Clinical Manifestations

Symptoms of shigellosis include abdominal pain, tenesmus, watery diarrhea, and/or dysentery (multiple scanty, bloody, mucoid stools). Other signs may include abdominal tenderness, fever, vomiting, dehydration, and convulsions.

Structure, Classification, and Antigenic Types

Shigellae are Gram-negative, nonmotile, facultatively anaerobic, non-spore-forming rods. Shigella are differentiated from the closely related Escherichia coli on the basis of pathogenicity, physiology (failure to ferment lactose or decarboxylate lysine) and serology. The genus is divided into four serogroups with multiple serotypes: A (S dysenteriae, 12 serotypes); B (S flexneri, 6 serotypes); C (S boydii, 18 serotypes); and D (S sonnei, 1 serotype).

Pathogenesis

Infection is initiated by ingestion of shigellae (usually via fecal-oral contamination). An early symptom, diarrhea (possibly elicited by enterotoxins and/or cytotoxin), may occur as the organisms pass through the small intestine. The hallmarks of shigellosis are bacterial invasion of the colonic epithelium and inflammatory colitis. These are interdependent processes amplified by local release of cytokines and by the infiltration of inflammatory elements. Colitis in the rectosigmoid mucosa, with concomitant malabsorption, results in the characteristic sign of bacillary dysentery: scanty,. unformed stools tinged with blood and mucus.

Host Defenses

Inflammation, copious mucus secretion, and regeneration of the damaged colonic epithelium limit the spread of colitis and promote spontaneous recovery. Serotype-specific immunity is induced by a primary infection, suggesting a protective role of antibody recognizing the lipopolysaccharide (LPS) somatic antigen. Other Shigella antigens include enterotoxins, cytotoxin, and plasmid-encoded proteins that induce bacterial invasion of the epithelium. The protective role of immune responses against these antigens is unclear.

Epidemiology

Shigellosis is endemic in developing countries were sanitation is poor. Typically 10 to 20 percent of enteric disease, and 50% of the bloody diarrhea or dysentery of young children, can be characterized as shigellosis, and the prevalence of these infections decreases significantly after five years of life. In developed countries, single-source, food or water-borne outbreaks occur sporadically, and pockets of endemic shigellosis can be found in institutions and in remote areas with substandard sanitary facilities.

Diagnosis

Shigellosis can be correctly diagnosed in most patients on the basis of fresh blood in the stool. Neutrophils in fecal smears is also a strongly suggestive sign. Nonetheless, watery, mucoid diarrhea may be the only symptom of many S sonnei infections, and any clinical diagnosis should be confirmed by cultivation of the etiologic agent from stools.

Control

Prevention of fecal-oral transmission is the most effective control strategy. Severe dysentery is treated with ampicillin, trimethoprim-sulfamethoxazole, or, in patients over 17 years old, a 4-fluorquinolone such as ciprofloxacin. Vaccines are not currently available, but some promising candidates are being developed.

Pathology

The rectosigmoidal lesions of shigellosis resemble those of ulcerative colitis. With frequencies indicated in Figure 22-2, there is proximal extension of erythema, edema, loss of vascular pattern, focal hemorrhage, and adherent layers of purulent exudate. Biopsy specimens from affected areas are typically edematous, with capillary congestion, focal hemorrhage, crypt hyperplasia, goblet cell depletion, mononuclear and polymorphonuclear (PMN) cell infiltration, shedding of epithelial cells and erythrocytes, and microulcerations.

Figure 22-2

Gross pathology of shigellosis.

The pathogenic mechanism that underlies these pathological manifestations is diagrammed in Figure 22-3. This cartoon incorporates experimental observations from tissue cultures and from animal models of shigellosis such as rabbit ligated ileal loops injected with virulent organisms. In the latter model, Shigella infection is initiated at the membranous (M) cells that are associated with macroscopic lymphoid follicles (Peyer's patches). Biopsy studies in rhesus monkeys suggest that shigellae also infect microscopic lymphoid follicles of the primate colon. During the early stages of infection, bacteria are transcytosed through the M cells into the subepithelial space. In the subepithelial space, the organisms are phagocytosed by resident macrophages. However, virulent shigellae are not killed and digested in the macrophage phagolysome. The bacteria lyse the phagosome and initiate apoptosis (programmed cell death). During this process, the infected macrophage releases the inflammatory cytokine IL-1, which elicits infiltration of PMN.

