ENTEROBACTERIACEAECHAPTER 33 (P. 579-605)

Dr. Jumana Abbadi

Enterobacteraceae

• Enterobacteraceae or enteric bacteria are a group of

bacteria that commonly colonize and infect the alimentary

tract (intestine).

• Most of these bacteria are part of the intestinal normal

flora; they are present in the intestine but they do not

cause disease. However, in certain conditions these

harmless bacteria may become harmful and cause

diseases such as urinary tract infections, wound site

infections, sepsis, etc… This is known as an opportunistic

infection.

• Enterobacteraceae include a large number of bacterial

Genera/species:

1. Escherichia

2. Klebsiella

3. Enterobacter

4. Citrobacter

5. Proteus

6. Salmonella

7. Shigella

8. Serratia

9. Yersinia

10. Morganella

11. Providencia

• Salmonella and Shigella species are not considered

members of the normal flora.

• The Enterobacteriaceae produce the widest variety of

infections of any group of microbial agents, including two

of the most common infectious states, urinary tract

infections (UTI) and acute diarrhea. Enterobacteriaceae

are by far the most common cause of UTIs.

• Culture is the primary method of diagnosis; all

Enterobacteriaceae are readily isolated on routine media

under almost any incubation conditions.

• The cell wall, cell membrane, and internal structures are

morphologically similar for all Enterobacteriaceae.

• Components of the cell wall and surface form the basis of

the system that divides species into serotypes:

1. The outer membrane lipopolysaccharide (LPS) is called

the O antigen.

2. Cell surface polysaccharides may form a well-defined

capsule or an amorphous slime layer and are termed the K

antigen.

3. Motile strains have protein flagella, which extend well

beyond the cell wall and are called the H antigen.

ESCHERICHIA COLI

• Escherichia Coli (E. coli) is the most commonly isolated

enterobacteriaceae.

• It is present as normal flora in the intestinal tract but under

certain circumstances E. coli can enter into sterile sites

and cause opportunistic infections.

• There are more than 700 different serotypes of E. coli.

Virulence Factors

• Fimbriae (Pili)

• Hemolysins

• Flagella (H antigen)

• Exotoxins

• Endotoxin (LPS or O antigen)

• Capsule (K antigen)

• Antigenic variation

• Drug resistance plasmids

Pili

• Main function: attachment

• There are different types of pili:

1. Type 1 or common pili: bind to d-mannose residues

commonly present on wide variety of cell types.

2. P pili: bind to di-galactoside (Gal–Gal) moieties on

kidney cells; leading to urinary tract infections.

3. Pili that mediate binding to enterocytes are found

among the diarrhea-causing E coli (BFP, CFA, etc)

Exotoxins

1. Alpha-hemolysin:

• a pore forming toxin that causes leakage of

cytoplasmic contents and eventually cell death.

2. Cytotoxic necrotizing factor (CNF)

• Often produced inconcert with α-hemolysin.

• It disrupts signaling pathways in the cell cytoplasm

leading to cytoskeleton rearrangement and apoptosis.

3. Heat-labile toxin (LT):

• Also an A–B toxin.

• Function: stimulates chloride secretion out of the cell

and the blockage of NaCl absorption.

• The net effect is the secretion of water and

electrolytes into the bowel lumen leading to diarrhea

• LT is less potent than CT (cholera toxin).

4. Heat-stable toxin (ST):

• is a small peptide that causes an LT-like net secretion

of fluid and electrolytes into the bowel

5. Shiga toxin (Stx)

• An A-B type toxin

• The B subunit directs binding

to a specific glycolipid receptor

(Gb3) present on eukaryotic

cells and is internalized in an

endocytotic vacuole.

• Inside the cell, the A subunit

enzymatically modifies the

ribosome site where tRNA

binds.

• This alteration blocks protein

synthesis, leading to cell

death.

E. Coli opportunistic infections

• E. coli can cause a wide variety of infections, such as:

a) Urinary tract infections:

Uropathogenic E. coli (UPEC)

b) Meningitis

c) Intestinal infections (diarrhea)

Urinary tract infections

• E. coli is the most common cause of urinary tract

infections

• Fewer than 10 E. coli serotypes account for the majority of

UTI cases. These E. coli with enhanced potential to

produce UTI are called uropathogenic E coli (UPEC).

