cholerae O1 strains. The non-O1 serogroups of V. cholerae cause cholera and dysentery but have not been linked to cholera epidemics. Vibrio cholerae secretes cholera toxin (CT), which is responsible for the characteristic secretory diarrhea. CT is an enterotoxin that binds to enterocytes via five B subunits that facilitate the entry of the enzymatically active A subunit. The A subunit then catalyzes the ADP-ribosylation of the GTP binding protein, Gs_, which results in activation of adenylate cyclase, accumulation of cAMP in enterocytes, and increase in secretion of chloride and water. Increased release of water into the intestinal lumen leads to secretory diarrhea. CT is encoded by the ctxAB genes carried on a filamentous bacteriophage. The receptor for the phage is a type IV fimbria, called the toxin-coregulated pilus (TCP), which is an essential colonization factor of V. cholerae. The gene encoding TCP is located on a 40-kb pathogenicity island (PI). This PI is associated with pandemic and epidemic strains of V. cholerae. Vibrio cholerae O1 also produces two other toxins, called the zonula occludens toxin (Zot) and the accessory cholera enterotoxin (Ace). Zot affects the structure of the intercellular tight junction, zonula occludens. 

Ace is postulated to form ion-permeable channels in the host cellular membrane. The role of these toxins in pathogenesis is unknown. B. Enteropathogenic E. coli Enteropathogenic E. coli (EPEC) is an important cause of diarrhea in infants less than 2 years of age. EPEC is transmitted by the fecal–oral route by person-to-person contact. The infection occurs more frequently during the warm seasons. Infection with EPEC is often severe and leads to a high mortality rate in developing countries. The symptoms of the disease include watery diarrhea, vomiting, and fever. All EPEC strains induce a characteristic attaching and effacing (A/E) lesion on the brush border of the intestine which can be mimicked in tissue culture (Fig. 34.2). Pedestal-like structures form beneath the intimately adhering bacteria, due to the polymerization of actin. The accumulation of actin beneath the bacteria can be detected by a fluorescent-actin staining (FAS) assay. In addition, EPEC adheres to epithelial cells in tissue culture in a localized pattern, which can be detected by light microscopy. Intimate attachment of EPEC to epithelial cells is mediated by an adhesin called intimin, which is encoded by the eae gene located on a 35-kb pathogenicity island, called the locus of enterocyte effacement (LEE). The LEE also encodes a type III secretion system called Esc, several proteins secreted by this secretion system called EspA, B, D, and F, and Tir. Tir becomes localized to the host cell membrane, where it serves as a receptor for intimin. The formation of the A/E lesions requires the Esc proteins, EspA, B, D, Tir, and intimin. The mechanisms that lead to diarrhea are unknown but may be related to changes in ion secretion and intestinal barrier function that have been detected in vitro and/or to loss of microvilli. Localized adherence of EPEC to epithelial cells is dependent on the presence of a 90-kb plasmid, which carries the genes required for the biogenesis of type IV fimbriae, called bundle-forming pili (BFP). 