Figure 22-3

Histopathology of acute colitis following peroral infection with shigellae. The organisms are initially ingested by membranous (M) cells that are associated with lymphoid microfollicles in the colon. After transcytosis through the M cell, the bacteria (more...)

Transmigration of infiltrating PMNs through the tight junctions of local epithelial cells and into the intestinal lumen allows the reverse migration of shigellae from the lumen into the subepithelial spaces. These organisms then infect the columnar epithelial cells by inducing endocytic uptake at the basolateral surface. Immediately after infection of enterocytes, intracellular shigellae lyse endocytic vacuoles and attach to the actin cytoskeleton in the area of the junctional complex. As these organisms multiply within the enterocyte cytoplasm, occasional daughter cells induce polar nucleation of filamentous actin resulting in a “tail” that propels the shigellae into protrusions impinging on contiguous enterocytes. Plasma membranes enveloping the organisms are again lysed, and the organisms are deposited within the contiguous host cell resulting in intercellular bacterial spread.

In summary, shigellosis can be characterized as an acute inflammatory bowel disease initiated by the uptake of only a few organisms into lymphoid follicles. Intracellular replication and intercellular spread leads to an amplified inflammatory cascade at the initial site of entry, and as this inflammation persists and expands, the infiltration of PMN facilitates the entry of additional bacteria into the epithelium. The inflammatory infiltrate can also cause detachment of sheets of epithelial cells in areas devoid of lymphoid structures or bacterial cells.

Genetics of Virulence

Shigella are exquisitely adapted for reproduction within the colonic epithelium of the human host. Many of the bacterial virulence determinants that mediate the complex interactions between these bacteria and mammalian host cells have been identified by genetic and immunological means. These virulence determinants are encoded by large extra-chromosomal elements (plasmids) that are functionally identical in all Shigella species and in EIEC. A complex of two plasmid-encoded determinants, designated Invasion Plasmid Antigens (Ipa) B and C, is recognized by antibody in the sera of convalescent patients. Ipa proteins are maximally expressed in conditions approximating the intestinal lumen (e.g., bile salts, high osmolarity, and human body temperature), and release of the IpaBC complex is triggered by contact with the mammalian host cell. This complex induces the endocytic uptake of shigellae by M cells, epithelial cells, and macrophages. IpaB also mediates lysis of endocytic vacuoles in epithelial cells or macrophages. In the latter case, Ipa proteins also cause release of the IL-1 cytokine and macrophage apoptosis. Another plasmid-encoded virulence determinant is secreted at the poles of Shigella daughter cells as these organisms multiply within the cytoplasm of infected host cells. This InterCellular Spread (IcsA) protein elicits polymerization of filamentous actin. Formation of this actin tail provides a motive force for shigellae impinging on the plasma membrane of the infected cell. The resulting protrusions deform the plasma membrane of contiguous cells. The IcsB plasmid-encoded protein then lyses the plasma membranes, resulting in intercellular bacterial spread. Biochemical characterization of the interaction between these Shigella virulence determinants and host cell components is a remaining research challenge. Characterizing and enhancing the neutralizing potential of antibody recognizing these protein virulence determinants is also an important research goal.

Toxins

Spent medium from S flexneri or EIEC cultures elicits fluid accumulation in rabbit ligated ileal loops and ion secretion in isolated ileal tissue. Using these assays, enterotoxins designated ShET1 and ShET2 have been identified, and the genetic loci encoding these toxins have been localized to the chromosome and plasmid, respectively. ShET1 is neutralized by convalescent sera from volunteers challenged with S flexneri 2a, suggesting that this toxic moiety is expressed by shigellae growing in the human intestine. The ShET1 locus is present on the chromosome of S flexneri2a, but it is only occasionally found in other serotypes. In contrast, ShET2 is more widespread and detectable in 80% of shigellae representing all four species. These enterotoxins may elicit the diarrheal prodrome that often precedes bacillary dysentery; however, their role in the disease process remains to be defined by controlled challenge studies using toxin-negative mutants.