• The ability of UPEC to produce UTI is due to:

1. Type 1 pili

2. P pili

3. Motility driven by flagellae also plays a role both in access to the

bladder and swimming up the ureter to the kidney.

Pathogenesis

A. A few E coli have gained

access to the bladder owing

to mechanical disruptions

such as sexual intercourse

or instrumentation

(catheters).

B. During voiding, the bladder

has expelled the E coli,

which have only type 1 pili.

The P pili-containing

bacteria remain behind due

to the strong binding to the

P pili receptor.

C. The remaining E coli

have multiplied and are

causing a UTI (cystitis)

with inflammation and

hemorrhage. In some

cases, the bacteria

ascend the ureter to

cause pyelonephritis in

the kidney where the P

pili receptor is most

abundant.

E. coli intestinal infections

• There are 5 types that cause diarrhea:1. Enterotoxigenic E. coli (ETEC)

2. Enteropathogenic E. coli (EPEC)

3. Enterohemorrhagic E.coli (EHEC)

4. Enteroinvasive E. coli (EIEC)

5. Enteroaggregative E. coli (EAEC)

• Food and water contaminated with human waste and

person-to-person contact are the principal means of

infection.

• Diarrhea is the universal finding with E coli strains that are

able to cause intestinal disease.

• The nature of the diarrhea varies depending on the

pathogenic mechanism.

• Enterotoxigenic and enteropathogenic strains produce a watery

diarrhea.

• Enterohemorrhagic strains produce a bloody diarrhea.

• Enteroinvasive strains may cause dysentery with blood and pus

(white blood cells) in the stool.

Enterotoxigenic E coli (ETEC)

• Virulence factors: LT, ST and colonizing factor (CF) pili

• Disease:

• Traveler’s diarrhea; non invasive watery diarrhea.

• Diarrhea in infants (developing countries)

• Food and water contamination, animals not infected.

Enteropathogenic E. coli

• Virulence factors:1. Bundle-forming (Bfp) Pili.

2. An injection (type III) secretion system; it injects over 30 E. coli

secretion proteins (Esps) into the host cell cytoplasm. The E. coli

secretion proteins disturb intracellular signal transduction

pathways. Other Esps also cause a host of other intracellular

disruptions, including mitochondrial injury and induction of

apoptosis.

• Disease: Acute or chronic diarrhea in infants. It accounts

for up to 20% of diarrhea in infants in nurseries.

• Feco-oral route of infection

• Pathogenesis:• Enteropathogenic E coli initially attach to small intestine

enterocytes using bundle-forming (Bfp) pili to form clustered

microcolonies on the enterocyte cell surface. In addition to the

actions of the injection secretion system, this leads to localized

degeneration of the brush border, loss of the microvilli, and

changes in the cell morphology.

Enterohemorrhagic E. coli (EHEC)• Virulence factors:

1. Shiga like toxin2. Adhesive pili (long polar fimbriae [Lpf])

• Disease:• Hemorrhagic colitis: crampy abdominal pain, little or no fever, bloody diarrhea.• Hemolytic uremic syndrome (HUS); Serotype: O157:H7

• Pathogenesis:• The interaction of EHEC with enterocytes is much the same as that of EPEC,

except that EHEC strains do not form localized microcolonies on the mucosaand have their own adhesive pili (long polar fimbriae [Lpf]) which mediateattachment in the colon rather than the small intestine. This is sufficient tocause nonbloody diarrhea

• On top of this, Shiga toxin (Stx) production causes capillary thrombosis andinflammation of the colonic mucosa, leading to a hemorrhagic colitis and/orHUS.

• HUS: begins with oliguria, edema, and pallor. It may

progress to the triad of hemolytic anemia,

thrombocytopenia and renal failure.

• Requires transfusion and hemodialysis for survival.

• The mortality rate is 5%, and up to 30% of those who

survive suffer sequelae such as renal impairment or

hypertension

Enteroinvsive E. coli

• The biochemistry, genetics, and pathogenesis of

Enteroinvasive E coli (EIEC) strains are so close to those

of Shigella.

