Enterobacteriaceae Notes

Enterobacteriaceae

• Digestive tube (colon) of humans and animals is the typical habitat.
• Includes approximately 150 species.
• Facultative anaerobes.
• Associated with diarrheal illnesses.
• Causes approximately 3 million deaths per year.
• Responsible for approximately 4 billion infections worldwide.

Classification

• More than 15 different genera.
• Key genera include:
  • Escherichia
  • Shigella
  • Edwardsiella
  • Salmonella
  • Citrobacter
  • Klebsiella
  • Enterobacter
  • Hafnia
  • Serratia
  • Proteus
  • Providencia
  • Morganella
  • Yersinia
  • Erwinia
  • Pectinobacterium

Genus and Species

• Certain E. coli strains can be considered true pathogens.
• True pathogens include:
  • Citrobacter (4 species)
  • Edwardsiella (4 species)
  • Enterobacter (13 species)
  • Escherichia (5 species)
  • Shigella (nonmotile, 4 species)
  • Salmonella (2)
  • Serratia (7 subgroups)
  • Yersinia (2)
  • Ewingella (4)
    • Hafnia (5)
    • Klebsiella (nonmotile - 10 species)
    • Kluyvera (11)
    • Morganella (1)
    • Proteus
    • Providencia

Morphology and General Characteristics

• Facultative anaerobes.
• Gram-negative, non-sporing, rod-shaped bacteria.
• Motility via peritrichous flagella (except Shigella and Klebsiella).
• Many are normal inhabitants of the intestinal tract.
• Some are enteric pathogens, while others infect the urinary or respiratory tracts.
• Differentiation based on biochemical reactions and antigenic structure.

Morphology and Physiology

• Short, gram-negative rods.
• Grow readily and rapidly on simple media.
• Klebsiella pneumoniae has a large capsule, forming large and very mucoid colonies.
• Enterobacter has a smaller capsule.
• Others produce diffusible slime layers, forming circular, convex, and smooth colonies.

Physiology

• Glucose is fermented with strong acid formation and often gas.
• Reduce nitrates to nitrite.
• Do not liquefy alginate.
• Oxidase negative.
• Speciation based on:
  • Differences in carbohydrate fermentation.
  • Variations in end-product production.
  • Variation in substrate utilization.

Pathogenicity

• Opportunistic pathogens:
  • Escherichia coli
  • Klebsiella pneumoniae
  • Enterobacter aerogenes
  • Serratia marcescens
  • Proteus spp.
  • Providencia spp.
  • Citrobacter spp.
• Obligate pathogens:
  • Salmonella spp.
  • Shigella spp.
  • Yersinia spp.
  • Some E. coli strains
• Diseases caused:
  • Sepsis
  • Meningitis
  • UTI (Urinary Tract Infection)
  • Diarrhea
  • Pneumonia

Incidence of Bacteremia

• Escherichia: 45%
• Klebsiella: 22%
• Enterobacter: 20%
• Proteus: 4%
• Serratia: 4%
• Citrobacter: 2%
• Other: 3%

Cultural Characteristics

• Non-differential/non-selective media (blood agar, infusion agar):
  • No species distinction; appear as moist, smooth, gray colonies.
• Selective media:
  • Used to isolate Shigella and Salmonella from fecal matter.
• Differential media:
  • Selectively inhibit Gram-positive organisms.
  • Separate enterics into broad categories.

Modes of Infection

• Contaminated food and water (Salmonella spp., Shigella spp., Yersinia enterocolitica, Escherichia coli O157:H7).
• Endogenous (urinary tract infection, primary bacterial peritonitis, abdominal abscess).
• Abnormal host colonization (nosocomial pneumonia).
• Transfer between debilitated patients.
• Insect (flea) vector (Yersinia pestis).

Specific Infections

• Urinary Tract Infection, Pneumonia
  • Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus mirabilis
• Pneumonia:
  • Enterobacter spp., Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis
• Wound Infection:
  • Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis
• Bacteremia:
  • Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis
• Intestinal Infection:
  • Shigella sonnei (serogroup)
  • Salmonella serotype enteritidis
  • Salmonella serotype typhimurium
  • Shigella flexneri (serogroup)
  • Escherichia coli O157:H7
  • Yersinia enterocolitica

Media

• MacConkey (MAC) Agar
• Eosin Methylene Blue (EMB) Agar (Levine)
• HE Agar
• XLD Agar

Antigenic Structure

• H antigens: flagella.
• K antigen: capsule (Vi for Salmonella).
• O antigen: cell envelope (wall) – LPS (endotoxin).
• Outer membrane proteins.
• Pili: various antigen types, some encoded by plasmids.