BFP form ropelike structures (Fig. 34.3) that are responsible for the aggregation of EPEC bacteria to each other and for the localized adherence of EPEC to host epithelial cells. In addition to BFP, some EPEC strains produce other types of fimbriae that may also contribute to localized adherence. Some EPEC strains also produce a low molecular heat stable toxin called EAST1, similar to EAST1 of enteroaggregative E. coli, but the importance of this toxin in disease is unknown. C. Enterotoxigenic E. coli Enterotoxigenic E. coli (ETEC) is a cause of infantile and childhood diarrhea in developing countries and travelers’ diarrhea in adults of industrialized countries visiting ETEC-endemic areas. Infection in travelers results in mild watery diarrhea in the majority of cases, whereas in infants from endemic areas, it can cause more severe diarrhea. ETEC has a short incubation period of 14–50 h. Epidemiological studies indicate that ETEC infection is more common in warm seasons and is transmitted through fecally contaminated food and water. The infectious dose for ETEC is approximately 108. ETEC colonizes the mucosal epithelial cells of the small intestine and produces at least one of the two enterotoxins known as heat-labile toxin (LT) and heat-stable toxin (ST); both are encoded on a plasmid. LT is similar to cholera toxin (CT) in structure, function, and mode of action (see preceding). ST is a small polypeptide that activates intestinal guanylate cyclase, leading to accumulation of GMP, secretion of chloride and water, and, thus, diarrhea. ST resembles a peptide, guanilyn, normally found in the intestinal epithelium.  ETEC strains produce multiple fimbriae that are host species-specific. Human ETEC fimbriae, called colonization factor antigens (CFAs), are associated with specific O serogroups. These fimbriae exhibit different morphological features, such as rigid rods similar to common fimbriae, bundle-forming flexible rods, and thin wavy filaments. In addition, many human ETEC strains produce a type IV fimbria, called Longus. D. Enterohemorrhagic E. coli Enterohemorrhagic E. coli (EHEC) is an emerging enteropathogen, which can cause watery diarrhea followed by bloody diarrhea, an illness designated hemorrhagic colitis (HC). EHEC is also associated with severe cases of hemolytic uremic syndrome (HUS), with a mortality rate of 5–10% The reservoir for this organism is the intestinal tract of cattle and, therefore, undercooked contaminated beef is the major source of infection. 

Contaminated milk, juice, lettuce, sprouts, and fast food have also caused outbreaks. Since the infectious dose is as low as 50–200 organisms, it is not surprising that EHEC is also spread by direct personto- person contact. The most important EHEC serotype, O157:H7, has been the cause of several food-borne outbreaks in the United States, Canada, Japan, and Europe since the 1980s. The mortality related to HUS has brought public attention to EHEC infections and has been the impetus for new regulations for handling and cooking of beef products. The major pathogenic feature of EHEC is production of the bacteriophage-encoded Shiga toxin (or verotoxin), that is closely related to the Shiga toxin of S. dysenteriae. HUS is thought to be the result of hematogenous dissemination of Shiga toxin, cytotoxicity to endothelial cells, and microscopic thrombosis in the kidneys and elsewhere. Shiga toxins are also essential for development of bloody diarrhea and hemorrhagic colitis. Many EHEC strains also produce EAST1 toxin, similar to the toxin of enteroaggregative E. coli. Similar to EPEC, EHEC strains also possess the LEE pathogenicity island on the chromosome (see preceding) and exhibit classic A/E histopathology. The A/E phenomenon is thought to be responsible for development of watery diarrhea, in a manner similar to EPEC diarrhea. E. Enteroaggregative E. coli Enteroaggregative E. coli (EAEC) is a cause of persistent diarrhea in children of developing and developed countries. EAEC infects the small bowel and causes diarrhea in less than 8 h, which may persist for _14 days. The diarrhea is usually mucoid and may be watery, with low-grade fever. EAEC has also been associated with diarrhea in human immunodeficiency virus-infected patients. More importantly, colonization with EAEC is linked to growth retardation in children, independent of the symptoms of diarrhea. 