• Enteroinvasive E coli disease is essentially a mild version

of shigellosis/dysentery.

• The occasional documented outbreaks in industrialized

nations are usually linked to contaminated food or water.

Humans are the only known reservoir.

Enteroaggregative E. coli (EAEC)

• Enteroaggregative E coli (EAEC) isassociated with a protracted (>14days) watery diarrhea.

• Virulence factors:• Enteroaggregative E coli pili (aggregative

adherence fimbriae [AAF]) mediate tightadherence to the intestinal mucosa

• The pathogenesis of diarrheainvolves formation of a thick mucus–bacteria biofilm on the intestinalsurface.

ETEC EPEC EHEC EIEC EAEC

mild watery

diarrhea

last few days

mild watery

diarrhea

last few days,

but may

become chronic

Begins with mild

watery diarrhea

but may progress

into dysentery;

bloody diarrhea.

Vomiting

Abdominal

cramps.

HUS

last few days

Begins with mild

watery diarrhea

but may progress

into dysentery

last few days

mild watery or

bloody diarrhea

last for weeks

Diagnosis

• E. coli are easily isolated on routine culture media (blood

and MacConkey agars).

• E coli ferment lactose rapidly and are positive for indole. E. coli are negative for both urease and citrate utilization.

TREATMENT

• Acute uncomplicated UTIs are often treated empirically

using trimethoprim/sulfamethoxazole (TMP-SMX) or

fluoroquinolones.

• As for intestinal E. coli:

• Because most E. coli diarrheas are mild and self-limiting, treatment

is usually not an issue. When it is, rehydration and supportive

measures are the mainstays of therapy, regardless of the causative

agent.

• Because the risk of HUS may be increased by the use of

antimicrobial agents, their use is contraindicated when EHEC is

even suspected.

ETEC EPEC EHEC EIEC EAEC

TMP-SMX or

fluoroquinolones

TMP-SMX or

fluoroquinolones

Hemodialysis

C/I TMP-SMX or

fluoroquinolones

C/I Antimotility

agents

TMP-SMX or

fluoroquinolones

C/I Antimotility

agents

TMP-SMX or

fluoroquinolones

SHIGELLA

Shigella species

• Similar to that of E. coli with the exception that they lack

flagella.

• All Shigella species are nonmotile.

• The genus is divided into four species, which are defined

by biochemical reactions and specific O antigens.

• The Shigella species are:

1. Shigella dysenteriae (serogroup A)

2. Shigella flexneri (serogroup B)

3. Shigella boydii (serogroup C)

4. Shigella sonnei (serogroup D)

• Shigella is an invasive bacterial pathogen. It invades

enterocytes and cause dysentery.

• All Shigella species produce various molecular forms and

quantities of Shiga toxin (Stx). However, Shigelladysenteriae type A1 is the most potent producer of Stx;thus it causes the most severe disease.

Epidemiology

• Shigellosis is a strictly human disease with no animal

reservoirs.

• Shigella is typically spread person to person (feco-oral

transmission) under poor sanitary and hygienic conditions.

• This spread by person-to-person contact is so effective

because the infecting dose is extremely low, as few as 10

organisms can cause disease.

• Worldwide, it is consistently one of the most common

causes of infectious diarrhea

Pathogenesis

• Shigella is acid-resistant; survives passage through the

stomach to reach the intestine where it invades and

destructs the human colonic mucosa. This triggers an

intense acute inflammatory response with mucosal

ulceration and abscess formation.

• Shiga toxin (Stx), which is not essential for bacterial

invasion, but contributes to the severity of the illness. This

is probably due to systemic effects of the toxin, which can

include HUS.

Mechanism of Shigella invasion

1. The Shigella adhere

selectively to M cells. After

they enter, the bacteria

transcytose through M cells

into the underlying collection

of macrophages.

2. Inside macrophages, Shigella

escape phagosomes and

activate programed cell death

(apoptosis) in the

macrophage.

3. Shigella is released from the dead macrophages and is

now in contact with the basolateral side of enterocytes.

