 Coliform bacilli (enteric rods)  Nonmotile gram-negative facultative anaerobes  Four species  Shigella sonnei (most common in industrial world)  Shigella flexneri (most common in developing countries)  Shigella boydii  Shigella dysenteriae  Non-lactose fermenting  Resistant to bile salts General Characteristics of Shigella

Epidemiology and Clinical Syndromes of Shigella Shigellosis = Generic term for disease Shigellosis is a strictly human disease with no animal reservoirs.  Low infectious dose ( CFU)  Humans are only reservoir  Transmission by fecal-oral route  Incubation period = 1-3 days  Watery diarrhea with fever; changing to dysentery  Major cause of bacillary dysentery (severe 2 nd stage) in pediatric age group (1-10 yrs) via fecal-oral route  Outbreaks in daycare centers, nurseries, institutions  Estimated 15% of pediatric diarrhea in U.S.

Leading cause of infant diarrhea and mortality (death) in developing countries The incidence and spread of shigellosis is directly related to personal and community sanitary practices. In developed countries, it is largely a pediatric disease.

. Shigellosis Two-stage disease:  Early stage:  Watery diarrhea attributed to the enterotoxic activity of Shiga toxin following ingestion and noninvasive colonization, multiplication, and production of enterotoxin in the small intestine  Fever attributed to neurotoxic activity of toxin  Second stage:  Adherence to and tissue invasion of large intestine with typical symptoms of dysentery  Cytotoxic activity of Shiga toxin increases severity Pathogenesis of Shigella

Virulence attributable to:  Invasiveness  Attachment (adherence) and internalization with complex genetic control  Large multi-gene virulence plasmid regulated by multiple chromosomal genes  Exotoxin (Shiga toxin)  Intracellular survival & multiplication Pathogenesis and Virulence Factors Pathogenic strians of Shigellae carry a plasmid of 100 to 140 MDa which encodes the pathogenic mechanisms invovlved with eukaryotic cell invasion

 M cells typically transport foreign antigens from the intestine to underlying macrophages, but Shigella can lyse the phagocytic vacuole (phagosome) and replicate in the cytoplasm  Note: This contrasts with Salmonella which multiplies in the phagocytic vacuole  Actin filaments propel the bacteria through the cytoplasm and into adjacent epithelial cells with cell-to-cell passage, thereby effectively avoiding antibody-mediated humoral immunity (similar to Listeria monocytogenes) Invasiveness in Shigella-Associated Dysentery (cont.) Pathogenesis and Virulence Factors (cont.)

Methods That Circumvent Phagocytic Killing

 Enterotoxic, neurotoxic and cytotoxic  Encoded by chromosomal genes  Two domain (A-5B) structure  Similar to the Shiga-like toxin of enterohemorrhagic E. coli (EHEC)  NOTE: except that Shiga-like toxin is encoded by lysogenic bacteriophage Characteristics of Shiga Toxin Pathogenesis and Virulence Factors (cont.)

Enterotoxic Effect:  Adheres to small intestine receptors  Blocks absorption (uptake) of electrolytes, glucose, and amino acids from the intestinal lumen  Note: This contrasts with the effects of cholera toxin (Vibrio cholerae) and labile toxin (LT) of enterotoxigenic E. coli (ETEC) which act by blocking absorption of Na +, but also cause hypersecretion of water and ions of Cl -, K + (low potassium = hypokalemia), and HCO 3 - (loss of bicarbonate buffering capacity leads to metabolic acidosis) out of the intestine and into the lumen Shiga Toxin Effects in Shigellosis Pathogenesis and Virulence Factors (cont.)

Shiga Toxin Effects in Shigellosis (cont.) Pathogenesis and Virulence Factors (cont.) Cytotoxic Effect:  B subunit of Shiga toxin binds host cell glycolipid  A domain is internalized via receptor-mediated endocytosis (coated pits)  Causes irreversible inactivation of the 60S ribosomal subunit, thereby causing:  Inhibition of protein synthesis  Cell death  Microvasculature damage to the intestine  Hemorrhage (blood & fecal leukocytes in stool) Neurotoxic Effect: Fever, abdominal cramping are considered signs of neurotoxicity

