Enterobacteriaceae II: Enterobacteriaceae II: Intestinal PathogensIntestinal Pathogens

Enterobacteriaceae II: Enterobacteriaceae II: Intestinal PathogensIntestinal Pathogens

Salmonella, Shigella, Salmonella, Shigella, YersiniaYersinia

Salmonella• Animals main reservoir of human

disease:– 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:

– 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 IThursday, 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!