Enterobacteriaceae II: Intestinal Pathogens Salmonella, Shigella, Yersinia

Salmonella Animals main reservoir of human disease: Transmission: Gastroenteritis Typhoid (Enteric) fever Septicemia Asymptomatic carriage Transmission: Ingest contaminated food (poultry, eggs, dairy products) Direct fecal-oral spread in children

Salmonella Genera Describe >2500 serotypes DNA homology studies: One Genus-species - Salmonella enterica Seven subspecies (1, 2 ,3a ,3b ,4 ,5, and 6) Subgroup 1 causes most human infections Clinically Salmonella isolates often still reported as serotypes by Kauffman-White classification: Based on O (cell) and H (flagella) antigens H antigens occur in two phases (antigenic variation) Polyvalent antisera used, followed by group specific antisera (A, B, C1, C2, D, and E) Salmonella Typhi (capitalize, not italics to designate Genus-Serotype) - also has capsular Vi antigen

Salmonella: Biochemical Test ID TSI = K/A + gas, H2S Salmonella Typhi small amount of H2S, no gas Salmonella Paratyphi no H2S LIA = K/K (+)lysine DC, +H2S Salmonella Paratyphi = K/A Urea(–) PA(-) Citrate(±) Indole(-)

Salmonella: Infection MO found in all animals – poultry, reptiles, livestock, rodents, domestic animals, birds, and humans Highly adapted strict human pathogen: Salmonella Typhi Salmonella Paratyphi Peak incidence of infections in warm months via ingesting contaminated food at outdoor social gatherings ~50,000 cases reported annually in U.S

Salmonella: Infection of GI Tract Ingest contaminated food Infect GI cells Blood (systemic infection, target organs) RES (Reticuloendothelial System) – macrophages, liver, spleen, bone marrow

Infection of GI Tract MO adheres to intestinal mucosa epithelium Invasion of enterocytes, M cells (microfold cells of Peyer patches) Induced endocytosis by bacteria effector proteins Salmonella multiplies within phagosome, released; may be phagocytosed by macrophages

Salmonella: Gastroenteritis Salmonellosis most common form of gastroenteritis Due to Salmonella Enteritidis in U.S. Symptoms 6-48 hours after consumption contaminated food: Nausea, vomiting, non-bloody diarrhea Also common with elevated temperature, abdominal cramps, myalgia, headache Persist 2-7 days, spontaneous resolution Supportive care, no antibiotics required Incidence greatest children <5 yrs. and adults >60 yrs.

Gastroenteritis MOs multiply, induce strong inflammatory response Causes disease symptoms (fever, diarrhea, abdominal cramps) Inflammatory response prevents spread beyond GI tract, eventually kills MOs

Salmonella: Typhoid ( Enteric) Fever Typhoid fever - Salmonella Typhi Paratyphoid fever (milder) - Salmonella Paratyphi 10-14 day incubation period following ingestion of contaminated food Fever, headache, myalgia, malaise, anorexia Persist 1 week, followed by GI symptoms (GI→blood→GI) Mortality ~20% if untreated

Typhoid Fever MO disseminate before high enough levels to stimulate inflammatory response Initial symptoms low fever, constipation MO move via lymphatics & bloodstream to liver & spleen; phagocytosis & multiplication occurs MO re-enter bloodstream, disseminate to all organs; fever, headaches, myalgia, GI problems Rose spots (erythema, maculopapular lesions) on abdomen Osteomyelitis, cystitis, gall bladder infections may occur

Salmonella: Septicemia Most frequently: Salmonella Typhimurium Salmonella Choleraesuis Salmonella Paratyphi At risk: children, elderly, AIDS patients Infection presents like Gram(-) bacteremia 10% develop osteomyelitis, endocarditis, arthritis

Salmonella: Asymptomatic Infection Salmonella Typhi Salmonella Paratyphi MO’s from enteric fever maintained by human carriage ~1-5% infected patients chronic carrier >1 year Reservoir in gall bladder

Lab Diagnosis: Typhoid Fever Blood cultures positive during first week, after second week Stool culture, sometimes urine culture positive after second week Widal Test (serology): Antibodies against Salmonella Typhi Look for 4-fold rise in titer between acute and convalescent stage (~one month)

Salmonella: Prevention Gastroenteritis: Public Health education Improved hygiene, especially food handlers Safe preparation of poultry & eggs, refrigerate food Antibiotics not recommended, may prolong disease Enteric fevers: Treat with fluoroquinolone (ciprofloxacin), chloramphenicol ( decrease mortality to <2%) Live oral vaccine (attenuated Salmonella Typhi) for travelers to endeminc countries Clean water supply ID, treat chronic carriers

Shigella: Gastroenteritis Shigellosis (Bacillary dysentery) ~150 million cases annually worldwide; 450,000 in U.S. Humans only reservoir, transmitted person-to-person by fecal-oral route Outbreaks in communities where sanitary standards, level of hygiene low Common in daycare centers, nurseries, custodial institutions

Shigella Genera Four species by O antigen serotype: S. dysenteriae (Group A) – most severe; Shiga toxin S. flexneri (Group B) – developing countries S. boydii (Group C) – not commonly isolated S. sonnei (Group D) – developed countries; U.S. By DNA analysis, now shown to be biogroup within E. coli

Shigella: Biochemical Test ID TSI = K/A with NO gas LIA = K/A (-)lysine DC Urea (–) Motility(–) S. sonnei may show delayed lactose fermentation