EAEC induces the formation of a mucous biofilm, which can trap and may protect the bacteria, leading to persistent colonization and diarrhea. Bacteria also elicit a cytotoxic effect on the intestinal mucosa. The cytotoxic effect is mediated by genes present on a 100-kb plasmid. EAEC exhibits an aggregative adherence phenotype, mediated in some strains by a flexible bundleforming fimbria called Aggregative Adherence Fimbriae I (AAF/I). AAF/I is a member of the Dr family of adhesins, present in uropathogenic E. coli. EAEC strains also produce a ST-like toxin, EAST1, which is linked to the AAF/1 gene cluster on the 100-kb plasmid. The role of EAST1 in diarrhea is unknown, In addition, EAEC strains produce a 108-kDa cytotoxin that belongs to the autotransporter family of proteins. This protein also exhibits enterotoxin activity. F. Bacillus cereus Bacillus cereus is a gram-positive spore-forming rod that resides in water, soil, and as part of the normal flora in humans. This organism produces several toxins and causes two forms of toxin-mediated food poisoning, one characterized by emesis and the other by diarrhea. The production of either the emesis or diarrheal toxin is dependent on the type of the food on which the bacteria grow. The emetic toxin is a small toxin that is resistant to heat, extreme pH, and proteolytic enzymes. The toxin acts on the enteric nervous system through unknown mechanisms. The emetic toxin is associated with fried rice in the majority of cases. The emetic form of the disease has an incubation period of 2–3 h and elicits symptoms of vomiting and abdominal cramps that last 8–10 h. The diarrheal toxin is a secretory cytotoxin consisting of a two- to three-component protein complex. This toxin induces secretion in the rabbit-ligated ileal loop and is cytotoxic in tissue culture. The mechanism of action of this toxin is unknown. The diarrheal toxin is associated with a variety of foods, such as sausage, vanilla sauce, and puddings. The incubation period for the diarrheal form ranges from 6 to 14 h. 

The illness is characterized by diarrhea and abdominal cramps, which may last 20–36 h.  G. Staphylococcus aureus Staphylococcus aureus is a gram-positive coccus that is among the most common causes of bacterial foodborne disease. Food poisoning usually occurs by contamination of food with infected wounds on the hands, from the normal flora of skin or from the respiratory tract of food handlers. Foods such as ham and custard, which have a high concentration of salt or sugar, provide a good growth medium for S. aureus. One to 6h after ingestion of the contaminated food, the symptoms of food poisoning begin, with severe vomiting and abdominal pain followed by diarrhea, which may last 24–48 h. Food poisoning with S. aureus results from ingestion of the small enterotoxins A, B, C, D, or E, which are superantigens. Enterotoxin A is the most common one associated with food poisoning. These toxins act on enteric nervous system through unknown mechanisms. As little as 100–200 ng of the toxins can cause food poisoning. The S. aureus toxins are resistant to heat, irradiation, pH extremes, and proteolytic enzymes. Therefore, even overcooking of the contaminated food does not prevent the food poisoning. Involvement of S. aureus in outbreaks of food-borne disease is confirmed by detection of enterotoxins in food and by phage typing. H. Clostridium perfringens Clostridium perfringens is a gram-positive spore-forming organism that can tolerate aerobic conditions, unlike other members of Clostridia. C. perfringens type A exists in soil and in the intestinal tracts of most animals. This organism can cause a relatively mild food poisoning, more frequently in winter. Diarrhea and abdominal cramps develop 6–24h following ingestion of preformed toxin and last up to 24 h. Toxigenic strains of C. perfringens usually grow on foods such as meat and poultry at temperatures between 15 and 50 _C, with a doubling time as short as 10 min. The spores are heatresistant and can survive cooking and germinate after cooling. C. perfringens type A produces a 35-kDa cytotoxin called C. perfringens enterotoxin (CPE) during sporulation. CPE binds irreversibly to cells and forms ion-permeable channels in intestinal epithelial cells and acts as a superantigen that reacts with human T cells. 

Clostridium perfringens can also cause non-foodpoisoning diarrhea. The diarrhea is more severe, with blood and mucus in feces, and lasts longer. The disease occurs predominantly in the elderly or results from antibiotic therapy, similar to the cases with C. difficile. Infections with C. perfringens type C can lead to necrotizing enteritis known as “pig-bel,” a syndrome described in New Guinea related to consumption of large undercooked pork meals in native feasts. The symptoms of pig-bel include severe abdominal pain, bloody diarrhea, vomiting, and death due to intestinal perforation. The symptoms of pig-bel are associated with a toxin called _ toxin. _ Toxin is a cytotoxin with an unknown mechanism of action. This toxin is usually inactivated by proteolytic enzymes in the intestine. However, lack of proteolytic enzymes in malnourished hosts or inhibition of these enzymes by certain foods, such as sweet potato, allows the activity of _ toxin and subsequent necrotizing enteritis. 

