1. Enterobacteriaceae

2. Introduction
The family Enterobacteriaceae is the largest, most heterogeneous collection of medically important gram –negative bacilli.
Their natural habitat is the intestinal tract of humans and animals (Enteric gram negative rods).
The Enterobacteriaceae are the most common group of gram negative bacilli isolated in the lab.

3. At present, at least 27 genera and 102 species as well as eight enteric groups ( isolates with undefined genus affiliation) have been described.
These genera have been classified on the basis of DNA homology, biochemical properties, serologic reactions, susceptibility to genus – specific and species – specific bacteriophages and antibiotic susceptibility patterns.
Despite the complexity of this family, more than 95% of medically important isolates belong to fewer than 25 species.

4. Family Enterobacteriaceae
Certain E .coli strains can be considered true pathogens
True pathogen
(nonmotile)
(nonmotile)
True pathogen
True pathogen

5. Enterobacteriaceae are ubiquitous organisms that are found worldwide in soil, water, vegetation and are part of the normal flora of the intestines of most animals including humans.
Some members of the family (Shigella, Salmonella) are always associated with disease when isolated from humans whereas others are members of the normal flora that can cause opportunistic infections.

6. Infections can originate from an animal reservoir (Salmonella), from human carriers (Shigella, Salmonella), or by endogenous spread of organisms in a susceptible patient (E. coli) involving virtually all body sites .
The Enterobacteriaceae are responsible for 30% to 35 % of all septicemias, more than 70% of urinary tract infections and many intestinal infections .

7. Physiology and Structure
The Enterobacteriaceae are gram negative bacilli, usually motile, aerobes or facultative anaerobes and non spore- forming.
The Enterobacteriaceae have simple nutritional requirements; ferment glucose, reduce nitrate and are oxidase negative and catalase positive.

8. Morphological characteristics on differential and selective media have been used to identify members of the family (lactose fermentation, resistance to bile salts).
Some members are encapsulated (Klebsiella and Enterobacter).
The serologic classification is based on three major group antigens; somatic(O antigen), Capsular(K antigen) and flagellar (H antigen).

9. Pathogenesis
Consistent with the large and diverse composition of the family is the observation of many virulence factors in the pathogenic strains.
Endotoxin (more than 150 somatic O antigens).
Capsule (more than 100 K (Vi) antigens).
Antigenic phase variation (protection from antibody –mediated cell death)

10. Exotoxin production
- Numerous, including heat stable and heat labile enterotoxins, shiga and shiga – like toxins, and hemolysins.
- The heat –labile enterotoxins as well as shiga and shiga-like toxins are produced primarily by E. Coli and occasional isolates of Klebsiella and Salmonella.
- The heat stable toxin is produced by E .coli and occasionally Citrobacter freundii.
- Shigella dysenteriae produces shiga toxin (neurotoxic, enterotoxic and cytotoxic) which inhibits protein synthesis by inactivation of 60'S ribosomes.

11. Expression of Adhesion Factors: Fimbriae with phase variation.
Intracellular Survival and Multiplication: can replicate intracellularly but do not require intracellular host factors for survival.
Sequestration of growth factors: (siderophores like enterobactin).
Resistance to Serum Killing.
Antimicrobial Resistance.

12. OPPORTUNISTIC INFECTIONS OF ENTEROBACTERIACEAE
GRAM NEGATIVE SEPSIS
URINARY TRACT INFECTIONS
PNEUMONIA
ABDOMINAL SEPSIS
MENINGITIS
SPONTANEOUS BACTERIAL PERITONITIS
ENDOCARDITIS

13. Endotoxin-Mediated Toxicity
Fever
Leukopenia (reduced # of WBCs) (<5000/mm3) followed by leukocytosis (increased # of WBCs)(>10-12,000/mm3)
Activation of complement
Thrombocytopenia (reduced # of platelets)
DIC (Disseminated intravascular coagulation)
Decreased peripheral circulation and perfusion (blood flow) to major organs
Shock
Death

14. Nonspecific Disease Entities E . Coli and Others
Urinary tract infection (UTI)
- E . coli is the most common cause.
- Infecting strains usually originate from the GIT.
- Signs and symptoms.
- Diagnosis.
Pneumonia (Esp. Klebsiella pneumonia)
Septicemia
- When normal host defenses are inadequate.
- Newborns may be highly susceptible to E. coli, Klebsiella, and Enterobacter.
- The focus of infection from which spread takes place is the UT or GIT.

