Enterobacteriaceae

Dr. Thanaa Rasheed

• The Enterobacteriaceae is a large family of gram-negative rods found

primarily in the intestinal tract of humans and animals. They cause a

variety of diseases.

• This family includes:

• Escherichia coli, Shigella, Salmonella, Klebsiella, Enterobacter,

Serratia ,Proteus , Yersinia and others in which some of them are

normal flora such as E. coli (coliform).

•

General properties

1.They are all facultative anaerobes. (2) They

all ferment glucose (3) None have

cytochrome oxidase (i.e., they are oxidase-

negative) and (4) they reduce nitrates to

nitrites as part of their energy-generating

processes.

Antigenic structure and Virulence factors

• Antigenic structure used for identification purposes both in the clinical laboratory

and in epidemiologic investigations.

O antigens

The cell wall antigen (also known as the somatic Ags) is the outer polysaccharide

portion of the cell wall lipopolysaccharide.

Is the basis for the serologic typing of many enteric rods.

O antigens are resistant to heat and alcohol and usually are detected by

agglutination test.

Antibodies to O antigens are predominantly IgM.

Antigenic structure

• H antigen

Is on the flagellar protein(flagellae) .

Only on flagellated organisms, such as Escherichia and Salmonella.

The H antigens denaturized or removed by heat and alcohol.

H Ags agglutinate with anti-H Ag Antibodies (IgG).

Antigenic structure

• K antigen

The capsular or K polysaccharide antigens are external to O Ag on

some but not all Enterobacteriaceae such as Klebsiella .

Some K Ag are polysaccharides and others are proteins.

The K antigen is identified by the quellung (capsular swelling)

reaction in the presence of specific antisera .

In Sal. typhi, the cause of typhoid fever, it is called the Vi (or

virulence) antigen.

This Ag may interfere with agglutination by O-Antisera.

K Ags of E coli cause attachment of the bacteria to the epithelial

cell prior to GIT or UT invasions.

Colicins (Bacteriocins)

Many gram-negative organisms produce bacteriocins.

These high-molecular-weight bactericidal proteins are produced by

certain strains of bacteria active against some other strains of the

same or closely related species.

Their production is controlled by plasmids.

e.g: Colicins are produced by E coli; marcescens by Serratia, and

pyocins by Pseudomonas.

Bacteriocin-producing strains are resistant to their own bacteriocin;

thus, bacteriocins can be used for "typing" of organisms.

Toxins

• Most gram-negative bacteria possess complex

lipopolysaccharides in their cell walls (endotoxins).

• These substances; cell envelope (cytoplasmic

membrane, peptidoglycan, outer membrane)

endotoxins, have a variety of pathophysiologic

effects.

• Many gram-negative enteric bacteria also produce

exotoxins of clinical importance.

Classification

• Genus and species designations are based on phenotypic

characteristics, such as

Patterns of carbohydrate fermentation and amino acid

breakdown.

• The O, K, and H antigens are used to further divide some

species into multiple serotypes. These types are expressed

with letter and number of the specific antigen, such as

Escherichia coli O157:H7.

Diseases Caused by Enterobacteriaceae other than Salmonella &

Shigella

1. E coli, Proteus, Enterobacter, Klebsiella, Morganella, Providencia, Citrobacter, and

Serratia spp all are normal flora of the upper respiratory and genetal tracts.

2. The bacteria become pathogenic only when they reach tissues outside of their

normal sites (urinary tract, biliary tract, and other sites in the abdominal

cavity).

3. Some of the enteric bacteria (eg, Serratia marcescens, Enterobacter aerogenes) are

opportunistic pathogens.

4. When normal host defenses are inadequate—particularly in infancy or old age, in

the terminal stages of other diseases, after immunosuppression, or with

indwelling venous or urethral catheters—localized clinically important

infections can result, and the bacteria may reach the bloodstream and cause

sepsis.

 

Escherichia coli (E coli)

• Antigenic structure of Escherichia coli

1. The O, H and K antigen (is the polysaccharide capsule present in

some strains).