III. BACTERIAL AGENTS OF ENTERIC FEVER 

A. Salmonella typhi Typhoid fever is a severe systemic disease caused by S. typhi. The disease is characterized by fever and abdominal symptoms. Infection can be transmitted by consumption of water or food contaminated with the feces of a patient or a chronic carrier. Humans are the only known reservoir for this organism, making the studies of typhoid infection difficult. However, S. typhimurium, which normally causes gastroenteritis in humans, causes a disease similar to typhoid in mice. Therefore, most of the studies of typhoid have focused on infection of mice and mouse macrophages with S. typhimurium. However, it is not clear that all of the conclusions from studies of S. typhimurium in mice apply to S. typhi in humans. Following oral inoculation in mice, S. typhimurium survives the gastric acid barrier and reaches M cells, specialized epithelial cells that cover lymphoid tissues of the small intestine. Bacteria use M cells to penetrate the intestinal mucosa, whereupon they are engulfed by macrophages. Within macrophages, Salmonella attenuates the acidification process and multiplies. Survival within macrophages results in spread of the organisms and systemic infection. Bacteria enter the blood through the thoracic duct. Finally, bacteria are taken up by tissue macrophages in the bone marrow, liver and spleen. The ability to multiply in macrophages and cause systemic infections is encoded by genes that reside on a second pathogenicity island, SPI-2, at 30 min on the S. typhimurium chromosome. (See preceding section on nontyphoidal Salmonella about SPI-I.) In addition to the products of these genes, intramacrophage survival is also modulated by the PhoP/PhoQ twocomponent regulatory system. These proteins regulate acid phosphatase synthesis and unknown genes, which are essential for survival in the acidic environment of the macrophage. B. Yersinia spp. 

Within the genus Yersinia, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica are pathogenic for humans. Based on DNA hybridization techniques, Y. enterocolitica has recently been subdivided into eight other species. Y. pestis is the cause of bubonic plague, the “Black Death,” which claimed one-fourth of Europe’s population in the fourteenth century. Y. pseudotuberculosis and Y. enterocolitica are the causes of yersiniosis, a disease more prevalent in developed countries. Yersiniosis is characterized by an enteric feverlike illness, which is accompanied by acute diarrhea. Mesenteric adenitis is a common manifestation of the disease, which causes an acute appendicitis-like syndrome, with fever and abdominal pain. Extra-intestinal manifestations can also include septicemia and nonpurulent arthritis. Infections with Y. pseudotuberculosis are more common in animals and less frequent in humans. Y. enterocolitica is carried by healthy pigs but is pathogenic for humans. It is transmitted by ingestion of contaminated water or food, more commonly, contaminated milk. The organisms can multiply at low temperatures, such as those of refrigerated food. The infectious inoculum may be 109 organisms and the incubation period may last 4–7 days. Following ingestion of Yersinia, bacteria adhere to and enter the intestinal epithelial cells. Infection then spreads to the mesenteric lymph nodes, where abscesses develop. Adherence of Yersinia to host cells is mediated by a plasmid-encoded adhesin, called YadA, and a chromosomally encoded protein, called Invasin. Invasin mediates entry into cells by interacting with _1 integrin receptors. This interaction leads to the extension of a pseudopod that forms a “zipper” around the bacterium, resulting in internalization. At 37 _C under low calcium conditions in vitro, Yersinia spp. secrete a set of proteins called Yops. Yops are virulence factors that enable bacteria to survive and multiply within lymphoid tissues of the host. The genes encoding Yops reside on a 70-kb virulence plasmid. Yops are secreted by a type III secretion system called Ysc and the secretion is regulated by temperature and contact with eukaryotic cells. Upon contact of bacteria with host cells, Yops are synthesized, secreted, and some are injected into the cytoplasm of host cells. Following injection, YopE and YopT act as cytotoxins that disrupt the actin microfilament structure. YopH is a protein tyrosine phosphatase that dephosphorylates certain proteins of macrophages. These Yops together inhibit phagocytosis by macrophages. YopP (YopJ) induces apoptosis of macrophages, which results in release of proinflammatory cytokines and subsequent inflammatory responses to infection. Yersincia enterocolitica also produces an enterotoxin similar to heat-stable toxin of E. coli, called Yst. This enterotoxin might be responsible for cases of food poisoning caused by this organism. 