Shigella: Virulence Factors - Enterotoxin Shiga toxin - S. dysenteriae Smaller amounts by S. flexneri, S. sonnei Inhibit protein synthesis by inactivating 60S ribosomal subunit Role in ulceration intestinal mucosa

Shigella: Virulence Factors - Cell Wall Proteins Expressed at body temperature Upon contact with intestinal mucosa M cells (microfold cells, Peyer’s patches), induce phagocytosis of bacteria into vacuoles Shigella destroys vacuoles, escape into cytoplasm Spread laterally (actin filaments propel MO through cytoplasm) to epithelial cells; multiply Do not usually disseminate beyond epithelium

Shigella: M Cell Attachment

Shigella: M Cell Penetration

Shigella: Epithelial Cell (CCEC) Invasion

Shigellosis Fecal-oral route, primarily contaminated hands Infective dose very low (100-200 MO) Incubation 1-7 days Fever, cramping, abdominal pain, watery diarrhea for 1-3 days (due to exotoxin) Followed by frequent, scant stools with blood, mucous, pus (invasion of intestinal mucosa) Rare for MO to disseminate, generally self-limited but may lead to death Severity of disease depends upon species: S. dysenteriae most pathogenic Followed by S. flexneri, S. sonnei, S. boydii

Shigella: Treatment and Prevention Trimethoprim-sulfamethoxazole (SXT) or fluoroquinolone (ciprofloxacin) - shortens course of disease & fecal shedding; recommended to reduce spread to contacts Resistant strains becoming increasingly common, so antibiotic sensitivity testing required Infection control by proper hand washing and disposal of soiled diapers/linens Public Health monitoring for clean water supply

Comparison of Invasion: Salmonella versus Shigella

Yersinia Genera Infections are zoonotic, humans accidental host Three species important pathogens in humans: Yersinia pestis – plague (bubonic or pneumonic) Yersinia enterocolitica – gastroenteritis, transfusion-related sepsis Yersinia pseudotuberculosis – mainly disease of animals, uncommon human gastroenteritis

Yersinia: Infection Y. pestis: Y. enterocolitica: Mammalian reservoir: rats, squirrels, rabbits, domestic animals Humans infected by: Fleas Direct contact with infected animal tissue Aerosols from patient with pulmonary disease Y. enterocolitica: Ingest contaminated food products Infusion of contaminated blood products (growth in blood stored at 4°C)

Yersinia pestis: Lab ID Non-motile - 37°C & 25°C Bipolar staining Slow growth, small colonies at 37°C (grows better 25°C) TSI = K/A Urea(-) ODC(-) Guinea pig LD50<10 Direct fluorescent antibody test New DNA probe test

Y. pestis: Bubonic Plague By flea bite infected animal (rat, squirrel, rabbit) to human Endemic in local So Cal mountains MO travel to nearest lymph node, engulfed by macrophage High fever, buboe (enlarged lymph node), MO proliferate, stimulate inflammatory response MO multiply in lymph node, leak into bloodstream Lysis of bacteria releases LPS causing septic shock, DIC Subcutaneous hemorrhage, disease named Black Death in Middle Ages High mortality rate (30-40%) if untreated

Pneumonic Plague Eventually bacteria reach lungs, engulfed by lung macrophages, cause pneumonic plague Transmit directly to others via aerosol Direct inhalation produces disease that progress more rapidly Mortality rate close to 100%

Y. pestis: Treatment and Prevention Streptomycin, tetracycline, SXT Control by reducing rodent population Vaccine (formalin killed bacteria) for individuals at risk; no longer available

Yersinia enterocolitica: Lab ID Non-motile at 37°C Motile at 25°C Bipolar staining Slow growth, small colonies at 37°C (grows better at 25°C) TSI = A/A (sucrose fermentation) Urea(+) ODC(+)

CIN Agar Isolation of Y. enterocolitica from stool specimen (also Aeromonas, Plesiomonas) Selective - antimicrobials (Cefsulodin-Irgasan-Novobiocin), bile, crystal violet inhibit NF Differential - mannitol, neutral red Incubate room temperature After 48 hours at RT, Y. enterocolitica produce typical pink colonies (mannitol fermentation) with bulls-eye appearance

Y. enterocolitica: Gastroenteritis Ingestion of contaminated food or water Common cause of human disease (mostly in children) involving fever, abdominal pain, watery diarrhea Intestinal epithelium invasion of M cells, transcytosed through M cells, released at basal surface Bacteria penetrate into underlying lymphoid tissue, multiply both inside and outside host cells

Gastroenteritis Inflammatory response to MO responsible for extreme pain (~acute appendicitis) Fever due to LPS endotoxin Sometimes drain into adjacent mesenteric lymph nodes, causing lymphadenitis Reactive arthritis may occur in some people; thought cross reacting T cells or antibodies that attack joint Usually self-limited If treatment indicated, susceptible to broad-spectrum cephalosporins

Summary: Yersinia Infections

Lab: Enterobacteriaceae II Group Unknowns TSI LIA Salmonella K/A, H2S K/K, H2S Proteus K/A, H2S R/A, H2S Citrobacter K/A, H2S K/A, H2S Yersinia K/A K/A

Lecture Exam I Thursday, Feb. 2, 2012 Introduction thru Enterobacteriaceae Lecture Reading (Chap. 14, 15, 17, 19) Key Terms, Learning assessment Questions Case Study 1,2,3 Exam Format: Multiple Choice Terms True/False Statements Short Essay Review, Review, Review!