15. A common cause of traveler’s diarrhea and a
Neonatal meningitis.
E. coli Associated Diarrheal Diseases
Enterotoxigenic E. coli (ETEC)
A common cause of traveler’s diarrhea and a
very important cause of diarrhea in infants in the
developing countries.
Secretes a heat labile exotoxin that acts on the small intestines causing activation of adenylate cyclase and increase of cAMP leading to hypersecretion of water.
Some strains produce a heat stable toxin and a colonization factor

16. Enteropathogenic E. coli (EPEC)
Damage the epithelium of the small intestine.
An important cause of diarrhea in infants especially in the developing countries.
Self limited watery diarrhea but can be chronic.
May cause outbreaks of diarrhea in nurseries.

17. Enterohemorrhagic E. coli (EHEC)
Produces verotoxin which has 2 antigenic types.
Has been associated with hemorrhagic colitis, a severe form of diarrhea with hemolytic uremic syndrome, a disease resulting in acute renal failure, microangiopathic hemolytic anemia and thrombocytopenia.
E. coli O157:H7 is the most common.
Can be prevented by thorough cooking of ground beef.

18. Enteroeinvasive E. coli (EIEC)
Invades the intestinal mucosal epithelial cells.
Disease is similar to shigellosis
Affects children in the developing countries and travelers to them.

19. Enteroaggregative E. coli (EAEC)
Causes acute and chronic diarrhea( >14 days) in the developing countries.
Causes a food-borne illness in the developed countries.
Characteristic pattern of adherence to human cells.
Elaborates an enterotoxin and cytotoxin.

20. The Salmonella: Classification and Taxonomy
Old: Serotyping & biochemical assays used to name individual species within genus (e.g., Salmonella enteritidis, S. choleraesuis, S. typhi)
Over 2400 O-serotypes (referred to as species) (Kauffman-White antigenic schema)
Bioserotyping (e.g., S. typhimurium)
New: DNA homology shows only two species Salmonella enterica (six subspecies) and S. bongori
Most pathogens in S. enterica ssp. enterica

21. The Salmonella
Serotypes Typhi, paratyphi A and B, and choleraesuis are primarily human pathogens.
The vast majority of salmonellae are chiefly animal pathogens (poultry, pigs, rodents, cattle, pets and others).
Infecting dose is variable depending on serotype.

22. Clinical Syndromes of Salmonella
Enteritis (acute enterocolitis)
Enteric fever (prototype is typhoid fever and less severe paratyphoid fever)
Septicemia (particularly S. choleraesuis, S. typhi, and S. paratyphi)
Asymptomatic carriage (gall bladder is the reservoir for Salmonella typhi)

23. Epidemiology and Clinical Syndromes of Salmonella
Enteritis
Most common form of salmonellosis with major foodborne outbreaks and sporadic disease
High infectious dose (108 CFU)
Poultry, eggs, etc. are sources of infection
6-48h incubation period
Nausea, vomiting, nonbloody diarrhea, fever, cramps, myalgia and headache are common
S. enteritidis bioserotypes (e.g., S. typhimurium)

24. Pathogenesis of Salmonella
Enteritis (cont.)
Virulence attributable to:
Invasiveness
Intracellular survival & multiplication
Endotoxin
Exotoxins: Effects in host have not been identified
Several Salmonella serotypes produce enterotoxins similar to both the heat-labile (LT) and heat-stable enterotoxins (ST), but their effect has not been identified
A distinct cytotoxin is also produced and may be involved in invasion and cell destruction

25. Pathogenesis of Salmonella (cont.)
Invasiveness in Enteritis (cont.)
Penetrate mucus, adhere to and invade into epithelial layer (enterocytes) of terminal small intestine and further into subepithelial tissue
Bacterial cells are internalized in endocytic vacuoles (intracellular) and the organisms multiply
PMN’s confine infection to gastrointestinal (GI) tract, but organisms may spread hematogenously (through blood, i.e., septicemia) to other body sites
Inflammatory response mediates release of prostaglandins, stimulating cAMP and active fluid secretion with loose diarrheal stools; epithelial destruction occurs during late stage of disease

26. Epidemiology & Clinical Syndromes (cont.)
Enteric Fevers
S. typhi causes typhoid fever S. paratyphi A, B (S. schottmuelleri) and C (S. hirschfeldii) cause milder form of enteric fever
Infectious dose = 106 CFU
Fecal-oral route of transmission
Person-to-person spread by chronic carrier
Fecally -contaminated food or water
10-14 day incubation period
Initially signs of sepsis/bacteremia with sustained fever (delirium) for > one week before abdominal pain and gastrointestinal symptoms

27. Enteric Fevers (cont.) Virulence attributable to Invasiveness
Pass through intestinal epithelial cells in ileocecal region,
infect the regional lymphatic system, invade the bloodstream,
and infect other parts of the reticuloendothelial system
Organisms are phagocytosed by macrophages and monocytes,
but survive, multiply and are transported to the liver, spleen,
and bone marrow where they continue to replicate
Second week: organisms reenter bloodstream and cause
prolonged bacteremia; biliary tree and other organs are
infected; gradually increasing sustained fever likely from
endotoxemia
Second to third week: bacteria colonize gallbladder, reinfect
intestinal tract with diarrheal symptoms and possible necrosis
of the Peyer’s patches