2. Pili: Most E coli have type 1 (common) hair-like pili extending

from the surface. Pili play a role in virulence as mediators of

attachment to specific receptor on human epithelial surfaces.

3. Toxins: Exotoxin , α -hemolysin ,cytotoxic necrotizing factor

(CNF) ,Shiga like toxin (Stx), Labile toxin (LT), Stable toxin (ST)

Pathogenesis & Clinical Findings

• E. coli

A. Urinary tract infection—E coli is the most common cause and accounts

for approximately 90% of first urinary tract infections in young women

• Symptoms

1. Urinary frequency, dysuria, hematuria, and pyuria.

2. Flank pain is associated with upper tract infection.

3. Bacteremia with clinical signs of sepsis.

4. Nephropathogenic E.coli typically produce hemolysin, which is

cytotoxic and facilitates tissue invasion.

B. E. coli-associated diarrheal diseases

E coli that cause diarrhea are common worldwide.

E coli are classified by the characteristics of their virulent properties as:

Enteropathogenic E coli (EPEC):

Is an important cause of diarrhea in infants, especially in developing countries. Previously was

associated with outbreaks of diarrhea in nurseries in developed countries.

Pathogenesis

1) EPEC adhere to the mucosal cells of the small bowel.

2) Chromosomally mediated factors promote tight adherence.

3) There is loss of microvilli (effacement)

4) Formation of filamentous actin pedestals or cup-like structures, and, occasionally, entry

of the EPEC into the mucosal cells.

5) The result of EPEC infection is watery diarrhea, which is usually self-limited but can be

chronic cured by antibiotic treatment.

Enterotoxigenic E coli (ETEC):

Is a common cause of "traveler's diarrhea" and a very important cause of diarrhea in infants in

developing countries.

• Pathogenesis

1. ETEC colonization factors specific for humans promote adherence of bacteria to

epithelial cells of the small bowel.

2. Some strains of ETEC produce a heat-labile exotoxin (LT).It is under the genetic control

of a plasmid. Its subunit B attaches to the ganglioside at the brush border of epithelial

cells of the small intestine and facilitates the entry of subunit A into the cell, where the

latter activates adenylyl cyclase. This markedly increases the local concentration of cyclic

adenosine monophosphate (cAMP), which results in intense and prolonged hypersecretion

of water and chlorides and inhibits the reabsorption of sodium. The gut lumen is

distended with fluid, and hypermotility and diarrhea ensue, lasting for several days.

3. Some strains of ETEC produce the heat-stable enterotoxin STa which is under the

genetic control of a heterogeneous group of plasmids. STa activates guanylyl cyclase in

enteric epithelial cells and stimulates fluid secretion.

4. Many STa-positive strains also produce LT. The strains with both toxins produce a more

severe diarrhea.

Enterohemorrhagic E coli (EHEC):

Also known as Shiga toxin producing E coli (STEC), Produce cytotoxic

toxins (verotoxins). There are at least two antigenic forms of the toxin referred

to as Shiga-like toxin 1 and Shiga-like toxin 2. E coli O157:H7 is the most

common serotype and is the one that can be identified in clinical specimens.

• Symptoms

1. E coli hemorrhagic colitis, a severe form of diarrhea.

2. Hemolytic uremic syndrome, a disease resulting in acute renal failure

•Microangiopathic hemolytic anemia and thrombocytopenia.

•Control

• Can be prevented by thoroughly cooking ground beef.

Enteroinvasive E coli (EIEC):

1) Produces a disease very similar to shigellosis.

2) The disease occurs most commonly in children in developing countries and in

travelers to these countries.

3) Like Shigella, EIEC strains are non-lactose or late lactose fermenters and are

nonmotile.

4) EIEC produce disease by invading intestinal mucosal epithelial cells.

Enteroaggregative E coli (EAEC):

1) Causes acute and chronic diarrhea in persons in developing countries.

2) Also cause food-borne illnesses in industrialized countries.

3) They are characterized by their specific patterns of adherence to human cells.