4. The bacteria then initiate a multistep invasion process

mediated by an injection (type III) secretion system

which induces cytoskeleton reorganization, actin

polymerization, and other changes particularly at the

cell surface.

5. This cytoskeleton modification process induces

engulfment and internalization of Shigella into the host

cell by endocytosis.

6. Almost immediately, they orient in parallel with the

filaments of the actin cytoskeleton of the cell and initiate

a process in which they control polymerization of the

monomers that make up the actin fibrils.• This process creates an actin “tail” at one end of the microbe, which

appears to propel it through the cytoplasm like a comet.

• This allows nonmotile Shigella to not only replicate in the cell but to

move efficiently through it.

7. The movement allows Shigella to invade the

neighboring enterocyte by forming finger-like

projections.

8. This cell-by-cell extension destroys enterocytes and

creates focal ulcers in the mucosa, particularly in the

colon.

9. Shigella eventually reach the lamina propria causing an

intense acute inflammatory response.• Extension of the infection beyond the lamina is unusual in healthy

individuals.

10. The diarrhea created by this process is almost purely

inflammatory, consisting of small-volume stools

containing WBCs, RBCs, bacteria, and little else. This

is classic dysentery.

Disease manifestations

• Acute inflammatory colitis and bloody diarrhea, which in

the most characteristic state presents as a dysentery

syndrome; a clinical triad consisting of:

1. Abdominal cramps

2. Painful straining to pass stools (tenesmus)

3. A frequent small-volume, bloody, mucoid, fecal discharge.

• Exception: shigellosis due to S. sonnei is a watery diarrhea.

1. The disease usually begins with fever and systemic

manifestations of malaise and anorexia.

2. These are followed by the onset of watery diarrhea containing

the large numbers of leukocytes detectable by light microscopy.

3. The diarrhea may turn bloody with or without the other classic

signs of dysentery.

Diagnosis

• All Shigella species are readily isolated using selective

media (Hektoen enteric agar), which are part of the

routine stool culture in all clinical laboratories.

• Isolates are identified with further biochemical tests such

as motility test; Shigella species are nonmotile.

• Slide agglutination tests using O group-specific antisera

(A, B, C, D) confirm both the species and the Shigella

genus.

Treatment

• Usually self-limiting, the beneficial effect of treatment is in

shortening the duration of the illness.

• Ampicillin, Ciprofloxacin, ceftriaxone, and azithromycin have

been used depending on susceptibility testing.

• Antispasmodic agents may aggravate the condition and are

contraindicated in shigellosis and other invasive diarrheas.

SALMONELLA

• The infecting dose of Salmonella species is generallyhigher than that of Shigella.

• Poultry products, including eggs, are most often

implicated in causing Salmonella gastroenteritis.

Virulence Factors

1. Type 1 pili

2. Motility through the action of their flagella.

3. Injection type III secretion system

4. Salmonella Typhi: has a surface polysaccharide capsule

called the Vi antigen.

Pathogenesis

• The invasion of enterocytes together with the associated

increased vascular permeability and inflammatory

response are the cause of the diarrhea.

• Pathogenesis:

1. Ingested S. enterica cells that pass the stomach acidand eventually reach the small bowel.

2. Initial contact there is with M cells, enterocytes or both,

and adherence is probably mediated by pili.

3. On engagement of S. enterica’sinjection (type III) secretion

systems, membrane “ruffles” are

created.

4. The ruffles seem to engulf the

organism in an endocytotic vacuole

and allow it to transcytose from the

apical surface to the basolateral

membrane.

These “ruffles”

are specialized

plasma

membrane

sites of

filamentous

actin

cytoskeletal

rearrangement.

5. Once in the cell, S enterica multiplies in a vacuoleand enters the lamina propria. There they induce

a profound inflammatory response and are

phagocytosed by neutrophils and macrophages.

6. However due to their ability to kill macrophages

by multiple mechanisms including induction of

apoptosis; S. enterica cells persist in the lamina

propria.

7. Although the process remains localized to the

mucosa and submucosa with most S entericastrains, some invade more deeply, reaching the

bloodstream and distant organs (Typhoid Fever).