Pathogenesis and Immunity

• Type III secretion systems: possessed by some Enterobacteriaceae pathogens (e.g., E. coli, Yersinia, Salmonella, and Shigella); facilitate transport of bacterial virulence factors directly into host cells.
• Endotoxin (Lipid A of LPS).
• Capsule
• Antigenic phase variation
• Acquisition of growth factors (e.g. Fe).
• Resistance to serum killing
• Antimicrobial resistance

Escherichia coli

• Toxins: two types of enterotoxin; Shiga-type toxin; Enteroaggregative ST-like toxin; Hemolysins; Endotoxin
• Virulence factors: Capsule/Iron capturing ability (enterochelin)

E. coli Characteristics

*May be hemolytic on CBA – more common in pathogenic strains

• KEY tests for the normal strain:
  • TSI is A/A + gas
  • Lysine Iron Agar K/K
  • Urea –
  • Indole +
  • Citrate –
  • Motility +
• There is an inactive biotype that is anaerogenic, lactose –, and nonmotile.

Enteropathogenic E. coli (EPEC)

• fever
• infant diarrhea
• vomiting
• nausea
• non-bloody stools
• Destruction of surface microvilli loose attachment mediated by bundle forming pili (Bfp);
• Stimulation of intracellular calcium level;
• rearrangement of intracellular actin,

Enterotoxigenic E. coli (ETEC)

• A watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days.
• Travellers diarrhea and diarrhea in children in developing countries
• Transmission is via contaminated food or water.

Enteroinvasive E. coli (EIEC)

• The organism attaches to the intestinal mucosa via pili
• Outer membrane proteins are involved in direct penetration, invasion of the intestinal cells, and destruction of the intestinal mucosa.
• There is lateral movement of the organism from one cell to adjacent cells.
• Symptoms- fever,severe abdominal cramps, malaise, and watery diarrhea followed by scanty stools containing blood, mucous,& pus.
• resembles shigellosis

Enteropathogenic E. coli (EPEC) (repeated content)

• Malaise and low grade fever diarrhea, vomiting, nausea, non-bloody stools
• Bundle forming pili are involved in attachment to the intestinal mucosa.
• This leads to changes in signal transduction in the cells, effacement of the microvilli, and to intimate attachment via a non-fimbrial adhesion called intimin.
• This is a problem mainly in hospitalized infants and in day care centers.

Enterohemorrhagic E. coli (EHEC)

• Hemorrhagic – bloody, copious diarrhea – few leukocytes – afebrile
• hemolytic-uremic syndrome:
  • hemolytic anemia
  • thrombocytopenia (low platelets)
  • kidney failure
• Usually O157:H7

Enteroaggregative E. coli (EAEC)

• a cause of persistent, watery diarrhea with vomiting and dehydration in infants.
• That is autoagglutination in a ‘stacked brick’ arrangement.
• the bacteria adheres to the intestinal mucosa and elaborates enterotoxins (enteroaggregative heat-stable toxin, EAST).
• The result is mucosal damage, secretion of large amounts of mucus, and a secretory diarrhea.

Shigella

• Contains four species that differ antigenically and, to a lesser extent, biochemically.
  • S. dysenteriae (Group A)
  • S. flexneri (Group B)
  • S. boydii (Group C)
  • S. sonnei (Group D)
• Causes bacillary dysentery, shigellosis, bloody feces, intestinal pain, pus
• Biochemistry:
  • TSI K/A with NO gas
  • LIA K/A
  • Urea –
  • Motility -
  • All ferment mannitol except S. dysenteriae
  • S. sonnei may show delayed lactose fermentation

Shiga toxin

1. Chromosomally encoded
2. Neurotoxic
3. Enterotoxic
4. Cytotoxic

• Enterotoxicity can make the disease clinically appear as a diarrhea.
• The toxin inhibits protein synthesis (acting on the 60S ribosome and lysing 28S rRNA).

Clinical significance of Shigella

• man only "reservoir"
• mostly young children:
  • fecal to oral contact
  • children to adults
• transmitted by adult food handlers
  • unwashed hands

Diagnosis of Shigella infection

• Specimen: stool.
• Culture and Identification
• Quick immunological methods:
  • Immunofluorescent “ball” test;
  • Coagglutination.

