9th Lect.

Bacteriology

Enterobacteriaceae
(Part 2)

Shigellae, Salmonella
and Pseudomonas

By: Prof. Dr. Thanaa Rasheed Abdulrahman

Objectives
To study the medically important species of
Shigellae and Salmonella and Pseudomonas with
special emphasis on the pathogenesis & pathology
and diagnosis of these bacteria
 Shigella
Shigellosis (Bacillary dysentery )
The natural habitat of shigellae is the intestinal tracts of humans and other
primates.

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 Gram-negative rods , non motile.

2) Facultative anaerobes but grow best aerobically.

3) They do not ferment lactose on MaCconkey agar

with the exception of Shigella sonnei.

4) Shigellae form acid but rarely produce gas.

5) Group A mannitol non ferment.

➢Pathogenesis Highly communicable; the infective dose is
102 organisms

1) Limited to the gastrointestinal tract, bloodstream invasion is

quite rare.

2) Shigella invade the mucosal epithelial cells (eg, M cells) by

phagocytosis, escape from the phagocytic vacuole,
multiplication and spread within the cytoplasm, and passage
to adjacent cells.
3) Microabscesses, necrosis, superficial ulceration, bleeding,
and formation of a "pseudomembrane“ in the large intestine
and terminal ileum
4) Then granulation and scar tissue fills the ulcers
Toxins
✓Endotoxin:
Upon autolysis, all shigellae release their toxic LPS contributes to the irritation
of the bowel wall.

✓Shigella dysenteriae Exotoxin :

a)S dysenteriae type 1 (Shiga bacillus) produces a heat-labile antigenic and
lethal exotoxin that affects both the gut and the central nervous system.

b)As an enterotoxin produces diarrhea. It inhibits sugar and amino acid

absorption in the small intestine.

c)As a "neurotoxin,“ cause meningismus, coma this contribute to the fatal

nature of S dysenteriae infections.
❖ Clinical Findings

1) Incubation period (1–2 days)

2) Abdominal pain, fever, and watery diarrhea. A
day or so later , stools increases; contain mucus
and sometimes blood, severe stomach cramping
or tenderness, straining and tenesmus (rectal
spasms), with lower abdominal pain.
3) Subside spontaneously in 2–5 days.
4) In children and the elderly, dehydration, acidosis,
and even death. On recovery, few remain chronic
intestinal carriers.
Diagnosis

❑Specimens: include fresh stool and rectal swabs for culture.

❑Smear: Large numbers of fecal leukocytes and red blood cells often are
seen microscopically.

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

or EMB agar) and on selective media (Hektoen enteric agar or Salmonella-
Shigella(SS agar) and Xylose Lysine Deoxycholate agar (XLD).
▪Biochemical tests e.g. triple sugar iron agar and that are non motile should
be subjected to slide agglutination by specific Shigella antisera.

▪API20 E and VITIK2 SYSTEM

❑ Serology: Serology is not used to diagnose Shigella infections.
➢Treatment

Ciprofloxacin and trimethoprim-sulfamethoxazole

Many cases are self-limited.

✓Control

Sanitary control of water, food, and milk; Sewage disposal;

and fly control.

Isolation of patients and disinfection of excreta.

Detection of subclinical cases and carriers, particularly food

handlers; and antibiotic treatment of infected individuals.
 Salmonella
oSalmonellae are often pathogenic for humans or animals.

oThey cause enterocolitis, enteric fevers known as

typhoid fever, and septicemia with osteomyelitis.

oThe typhoidal species are : Salmonella enterica serovar

Typhi and Paratyphi

Sal. typhi and Sal. paratyphi A, B.

oThe nontyphoidal species: Salmonella enterica serovar

Typhimurium (S. typhimurium), many strains of Sal.
enteritidis and Sal. choleraesuis
They are transmitted from animals and animal products to
humans.
✓General properties
Motile with peritrichous flagella.

Salmonellae grow on simple media, never ferment lactose

or sucrose. They form acid and sometimes gas from
glucose and mannose.
Produce H2S.
They survive freezing in water for long periods.
Are resistant to certain chemicals (eg, brilliant green,
sodium tetrathionate, sodium deoxycholate) that inhibit
other enteric bacteria; such compounds inclusion in media
to isolate salmonellae from feces.
✓Epidemiology

Salmonella typhi, Salmonella choleraesuis, and perhaps

Salmonella paratyphi A and B are 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 others.
The entery via the oral route with contaminated food or
drink.
The mean infective dose 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

A.Enteric Fevers (Typhoid fever)

Salmonella typhi is the most important.

The ingested salmonellae reach the small intestine ,they enter

the lymphatics and then the bloodstream. The organisms
multiply in intestinal lymphoid tissue and are excreted in
stools.

They are carried by the blood to many organs, including the

intestine.
After an incubation period of 10–14 days, fever rises to a high
plateau, malaise, headache, constipation, bradycardia, and
myalgia occur.

The spleen and liver become enlarged.

Rose spots in rare cases on the skin of the abdomen or chest

The principal lesions are hyperplasia and necrosis of lymphoid

tissue (eg, Peyer's patches), hepatitis with focal necrosis of the
liver, and inflammation of the gallbladder, periosteum, lungs,
and other organs.