IV. BACTERIAL AGENTS OF GASTRITIS 

A. Helicobacter pylori Helicobacter pylori is a spiral, microaerophilic gramnegative bacterium with two to six polar sheathed flagellae that endow the bacterium with a corkscrew mode of motility. It has a relatively small genome of 1.7 _106bp which is highly AT-rich, similar to that of Campylobacters. The complete sequence of the genome has been determined for two strains. This organism is extremely prevalent, residing in large numbers (108–1010 organisms per stomach) in the stomachs of at least half of the human population. H. pylori probably is not found in the environment or in animals and, therefore, person-to-person contact and the fecal–oral route are the likely means of transmission. Once inoculated, the incubation period is estimated to be 3–7 days and infection can last for the lifetime of the host. H. pylori exhibits tissue specificity exclusively for gastric mucosal epithelial cells and does not invade beyond these tissues. Detection of the organisms is best accomplished by biopsy of stomach tissue and subsequent testing for urease activity, by serologic testing, or by culture. Infections with H. pylori result in acute and chronic gastric inflammation, which, when untreated, can lead to peptic ulcers or stomach carcinoma. The majority of cases of gastric and duodenal ulcers are caused by H. pylori. The outcome of infection with H. pylori depends on a variety of bacterial, host, and environmental factors. Gastric inflammation by H. pylori is mediated by several virulence factors. All H. pylori strains produce urease in very high amounts. Urease is a nickel-containing hexameric enzyme, which catalyzes hydrolysis of urea to ammonia. Ammonia neutralizes gastric acid of the stomach, allowing the organism to colonize. Flagellae are another important colonization factor of this organism, allowing the bacteria to move along the mucous layer of the stomach. Most H. pylori strains produce a vacuolating cytotoxin, called VacA, which is an autotransporter protein. This cytotoxin induces acidic vacuoles in the cytoplasm of eukaryotic cells, Also, the H. pylori strains that are more associated with duodenal ulcer and stomach cancer carry the cytotoxin-associated gene (cag) pathogenicity island. Genes on the cag PI are required for secretion of IL-8 and tyrosine phophorylation of host proteins. V. MISCELLANEOUS BACTERIAL ENTERIC PATHOGENS Thorough coverage of all enteric bacterial pathogens is beyond the scope of this article. However, we briefly describe a few other common enteropathogenic bacteria in the following section: A. Non-cholerae vibrios Vibrios parahaemolyticus, V. vulnificus, V. mimicus, V. fluvialis, V. furnissii, and V. hollisae reside in aquatic environments and prefer high salt and warm temperature habitats. These organisms have been implicated in intestinal infections following consumption of contaminated raw or undercooked shellfish. V. parahaemolyticus and V. vulnificus can also cause wound infections and septicemia. V. parahaemolyticus produces the thermostable direct hemolysin (TDH), which acts as an enterotoxin to stimulate intestinal secretion and subsequent diarrhea. Hemolysin may also be involved in tissue damage observed in wound infection. This hemolysin elicits the Kanagawa phenomenon on Wagatsuma agar, a phenotype specific to pathogenic strains. Vibrios vulnificus is an invasive pathogen. Eating contaminated raw oysters leads to septicemia 24–48 h later, particularly in people with underlying liver disease. This organism produces several proteolytic enzymes and a hemolysin, which contribute to overt damage to epithelial cells in wound infections. V. vulnificus also produces a capsule, which has been associated with virulence. B. Aeromonas spp. Aeromonas spp. are widely distributed in marine environments and can be transmitted to humans via contaminated food, especially during summer. Three species, A. hydrophila, A. sobria, and A. caviae, cause diarrhea in humans. A. hydrophila produces a hemolysin called aerolysin or _-hemolysin, which, in addition to cytolytic activity, acts as an enterotoxin and induces diarrhea. It also produces other enterotoxins and cytoskeleton-altering toxins. In addition, A. hydrophila produces a type IV fimbria that might contribute to virulence. C. Plesiomonas shigelloides Plesiomonas shigelloides has biochemical similarities to Aeromonas and antigenic similarities to Shigella spp. It is primarily a marine microorganism, which can be transmitted to humans by raw or undercooked seafood. Diarrhea, accompanied with relatively severe abdominal cramps, occurs 24 h after ingestion of the organism. The stool may contain mucus, blood, and pus, suggesting an invasive mechanism of the disease. P. shigelloides produces a heat-labile enterotoxin with cytoskeletonaltering activity and a heat-stable enterotoxin with unknown mechanism of action. Plesiomonas shigelloides also causes extraintestinal infections, such as meningitis in neonates, septicemia in immunocompromised hosts, and septic arthritis. 

