Download Gram-Negative Rods

Source or site of infection (continued)
Respiratory tract:
 Haemophilus, Legionella, Bordetella
Animal sources:
Brucella, Francisella, Pasteurella, Yersinia
Gram-Negative rods related to the respiratory
tract
Haemophilus
Bordetella
Legionella
Francisella
Haemophilus
Coccobacillus
Non-motile
Blood needed for growth
Polymorphism
Able to ferment
Growth in BHIA
Haemophilus influenzae & parainfluenzae
Colonies: bulging, small and circular
Colonies on chaculate agar after 36 – 48 hours
Mostly having capsule loosing it on culture
No maemolysis on blood
Adding isovitales increases the growth
Two cell wall antigens: P and M
Haemophilus influenzae
vs
parainfluenzae
Two growth factors: 'X' (haem) and 'V' (NAD):
H. influenzae requires both, while H. parainfluenzae
requires only the 'V' factor, since it can make its own
haem.
Haemophilus influenzae
Mirobiology properties
- Small gram-negative rod (coccobacillus)
- Capsule (one of the 3 important encapsulated
pyogens along with pneumococcus and
meningococcus).
- 6 serotypes based on the capsule antigenic structure
(Hib) containing a polyribitol phosphate capsule)
causes most of the severe invasive diseases). Nonencapsulated strains are not included.
Haemophilus influenzae
For lab growth, the bacterium needs 2 components:
heme (Factor X) and NAD (Factor V).
Virulence factors
Antiphagocytic capsule
Endotoxin
No exotoxin
IgA protease
Pathogenesis & Epidemiology
IgA protease degrading secretory IgA  facilitating
attachment to the respiratory mucosa  entering the
bloodstream  spreading to the meninges.
It also causes otitis media, sinusitis or pneumonia.
In some people resulting in asymptomatic colonization.
Clinical finding
Meningitis:
fever, headache, stiff neck along with drowsiness.
Sinusitis and otitis:
Pain in the affected area, opacification of the infected
sinus, redness with bulging of the tympanic
membrane.
Clinical finding
H. Influenza is second causative agent for
Meningitis, Sinusitis and Otitis infections after
pneumococcus.
Pneumonia in eldely adults can be caused by
untypeable (non-encapsulated) strains.
Epiglottitis
Most infections occur in children between the
ages of 6 months and 6 years.
The peak age:
0.5-1 year old due to a decline in maternal IgG.
Lab diagnosis
Coccobacillus
Isolation on heated-blood (chocolate agar)
Capsular swelling (quellung) reaction test.
Fluorescent-antibody staining
Haemophilus satellite phenomenon
On fresh blood agar, colonies of Haemophilus
influenzae grow as satellite colonies around
Staphylococcus aureus. The fresh blood acts as a
supply of haem and the staphylococci provide NAD
(V factor).
Treatment
Untreated H. influenzae meningitis has a fatality rate
of 90%
Ampicillin, Cephalosporin, Ceftriaxone
Prevention
Antibiotic prophylaxis by Rifampin to reduce
respiratory carriage.
Vaccination:
contains capsular polysaccharide of H. influenza type
b given between the ages of 2-15 months.
Bordetella
Coccobacillus (approximately 0.8 µm by 0.4 µm)
Gram negative
Strictly aerobic
Fastidious
Capsule
Motile or nonmotile
Bipolar in staining
Colonies: haemolyses, small, smooth and convex
Mucoidal colony changing to R after a while
Bordetella pertussis
A non-motile Gram negative bacillus
Urease-negative, Oxidase-positive
Fastidious
Incubation: high CO2 concentration, 35 c, 7 days
incubation time
Needs: blood (20-30%), potato extract, and glycerol
Bordetella pertussis causes pertussis or whooping
Virulence factors
Endotoxin
Pertussis toxin (PTx) (Helps to colonize the respiratory
cilia)
Filamentous haemagglutinin (FHA) (binds to
galactose residues on cilia)
Pertactin (an outer membrane protein that promotes
adhesion to tracheal epithelial cells)
Fimbriae
Tracheal cytotoxin factor (TCF)(stops the cilia from
beating)
Dermonecrotic toxin
Adenylate cyclase toxin
Pathogenesis
All virulence factors together causing:
1. Cilia paralysis and prevent them from clearing
debris from the lungs, so the body responds by
sending the host into a coughing fit. These coughs
expel some bacteria into the air, which are free to
infect other hosts.