Salmonella

• Only a few types that are commonly associated with characteristic human diseases
  • S. enteritidis
  • S. cholerae-suis
  • S. typhi
• Causes several syndromes including gastroenteritis, enteric (typhoid) fever or septicemia

Salmonella Biochemistry

• TSI K/A + gas and H2S: S. typhi produces only a small amount of H2S and no gas , and S. paratyphi A produces no H2S
• LIA K/K with H2S with S. paratyphi A giving K/A results
• Urea –
• Motility +
• Citrate +/-
• Indole -
• Virulence factors
  • Endotoxin – may play a role in intracellular survival
  • Capsule (for S. typhi and some strains of S. paratyphi)
  • Adhesions – both fimbrial and non-fimbrial

Salmonella typhi Transmission

• The organism is transmitted from:
  1. a human reservoir
  2. in the water supply (if poor sanitary conditions)
  3. in contaminated food
• The antigenic structures of salmonellae used in serologic typing

Salmonella - Virulence factors

• Endotoxin – may play a role in intracellular survival
• Capsule (for S. typhi and some strains of S. paratyphi)
• Adhesions – both fimbrial and non-fimbrial
• Type III secretion systems and effector molecules
  • 2 different systems may be found:
    • One type is involved in promoting entry into intestinal epithelial cells
    • The other type is involved in the ability of Salmonella to survive inside macrophages
• Outer membrane proteins - involved in the ability of Salmonella to survive inside macrophages
• Flagella – help bacteria to move through intestinal mucous
• Enterotoxin - may be involved in gastroenteritis
• Iron capturing ability

Salmonella Diagnosis

• A. Specimens
  • a) Enteric fever: blood, bone marrow, stool, urine.
  • b) Food poisoning: stool, vomitus, suspected food.
  • c) Septicemia: blood.
• B. Culture and identification
• C. Widal test

Klebsiella

• NF of GI tract, but potential pathogen in other areas
• TSI A/A + gas
• LIA K/K
• Urea +
• Citrate +
• MR-, VP+
• Motility -
• Has both O and K antigens

Klebsiella Virulence and Clinical Significance

• Virulence factors
  • Capsule
  • Adhesions
  • Iron capturing ability
• Clinical significance
  • Causes pneumonia, mostly in immunocompromised hosts.
  • Permanent lung damage is a frequent occurrence (rare in other types of bacterial pneumonia)
  • A major cause of nosocomial infections such as septicemia and meningitis

Proteus

• General characteristics: “swarming” phenomenon on nonselective agar (P.vulgaris; P.mirabilis and P.myxofaciens)
• P.vulgaris strains (OX-19, OX-K, OX-2)have common antigen with Rickettsia (Weil-Felix test).
• urinary tract infections; food poisoning.

Proteus spp.

• Some enteric bacteria are motile. Klebsiella species are not motile, while Proteus species move very actively by means of peritrichous flagella, resulting in "swarming" on solid medium. Some strains of E. coli produce hemolysis on blood plates.

CITROBACTER

• Resident of soil, water, stool
  • C.freundii
  • UTI and bacteremia

Serratia

• A free-living saprophyte
• TSI A/A or K/A; +/- gas (does not ferment lactose)
• LIA usually K/K
• Citrate +
• Motility +
• Urea +/-
• Has been found in RT and UT infections
• Is resistant to many antimicrobics

Citrate Utilization Test

• Special Features: Used in the differentiation o genera and species. e.g E. coli (+) from Klebsiell -).
  • Positive test e.g. E. coli
  • Negative test e.g. Klebsiella

Indole test

• Positive Klebsiella, Enterobacter
• Negative E. coli

Urease Test

• Positive test
• Negative test

MR/VP test Results

• Methyl Red test
• Voges-Proskauer test
  • Red: Positive MR (E. coli)
  • Yellow or orange: Negative MR (Klebsiella)
  • Pink: Positive VP (Klebsiella)
  • No pink: Negative VP (E. coli)

Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

• LF: Lactose fermenter
• NLF: Non-lactose fermenter

Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae(continued)

Oxidase Test

• Positive:
  • Pseudomonas
• Negative:
  • Enterobacteriaceae

Additional Tests

• Nitrate test: +ve further reduction to N_2
• Growth on cetrimide agar: Pale colonies with green pigmentation
• MacConkey’s agar & TSI
  • Lactose fermenter: Pink colonies on MacConkey & acidic butt and slant on TSI
  • Lactose non-fermenter: colorless colonies on MacConkey & acidic butt alkaline slant onTSI
• IMViC test & EMB
  • IMViC ++ - - & black colonies with metalic shines on EMB E.coli
  • IMViC - - ++ Klebsiella

H2S Production

• No H2S production (no blacking in TSI)-Shigella
• H2S production (blacking in TSI)- Proteus, Salmonella
• Urease production
  • +ve (Proteus)
  • -ve (Salmonella, Shigella)
• SS agar colorless colonies with black centers- Salmonella
• O/F test: O+/F-
• Motility
  • Not motile-Enterobacter
  • Motile