The complications were intestinal hemorrhage and perforation

Treatment with antibiotics reduced the mortality rate from 10–

15% to less than 1%.
B. Bacteremia with Focal Lesions
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 (Formerly gastroenteritis)

Salmonella typhimurium and Salmonella enteritidis are prominent

1.8 to 48 hours after ingestion of salmonellae, there is nausea, headache,

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

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

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

4.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 – (stool) selenite F or tetrathionate broth,

b)Differential medium: EMB, MacConkey, or deoxycholate medium .

c) Selective medium: salmonella-shigella (SS) agar, Hektoen enteric agar,

XLD, or deoxycholate-citrate agar.

d)Bismuth sulfite medium permits rapid detection of Salmonella typhi which

form black colonies because of H2S production.

❑ Biochemical reactions
(TSI agar ) produce acid and gas +H2S API E20 and
VITIK 2 System
❑Serologic Methods
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)There are commercial kits available to agglutinate

serogroup salmonellae by their O antigens.
b) Tube dilution agglutination test (Widal test)
•Serum agglutinins rise sharply during the second and third weeks of
Salmonella typhi infection.
• At least two serum specimens, obtained at intervals of 7–10 days.
•Serial dilutions of unknown sera (antibodies) are tested against antigens
from representative salmonellae (O , Vi and H antigens).
•False-positive and false-negative results occur.
❑Results
•Rising titer against the O antigen of >1: 160 active infection
• Rising titer against the H antigen of >1:640 positive.
•High titer of antibody to the Vi antigen carriers.

❑ Enzyme immunoassay methods.

❑PCR
❑Treatment

•Enterocolitis does not treated except in neonates.

• Ampicillin, trimethoprim-sulfamethoxazole, or a third-

generation cephalosporin.

•Replacement of fluids and electrolytes is essential.

•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

Pseudomonas
The most important is Pseudomonas aeruginosa.

Cause opportunistic infections of multiple sites in

immunosuppressed. Strains of Pse. aeruginosa in cystic fibrosis
patients have a prominent slime layer (glycocalyx) mediates
adherence the organism to the respiratory tract and prevents
antibody from binding .

Burkholderia cepacia and Burkholderia pseudomallei

Respiratory tract infection in cystic fibrosis patients, UTI, and
sepsis
 Important Properties

• Pseudomonads are Gram-negative rods that resemble the members

of the Enterobacteriaceae but they are strict aerobes, i.e., derive
energy only by oxidation of sugars rather than by fermentation(
Oxidase-positive).

• Not ferment lactose and glucose

• Pse. aeruginosa produces two pigments useful in clinical and

laboratory diagnosis:

1. Pyocyanin, give blue pus color in skin infection and burn

patients.

2. Pyoverdin (fluorescein), a yellow-green pigment in the laboratory.

 Pathogenesis
Pathogenesis is based on multiple virulence factors:

• Endotoxin, causes the symptoms of sepsis and septic shock.

•Exotoxin A, which causes tissue necrosis. It inhibits eukaryotic

protein synthesis by ADP-ribosylation of elongation factor 2.

•Enzymes, such as elastase and proteases, facilitate invasion of the

organism into the bloodstream.

•Pyocyanin damages the cilia and mucosal cells of the respiratory

tract.

•Pyocin is a type of bacteriocin.

Epidemiology
Normal flora of the colon in 10% .

•Found on moist areas of skin and upper respiratory tract of hospitalized

patients.

•Pse. aeruginosa has ability to withstand disinfectants, antiseptics and

detergents in hospitals causes contamination of respiratory therapy and
anesthesia equipment, intravenous fluids, and even distilled water.

• In immunosuppressed. In those with neutrophil counts of less than

500/µL;

• And in those with indwelling catheters.

• It causes 10% to 20% of nosocomial pneumonia.

 Clinical Findings
Urinary tract infections, pneumonia (especially in cystic
fibrosis patients), Burns and wound infections, sepsis.
Mortality rate of greater than 50% in sepsis,

Cause black, necrotic lesions in skin called ecthyma

gangrenosum and osteochondritis of the foot. It cause
endocarditis in intravenous drug users. Corneal infections
in contact lens users. Severe malignant otitis externa.
Folliculitis occur in users of swimming pools and hot tubs.
Laboratory Diagnosis
•Pse. aeruginosa grows as non–lactose-fermenting (colorless)
colonies on MacConkey's or EMB agar.

•Biochemical reactions It is oxidase-positive.

• A typical metallic sheen of the growth on TSI agar, coupled with

the blue-green pigment on ordinary nutrient agar and Tryptic Soy
Agar and a fruity aroma,.

• Identification for epidemiologic purposes is done by

bacteriophage or pyocin typing.
Tryptic Soy Agar - Pseudomonas
aeruginosa. Note the blue-green
color due to pyocianin production
by the bacteria
Treatment

The treatment of choice is antipseudomonal penicillin, e.g.,

piperacillin/tazobactam or ticarcillin/clavulanate, plus an
aminoglycoside, e.g., gentamicin or amikacin.
The drug of choice for urinary tract infections is ciprofloxacin.

Prevention
Prevention involves: keeping neutrophil counts above 500/µL,
removing indwelling catheters, taking special care of burned skin,
Thank you