4) EAEC produce ST-like toxin and a hemolysin.

C. Sepsis1) When normal host defenses are inadequate, E coli may reach the

bloodstream and cause sepsis.

2) Newborns may be highly susceptible to E coli sepsis because they

lack IgM antibodies.

3) Sepsis may occur secondary to urinary tract infection.

D. Meningitis

4) E coli is the leading causes of meningitis in infants.

5) Approximately 75% of E coli from meningitis cases have the K1

antigen.

Klebsiella, Enterobacter, Serratia, Proteus, Morganella, Providencia; &

Citrobacter

Klebsiella

1) K pneumoniae is present in the respiratory tract and feces of about 5% of normal

individuals.

2) It causes a small proportion (about 1%) of bacterial pneumonias.

3) K pneumoniae can produce extensive hemorrhagic necrotizing consolidation of the

lung.

4) It produces urinary tract infection and bacteremia with focal lesions in debilitated

patients.

5) Klebsiella sp. rank among the top ten bacterial pathogens responsible for hospital-

acquired infections.

6) Klebsiella ozaenae is the other klebsiellae are associated with inflammatory

conditions of the upper respiratory tract has been isolated from the nasal mucosa. .

Enterobacter

1) Three species of Enterobacter, E cloacae, E aerogenes, and E sakazakii .

2) These bacteria ferment lactose, it has small capsules that produce

mucoid colonies and they are motile.

3) These organisms cause a broad range of hospital acquired infections

such as pneumonia, urinary tract infections, wound and device

infections.

Serratia

1) S marcescens is a common opportunistic pathogen in hospitalized patients.

2) Serratia (usually nonpigmented) causes pneumonia, bacteremia and

endocarditis especially in narcotics addicts and hospitalized patients.

Enterobacter Serratia

Proteus

1) Proteus species produce infections in humans only when the bacteria leave the

intestinal tract.

2) They are found in urinary tract infections and produce bacteremia, pneumonia,

and focal lesions in debilitated patients or those receiving intravenous infusions.

3) P mirabilis causes urinary tract infections and occasionally other infections.

4) Proteus vulgaris and Morganella morganii are important nosocomial pathogens.

5) Proteus species produce urease, resulting in rapid hydrolysis of urea with

liberation of ammonia. Thus, in urinary tract infections with Proteus, the urine

becomes alkaline, promoting stone formation.

6) The rapid motility of Proteus may contribute to its invasion of the urinary tract.

Acinetobacter baumannii 

1) Is an opportunistic human pathogen.

2) Part of the normal microflora of between 25-70% of the population .

3) Infections appear to be concentrated in intensive care units (ICUs),

burns wards, high dependency units (HDUs) and other areas where

very sick patients reside.

4) There has also been a trend of military personnel in Iraq and

Afghanistan acquiring A. baumannii infections upon repatriation.

5) It is thought that A. baumannii can also be spread by airborne

transmission, person-to-person contact, through contaminated

environmental surfaces and medical equipment.

Providencia1) Providencia species (Providencia rettgeri, Providencia

alcalifaciens, and Providencia stuartii) are members of the

normal intestinal flora.

2) All cause urinary tract infections and occasionally other

infections and are often resistant to antimicrobial

therapy.

Citrobacter

Cause urinary tract infections and sepsis.

Diagnostic Laboratory Tests

Specimens: Specimens include urine, blood, pus, spinal fluid, sputum, or other material, as indicated by the localization of the disease process.

Smears :For Gram stain. The Enterobacteriaceae resemble each other morphologically. The presence of large capsules is suggestive of Klebsiella.

Culture :Specimens are plated on enrichment ,blood agar and differential media.

Biochemical tests : Very important.

Serotyping.

Treatment The sulfonamides, ampicillin, cephalosporins, fluoroquinolones,

and aminoglycosides have marked antibacterial effects against the

enteric bacteria with variation in the susceptibility so the laboratory

tests for antibiotic susceptibility are essential.

Multiple drug resistance is common and is under the control of

transmissible plasmids.