Manifestations

• The clinical patterns of salmonellosis can be divided into:

1. Gastroenteritis; diarrhea, vomiting and abdominal

cramps, self-limited

2. Enteric fever.

3. Bacteremia (bacteria in bloodstream); most common

in immunocompromised individuals.

4. Asymptomatic carrier state.

Enteric fever

• Salmonella serotype Typhi and related serotypes

(Paratyphi) cause enteric fever.

• It is a multi-organ Salmonella infection characterized byprolonged fever, sustained bacteremia, and profound

involvement of the mesenteric lymph nodes, liver, and

spleen.

• The first sign of disease is fever associated with a

headache. The fever rises in a stepwise fashion over the

next 72 hours.

• A faint rash (rose spots) appears during the first few days

on the abdomen and chest.

• Many patients are constipated, although some patients

have a mild diarrhea.

• The persistent bacteremia (for 2 weeks or more) can

cause infection at other sites:

• Of particular importance is the biliary tree, with reinfection of

the intestinal tract and diarrhea late in the disease.

• However, the most important complication of typhoid fever is

hemorrhage from perforations through the wall of the colon.

Salmonella serotype Typhi

• It is the main cause of enteric Typhoid Fever.

• Typhoid fever is a strictly human disease.

• Transmission is by the fecal–oral route

• Chronic carriers of serotype Typhi are the primary

reservoir for example the infamous “Typhoid Mary”

Mallon.

Pathogenesis of Salmonella Typhi

• The invasion and killing of intestinal M cells and

macrophages are presumed to follow the same pattern as

that of other S enterica species.

• Two important differences add to the virulence of

Salmonella serotype Typhi:

1. Vi surface polysaccharide

2. Extended multiplication of Salmonella serotype Typhi in

macrophages.

• In the submucosa, Vi (for virulence) retards neutrophil

phagocytosis of Salmonella serotype Typhi. In addition,

the Vi positive phenotype favors intracellular multiplication

of the bacteria.

• Another primary difference between Typhi and the other

serotypes is the prolonged intracellular survival in

macrophages. This is due to the organism’s ability to

inhibit the oxidative metabolic burst and continue to

multiply.

• As the bacteria proliferate in macrophages, they are

carried through the lymphatic circulation to the mesenteric

nodes, spleen, liver, and bone marrow, all elements of the

reticuloendothelial system (RES).

• At the RES sites, Typhi continues to multiply, infecting

new host macrophages. Eventually, the increasing

bacterial population begins to overflow into the

bloodstream.

• The entry of Gram-negative bacteria and their LPS

endotoxin into the blood starts the fever, which slowly

increases and persists with the continued seeding of STyphi.

• This sometimes results in metastatic infection of other

organs including the urinary tract and the biliary tree. The

latter causes reinfection of the bowel.

• This cycle beginning and ending in the small intestine

takes approximately 2 weeks to complete.

Diagnosis of Salmonella species

• Culture of Salmonella from the blood or feces is theprimary diagnostic method. Blood culture is very effective

in diagnosing Typhoid fever causing Salmonellae.

• The media used for stool culture are the same as those

used for Shigella (Hektoen Enteric Agar).• Salmonella; does not ferment lactose and produces hydrogen

sulfide (H2S)

• O/Vi serogroup antisera are available in laboratories for

confirmation.

Motility Test Hektoen Enteric Agar

Shigella Salmonella

Treatment

• Treatment of Salmonella gastroenteritis:

• Fluid and electrolyte replacement and the control of nausea

and vomiting.

• Antibiotic therapy is usually not appropriate because it has a

tendency to increase the duration and frequency of the

carrier state. In these instances, antimicrobials are used only

as a measure to prevent systemic spread in very severe

infections.

• Treatment of Typhoid Fever:

• Antimicrobials must be used to treat Typhoid fever.

• Antimicrobials used for Salmonella species:

1. Ampicillin

2. Cephalosporins (ceftriaxone, cefixime)

3. Ciprofloxacin.

• These antimicrobials must be checked for susceptibility due

to an increase in resistance towards them between

Salmonella species.

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