S dysenteriae serotype 1 expresses Shiga toxin, an extremely potent, ricin-like, cytotoxin that inhibits protein synthesis in susceptible mammalian cells. This toxin also has enterotoxic activity in rabbit ileal loops, but its role in human diarrhea is unclear, since shigellae apparently express a number of enterotoxins. Experimental infection of rhesus monkeys with S dysenteriae 1, and with a Shiga toxin-negative mutant, suggests that this cytotoxin causes capillary destruction and focal hemorrhage that exacerbates dysentery (see Table 22-1). More importantly, Shiga toxin is associated with the hemolytic-uremic syndrome, a complication of infections with S dysenteriae 1. Closely related toxins are expressed by enterohemorrhagic E coli (EHEC) including the potentially lethal, food-borne O157-H7 serotype.

Clinical

Patients presenting with watery diarrhea and fever should be suspected of having shigellosis. The diarrheal stage of the infection cannot be distinguished clinically from other bacterial, viral, and protozoan infections. Nausea and vomiting can accompany Shigella diarrhea, but these symptoms are also observed during infections with nontyphoidal salmonellae and enterotoxigenic E coli. Bloody, mucoid stools are highly indicative of shigellosis, but the differential diagnosis should include EIEC, Salmonella enteritidis, Yersinia enterocolitica, Campylobacter species, and Entamoeba histolytica. Although blood is common in the stools of patients with amebiasis, it is usually dark brown rather than bright red, as in Shigella infections. Microscopic examination of stool smears from patients with amebiasis should reveal erythrophagocytic trophozoites in the absence of PMN, whereas bacillary dysentery is characterized by sheets of PMN. Sigmoidoscopic examination of a shigellosis patient reveals a diffusely erythematous mucosal surface with small ulcers, whereas amebiasis is characterized by discrete ulcers in the absence of generalized inflammation.

Laboratory

Although clinical signs may evoke the suspicion of shigellosis, diagnosis is dependent upon the isolation and identification of Shigella from the feces. Positive cultures are most often obtained from blood-tinged plugs of mucus in freshly passed stool specimens obtained during the acute phase of disease. Rectal swabs may also be used to culture shigellae if the specimen is processed rapidly or is deposited in a buffered glycerol saline holding solution. Isolation of shigellae in the clinical laboratory typically involves an initial streaking for isolation on differential/selective media with aerobic incubation to inhibit the growth of the anaerobic normal flora. Commonly used primary isolation media include MacConkey, Hektoen Enteric Agar, and Salmonella-Shigella (SS) Agar. These media contain bile salts to inhibit the growth of other Gram-negative bacteria and pH indicators to differentiate lactose fermenters (Coliforms) from non-lactose fermenters such as shigellae. A liquid enrichment medium (Hajna Gram-negative broth) may also be inoculated with the stool specimen and subcultured onto the selective/differential agarose media after a short growth period. Following overnight incubation of primary isolation media at 37° C, colorless, non-lactose-fermenting colonies are streaked and stabbed into tubed slants of Kligler's Iron Agar or Triple Sugar Iron Agar. In these differential media, Shigella species produce an alkaline slant and an acid butt with no bubbles of gas in the agar. This reaction gives a presumptive identification, and slide agglutination tests with antisera for serogroup and serotype confirm the identification.

Some E coli biotypes of the normal intestinal flora closely resemble Shigella species (i.e. they are nonmotile, delayed lactose fermenters). These coliforms can usually be differentiated from shigellae by the ability to decarboxylate lysine. However, some coliforms cause enteroinvasive disease because they carry the Shigella-like virulence plasmid, and these pathogens are conventionally identified by laborious serological screening for EIEC serotypes. Sensitive and rapid methodology for identification of both EIEC and Shigella species utilizes DNA probes that hybridize with common virulence plasmid genes or DNA primers that amplify plasmid genes by polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) using antiserum or monoclonal antibody recognizing Ipa proteins can also be used to screen stools for enteroinvasive pathogens. These experimental diagnostic techniques are useful for epidemiological studies of enteroinvasive infections, but they are probably too specialized for routine use in the clinical laboratory.