Certain conditions predisposing to infection by these organisms

require surgical correction, eg, relief of urinary tract obstruction,

closure of a perforation in an abdominal organ, or resection of a

bronchiectatic portion of lung.

Treatment of gram-negative bacteremia and impending septic shock

requires rapid institution of antimicrobial therapy, restoration of

fluid and electrolyte balance, and treatment of disseminated

intravascular coagulation.

The Shigellae

• The natural habitat of shigellae is the intestinal tracts of humans and

other primates, where they produce bacillary dysentery. Shigellae are

transmitted by "food, fingers, feces, and flies" from person to person.

Most cases of Shigella infection occur in children under 10 years of age.

There are four Groups and Types of shigellae:

• Group A Shigella dysenteriae

• Group B Shigella flexneri

• Group C Shigella boydii

• Group D Shigella sonnei

General properties

1) Shigellae are slender gram-negative rods; coccobacillary forms

occur in young cultures.

2) Culture: Shigellae are facultative anaerobes but grow best

aerobically. Convex, circular, transparent colonies with intact

edges reach a diameter of about 2 mm in 24 hours.

3) Growth Characteristics: All shigellae ferment glucose. With the

exception of Shigella sonnei, they do not ferment lactose on

differential media.

4) Shigellae form acid from carbohydrates but rarely produce gas.

They may also be divided into those that ferment mannitol and

those that do not.

Pathogenesis & Pathology

1) Shigella infections are almost always limited to the gastrointestinal tract;

bloodstream invasion is quite rare.

2) Shigellae are highly communicable; the infective dose is on the order of 102

organisms

3) The essential pathologic process is invasion of the mucosal epithelial cells

(eg, M cells) by induced phagocytosis, escape from the phagocytic vacuole,

multiplication and spread within the epithelial cell cytoplasm, and passage

to adjacent cells.

4) Microabscesses in the wall of the large intestine and terminal ileum lead to

necrosis of the mucous membrane, superficial ulceration, bleeding, and

formation of a "pseudomembrane" on the ulcerated area.

5) Pseudomembrane consists of fibrin, leukocytes, cell debris, a necrotic

mucous membrane, and bacteria.

6) As the process subsides, granulation tissue fills the ulcers and scar tissue

forms.

• Toxins

Endotoxin :

a) Upon autolysis, all shigellae release their toxic lipopolysaccharide.

b) This endotoxin probably contributes to the irritation of the bowel wall.

Shigella dysenteriae Exotoxin :

a) S dysenteriae type 1 (Shiga bacillus) produces a heat-labile exotoxin that affects

both the gut and the central nervous system.

b) The exotoxin is a protein that is antigenic (stimulating production of antitoxin)

and lethal for experimental animals.

c) Acting as an enterotoxin, it produces diarrhea as does the E coli Shiga-like toxin.

d) It inhibits sugar and amino acid absorption in the small intestine.

e) Acting as a "neurotoxin," this material may contribute to the extreme severity

and fatal nature of S dysenteriae infections and to the central nervous system

reactions observed in them (ie, meningismus, coma).

Clinical Findings

1) After a short incubation period (1–2 days), there is a sudden onset

of abdominal pain, fever, and watery diarrhea.

2) A day or so later, as the infection involves the ileum and colon, the

number of stools increases; they are less liquid but often contain

mucus and blood.

3) Each bowel movement is accompanied by straining and tenesmus

(rectal spasms), with resulting lower abdominal pain.

4) Fever and diarrhea subside spontaneously in 2–5 days.

5) In children and the elderly, loss of water and electrolytes may lead

to dehydration, acidosis, and even death.

6) The illness due to S dysenteriae may be particularly severe.

7) On recovery, most persons shed dysentery bacilli for only a short

period, but a few remain chronic intestinal carriers.

• Diagnosis

Specimens: include fresh stool, mucus flecks, and rectal swabs for culture. Large

numbers of fecal leukocytes and some red blood cells often are seen

microscopically.

Serum specimens, if desired, must be taken 10 days apart to demonstrate a rise in

titer of agglutinating antibodies.