D. Listeria monocytogenes Listeria monocytogenes is the only species of Listeria that is pathogenic to humans. This organism is a gram-positive rod, which is present in soil, as part of the fecal flora of many animals, and on many foods, such as raw vegetables, raw milk, cheese, fish, meat, and poultry. Diarrhea can occur following ingestion of 109 organisms via contaminated food. The incubation period is long and can last between 11 and 70 days. Infections with L. monocytogenes are uncommon in the normal population, but in immunocompromised patients, neonates, the elderly, and pregnant women, infection can lead to encephalitis, meningitis, and stillbirth. Listeria monocytogenes invades both epithelial cells and phagocytes. Entry involves a “zippering” mechanism, similar to that described for Yersinia entry (see preceding). Interaction between Internalin, a bacterial protein, and E-cadherin, a receptor on epithelial cells, induces phagocytosis. Once inside the phagocytic vacuole, bacteria lyse the vacuolar membrane by a hemolysin called listeriolysin O and spread within the cytoplasm. In the cytoplasm, a bacterial protein called ActA induces assembly of actin microfilaments behind the bacterium, which results in movement of bacteria in a manner similar to movement of Shigella inside the cytoplasm (see preceding). Bacteria move to adjacent cells and spread. Genes involved in escape from the vacuole and in intra/intercelullar spread are carried on a pathogenicity island in the L. monocytogenes chromosome. E. Enterotoxigenic Bacteroides fragilis Bacteroides fragilis is a gram-negative non-sporeforming anaerobic rod that comprises a part of the normal intestinal flora of nearly all humans. Enterotoxigenic B. fragilis (ETBG) strains produce a toxin that stimulates fluid secretion in the intestinal lumen and causes rounding of epithelial cells and loss of intestinal microvilli in an in vivo intestinal model. These strains have been linked to diarrhea in animals and in a small number of studies of humans. F. Clostridium botulinum Clostridium botulinum is the cause of food poisoning, which can occur by ingestion of preformed toxins in inadequately processed food, such as home-canned vegetables and fish. Vomiting and diarrhea occur before neurological symptoms begin. The disease is caused by neurotoxins A, B, or E. Infant botulinum is another manifestation, which is different from food poisoning in that toxins are produced after germination of spores in gut. Clostridium botulinum also produces a cytotoxin called C2 toxin, which can alter the cell cytoskeleton by ADP-ribosylation of G actin and preventing polymerization of G to F actin. The role of this toxin in disease is unknown.