DIAGNOSIS AllShigella species are readily isolated using selective media (e.g. Hektoen enteric agar)which are part of the routine stool culture in clinical laboratories. These media containchemical additives empirically shown to inhibit facultative flora (eg,E. coli,Klebsiella),with relatively little effect on Shigella (orSalmonella). They also contain indicator sys-tems which utilize typical biochemical reactions to mark suspect Shigella colonies amongthe other flora. Isolates are identified with further biochemical tests. Slide agglutinationtests using O group specific antisera (A,B,C,D) confirm both the species and theShigella genu

TREATMENTSeveral antimicrobics have proved effective in the treatment of shigellosis. Because thedisease is usually self-limiting,the beneficial effect of treatment is in shortening the ill-ness and the period of excretion of organisms. Ampicillin was once the treatment of

Prevention Sanitation,insect control,handwashing,and cooking blocktransmissionLive attenuated vaccines are underinvestigation

. A life-threatening secretory diarrhea induced by enterotoxin secreted by V. cholerae Water-borne illness caused by ingesting water/food contaminated by copepods infected by V. cholerae An enterotoxic enteropathy (a non-invasive diarrheal disease) A major epidemic disease Cholera

Vibrio Vibrios are curved,Gram-negative rods commonly found in saltwater. They are highly motile with a single polar flagellum,non–spore forming, oxidase positive, and can grow under aerobic or anaerobic conditions.

CHOLERA TOXIN CT molecule is an aggregate of multiple polypeptide chains organized into two toxic subunits (A1, A2) and five binding (B) units. The B units bind to a GM1-ganglioside receptor found on the surface of many types of cells. Once bound,the A1 subunit is released from the toxin molecule by reduction of the disulfide bond that binds it to the A2 subunit, and it enters the cell by translocation. In the cell, it exerts its effect on the membrane-associated adenylate cyclase system. The target of the toxic A1 subunit is a guanine nucleotide (G) protein, Gs, that regulates activation of the adenylate cyclase system. CT catalyzes the ADP ribosylation of the G protein, rendering it unable to dissociate from the active adenylate cyclase complex. This causes persistent activation of intracellular adenylate cyclase, which in turn stimulates the conversion of adenosine triphosphate to cyclicadenosine 3,5-monophosphate (cAMP). The net effect is excessive accumulation of cAMP at the cell membrane, which causes hypersecretion of chloride, potassium, bicarbonate, and associated water molecules out of the cell. Strains of V. cholerae other than

Transmission is through untreated water supply Incubation period is 2 daysCholera is endemic in India andAfricaPandemics span decades Gulf Coast cases result fromundercooked shellfish Latin American epidemic is widespread El Tor biotype dominated 20thcentury New O139 serotype is spreading

PATHOGENESIS In healthy people,ingestion of large numbers of bacteria is required to offset the acid barrier of the stomach. Colonization of the entire intestinal tractfrom the jejunum to the colon by V. cholerae requires organism adherence to the epithelial surface, most probably by surface pili. The outstanding feature of V. cholerae pathogenicity is the ability of virulent strains to secrete CT, which is responsible for the disease cholera. The water and electrolyte shift from the cell to the intestinal lumen.

Genetic Regulation of Virulence The expression of the multiple virulence factors of V. cholerae is controlled in a complex but coordinated system involving environmental sensors and as many as 20 chromosomal genes divided between a pathogenicity island (PAI) containing CT and one containingTCP. The chief regulator is a transmembrane protein (ToxR ) that “senses”environmental changes in pH, osmolarity, and temperature which convert it to an active form. In the active state,ToxR can directly turn on CT genes.

CLINICAL ASPECTS MANIFESTATIONS Clinical features of cholera result from the extensive fluid loss and electrolyte imbalance, which can lead to extreme dehydration,hypoten-sion,and death within hours if untreated

DIAGNOSIS A bacteriologic diagnosis is accomplished by isolation of V. cholerae from the stool. The organism grows on common clinical laboratory media such as blood agar and MacConkey agar, but its isolation is enhanced by the useofa selective medium (thiosulfate- citrate-bile salt-sucrose agar). Once isolated, the or-ganism is readily identified by biochemical reactions. Outside cholera endemic areas,the

Treatment and prevention Oral or intravenous fluid and electrolyte replacement is crucial Antimicrobic therapy can reduced uration and severity Prevention Water sanitation and cookingshellfish prevent infectionVaccines are disappointing