Culture: The materials are streaked on differential media (eg, MacConkey or

EMB agar) and on selective media (Hektoen enteric agar or Salmonella-Shigella

agar and XLD), which suppress other Enterobacteriaceae and gram-positive

organisms.

Colorless (lactose-negative) colonies are inoculated into triple sugar iron agar.

Organisms that fail to produce H2S, that produce acid but not gas in the butt

and an alkaline slant in triple sugar iron agar medium, and that are nonmotile

should be subjected to slide agglutination by specific Shigella antisera.

Serology: Serology is not used to diagnose Shigella infections.

Treatment

• Ciprofloxacin, ampicillin, doxycycline, and trimethoprim-

sulfamethoxazole can suppress acute clinical attacks of dysentery and shorten the duration of symptoms. Multiple drug resistance can be transmitted by plasmids, and resistant infections are widespread. Many cases are self-limited.

Control

Sanitary control of water, food, and milk; sewage disposal; and fly control; (2) isolation of patients and disinfection of excreta; (3) detection of subclinical cases and carriers, particularly food handlers; and (4) antibiotic treatment of infected individuals.

The Salmonellao Salmonellae are often pathogenic for humans or animals

when acquired by the oral route.

o They are transmitted from animals and animal products to

humans, where they cause enterocolitis, enteric fevers such

as typhoid fever, and septicemia with metastatic infections

such as osteomyelitis.

o The typhoidal species are Sal. typhi and Sal. Paratyphi A,

B. The nontyphoidal species are the many strains of Sal.

enteritidis. Sal. choleraesuis is the species most often

involved in metastatic infections.

General properties

1. Salmonellae vary in length. Most isolates are motile with peritrichous

flagella.

• Salmonellae grow readily on simple media, but they almost never ferment

lactose or sucrose.

• They form acid and sometimes gas from glucose and mannose.

• They usually produce H2S.

• They survive freezing in water for long periods.

• Salmonellae are resistant to certain chemicals (eg, brilliant green, sodium

tetrathionate, sodium deoxycholate) that inhibit other enteric bacteria;

such compounds are therefore useful for inclusion in media to isolate

salmonellae from feces.

Epidemiology

• Salmonella Typhi, Salmonella Choleraesuis, and perhaps Salmonella

Paratyphi A and Salmonella Paratyphi B are primarily infective for humans

and from a human source (carriers).

• The reservoir for human infection are poultry, pigs, rodents, cattle, pets

(from turtles to parrots), and many others.

• The organisms almost always enter via the oral route, usually with

contaminated food or drink.

• The mean infective dose to produce clinical or subclinical infection in

humans is 105–108 salmonellae (but perhaps as few as 103 Salmonella Typhi

organisms).

• Among the host factors that contribute to resistance to salmonella infection

are gastric acidity, normal intestinal microbial flora, and local intestinal

immunity.

Clinical Findings & Pathogenesis

• Salmonellae produce three main types of disease in humans, but mixed

forms are frequent:

A. Enteric Fevers (Typhoid fever)

• Salmonella Typhi (typhoid fever) is the most important.

• The ingested salmonellae reach the small intestine, from which they

enter the lymphatics and then the bloodstream.

• They are carried by the blood to many organs, including the intestine.

• The organisms multiply in intestinal lymphoid tissue and are excreted

in stools.

• After an incubation period of 10–14 days, fever, malaise, headache,

constipation, bradycardia, and myalgia occur.

• The fever rises to a high plateau, and the spleen and liver become

enlarged.

• Rose spots, usually on the skin of the abdomen or chest, are seen briefly

in rare cases.

• The white blood cell count is normal or low.

• In the preantibiotic era, the chief complications of enteric fever were

intestinal hemorrhage and perforation,

• The mortality rate was 10–15%. Treatment with antibiotics has reduced

the mortality rate to less than 1%.

• The principal lesions are hyperplasia and necrosis of lymphoid tissue (eg,

Peyer's patches), hepatitis, focal necrosis of the liver, and inflammation of

the gallbladder, periosteum, lungs, and other organs.