28. Epidemiology & Clinical Syndromes (cont.)
Septicemia
Can be caused by all species, but more commonly associated with S. choleraesuis, S. paratyphi, S. typhi, and S. dublin
Old, young and immunocompromised (e.g., AIDS patients) at increased risk

29. Epidemiology & Clinical Syndromes (cont.) Asymptomatic Carriage
Chronic carriage in 1-5% of cases following S. typhi or S. paratyphi infection
Gall bladder usually the reservoir
Chronic carriage with other Salmonella spp. occurs in <<1% of cases and does not play a role in human disease transmission

30. Treatment, Prevention and Control
Enteritis:
Antibiotics not recommended for enteritis because they prolong duration of illness
Control by proper preparation of poultry & eggs
Enteric fever:
Antibiotics to avoid carrier state
Identify & treat carriers of S. typhi & S. paratyphi
Vaccination can reduce risk of disease for travellers in endemic areas

31. The Shigellae: Epidemiology and Clinical Syndromes
Four species: S. sonnei, S. flexneri, S. boydii, and S. dysenteriae
Low infectious dose ( CFU)
Humans are the 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 2nd stage) in pediatric age group (1-10 yrs) via fecal-oral route
Outbreaks in daycare centers, nurseries, institutions
Leading cause of infant diarrhea and mortality (death) in developing countries

32. Pathogenesis of 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

33. Pathogenesis and Virulence Factors (cont.)
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

34. Pathogenesis and Virulence Factors (cont.)
Invasiveness in Shigella -Associated Dysentery
Penetrate through mucosal surface of colon (colonic mucosa) and invade and multiply in the colonic epithelium but do not typically invade beyond the epithelium into the lamina propria (thin layer of fibrous connective tissue immediately beneath the surface epithelium of mucous membranes)
Preferentially attach to and invade into M cells in Peyer’s patches (lymphoid tissue, i.e., lymphatic system) of small intestine

35. Pathogenesis and Virulence Factors (cont.)
Characteristics of Shiga Toxin
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

36. Pathogenesis and Virulence Factors (cont.)
Shiga Toxin Effects in Shigellosis
Enterotoxic Effect:
Adheres to small intestine receptors
Blocks absorption (uptake) of electrolytes, glucose, and amino acids from the intestinal lumen

37. Pathogenesis and Virulence Factors (cont.)
Shiga Toxin Effects in Shigellosis (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

38. Yersenia
Most important species is Yersenia pestis, the causative agent of the most devastating disease in history; plague (black death).
Two epidemiologic forms, urban and sylvatic plague.
Urban plague is maintained in rat populations and spread among rats by infected fleas (humans are accidentally infected). It has been controlled

39. Sylvatic plague is impossible to eliminate because the mammalian reservoirs ( prairie dogs, mice, rabbits, rats) and flea vectors are wide spread.
Infection can be acquired by ingestion of contaminated animals or handling contaminated animal tissues.
Person to person transmission in pneumonic plague.
Classically bubonic plague.

40. Epidemiology and History of Plague
Zoonotic infection; Humans are accidental hosts
Outbreaks are cyclical corresponding to rodent reservoir and arthropod vector populations
Plague was recorded more than 2000 years ago
Three pandemics
1st 542 AD; 100 million dead in 60 years; from N.Africa
2nd 14th century; Black Death; 25 million dead in Europe alone (>1/4 of entire population); from central Asia; disease became endemic in urban rat population and smaller epidemics occurred through 17th century
3rd ended in 1990s; Burma to China (1894) & Hong Kong to other continents including N. America via rat-infected ships; 20 million dead in India alone; foci of infection firmly established in wild rodents in rural areas

41. Arthropod-Borne Transmission of Plague
Fleas required for perpetuation of plague vary greatly in vector efficiency and host range
Organisms ingested during blood meal from bacteremic host
Coagulase of flea may cause fibrin clot of organism in stomach which fixes to spines of proventriculus (throat parts of flea)
Organisms multiply causing blockage
Flea regurgitates infectious material into new host during subsequent attempts at blood meal
Flea remains hungry & feeds more aggressively
Sudden eradication of rats could lead to outbreak

42. Summary of Yersinia Infections
Clinical Forms of Plague (Black Death):
Bubonic plague with swollen and painful axillary (armpit) & inguinal (groin) lymph nodes (buboes)
Transmitted from mammalian reservoirs by flea (arthropod) bites or contact with contaminated animal tissues
Pneumonic plaque
Person-to-person spread
Yersinia enterocolitica
Enterocolitis
Transfusion-related septicemia