B. Bacteremia with Focal Lesions

• This is associated commonly with S choleraesuis but may be caused by any

salmonella serotype.

• Following oral infection, there is early invasion of the bloodstream (with

possible focal lesions in lungs, bones, meninges, etc), but intestinal

manifestations are often absent.

C. Enterocolitis (Formely gastroenteritis)

1. This is the most common manifestation of salmonella infection.

2. Salmonella Typhimurium and Salmonella Enteritidis are prominent, but enterocolitis

can be caused by any serotypes of salmonellae.

3. Eight to 48 hours after ingestion of salmonellae, there is nausea, headache,

vomiting, and profuse diarrhea, with few leukocytes in the stools.

4. Low-grade fever is common, but the episode usually resolves in 2–3 days.

5. Inflammatory lesions of the small and large intestine are present.

6. Bacteremia is rare (2–4%) except in immunodeficient persons.

Diagnosis

Specimens

Blood for culture must be taken repeatedly. In enteric fevers

and septicemias, blood cultures are often positive in the first

week of the disease.

Bone marrow cultures may be useful.

Urine cultures may be positive after the 2nd week.

Stool specimens also must be taken repeatedly. In enteric

fevers, the stools yield positive results from the 2nd or 3rd

week and in enterocolitis, during the 1st week.

A positive culture of duodenal drainage establishes the

presence of salmonellae in the biliary tract in carriers.

Bacteriologic Methods for Isolation of Salmonellae

a) Enrichment cultures—The specimen (usually stool) also is put into

selenite F or tetrathionate broth, both of which inhibit replication of

normal intestinal flora . After incubation for 1–2 days, this is plated on

differential and selective media.

b) Differential medium cultures—EMB, MacConkey, or deoxycholate

medium permits rapid detection of lactose nonfermenters.

c) Bismuth sulfite medium permits rapid detection of Salmonella typhi

which form black colonies because of H2S production.

d) Selective medium cultures—The specimen is plated on salmonella-

shigella (SS) agar, Hektoen enteric agar, XLD, or deoxycholate-citrate

agar.

Biochemical reaction Final identification—Suspect colonies from solid media

are identified by biochemical reaction patterns

Serologic Methods Are used to identify unknown cultures with known sera and may also be used to

determine antibody titers in patients with unknown illness and include:

a) Slide Agglutination Test

1)Known sera and unknown culture are mixed on a slide.

2) Clumping can be observed within a few minutes (+ve).

3)This test is particularly useful for rapid preliminary identification.

4)There are commercial kits available to agglutinate and serogroup

salmonellae by their O antigens: A, B, C1, C2, D, and E.

b) Tube dilution agglutination test (Widal test) Serum agglutinins rise sharply during the second and third weeks of

Salmonella Typhi infection.

The Widal test to detect these antibodies against the O , Vi and H antigens.

At least two serum specimens, obtained at intervals of 7–10 days, are needed to

prove a rise in antibody titer.

Serial dilutions of unknown sera are tested against antigens from

representative salmonellae.

False-positive and false-negative results occur.

Results

Rising titer against the O antigen of >1:320 active infection

Rising titer against the H antigen of >1:640 positive.

High titer of antibody to the Vi antigen carriers.

Rapid colorimetric and enzyme immunoassay

methods.

Treatment

Enteric fevers and bacteremias with focal lesions require antimicrobial

treatment while enterocolitis does not.

Antimicrobial therapy of invasive Salmonella infections is with ampicillin,

trimethoprim-sulfamethoxazole, or a third-generation cephalosporin.

Antimicrobial treatment of Salmonella enteritis in neonates is important.

In severe diarrhea, replacement of fluids and electrolytes is essential.

Susceptibility testing is an important adjunct to selecting a proper antibiotic.

In most carriers, the organisms persist in the gallbladder (particularly if

gallstones are present) and in the biliary tract. Some chronic carriers have

been cured by ampicillin alone, but in most cases cholecystectomy must be

combined with drug treatment.