2. Inflammation and necrosis and reduction of
phagocytic activity to help promote infection.
Transmission
Only a human pathogen
It typically attacks children, since adults appear to
develop natural immunities to it through minor
exposure.
This disease is highly contagious.
Clinical findings Stage 1 (Cataral)
(about 1-2 weeks)
During the first stage, common symptoms of
whooping cough are similar to those of a cold or flu
and can include:
Sneezing
Runny nose
Nasal congestion
Slight fever
Dry cough that may get worse at night.
Stage 2 (Paroxysmal)
(Whooping Cough)
Lasts about 2 weeks and the symptoms get worse:
Fits of coughing that seem to go on and on
Skin turning blue during coughing episodes.
Severe coughing leads to vomiting, which may make
it difficult for a person to eat or drink
Severe coughing may end in a "whooping" sound
when the infected person inhales
Clinical findings
The organism is restricted to the respiratory tract and
blood culture is negative.
A pronounced leukocytosis with up to 70%
lymphocytes is seen.
Anoxia and exhaustion can occur.
Death is mainly due to pneumonia.
Clinical findings
Stage 3
Recovery, still caughing for a while.
Lab Samples
Isolation from throat/nasopharynx swabs taken
during the cataral and paroxysmal stage.
Lab diagnosis
Lab diagnosis
Culture on Bordet-Gengou medium (contains a high
percentage of blood (20-30%), potato extract, and
glycerol, with an antibiotic such as cephalexin or
penicillin
Biochemical tests
Agglutination with specific antiserum
Fluorescent-antibody staining
Treatment
Erythromycin
Inhalation of warm moist air, and tapping of the
chest to loosen debris in the lungs
Gamma globulin
Prevention
Active vaccination (killed B. pertusis and acellular):
D.P.T. vaccine in ages 2, 4, 6, 18 monthes, and then at
6 year ages
Prophylaxis by erythromycin
Bordetella parapertussis
Non-motile, fastidious
Urease-positive, Oxidase-negative
Adapted to colonies the mammalian respiratory tract.
The Pertussis manifests with similar symptoms to B.
pertussis - derived disease but tends to be generally
less severe.
Bordetella parapertussis may also cause pneumonia in
sheep.
Bordetella bronchiseptica
Bordetella bronchiseptica
Humans are not natural carriers of B. bronchiseptica.
It typically infects the respiratory tracts of smaller
mammals (cats, dogs, rabbits, etc).
Legionella
Gram negative, stain faintly with standard Gram stain
(should increase the time of safranin).
For growth it needs iron and cysteine.
Most important species are L. pneumophila and L.
micdadei but also > 22 other species.
Causes Legionellosis
Pathogenesis and epidemiology
Associated chiefly with environmental water
sources, such as water-cooling towers and air
conditioners.
Portal of entry is the respiratory tract.
Incubation period: 2-10 days
Pathologic changes occur primarily in the lung.
High risk factors: elder people, smokers, alcohol
consumers, cancer, immunosuppression (renal
transplants)
Person-to-person spread does not occur.
Clinical findings
Clinical picture can vary from a mild influenzalike
illness to a severe pneumonia accompanied by
mental confusion, nonbloody diarrhea, proteinuria,
and microscopic hematuria.
Cough is a prominent symptom but sputum is
frequently scanty and nonpurulent.
Most cases resolve spontaneously in 7-10 days.
Legionellosis is an atypical pneumonia and must be
distinguished from other similar pneumonias, such as
Mycoplasma pneumonia, viral pneumonia,
psittacosis, and Q fever.
Pontiac fever is a mild flulike form of Legionella
infection that does not result in pneumonia.
Lab diagnosis
Samples can be sputum, blood or lung biopsy.
The organism fails to grow on ordinary media.
Significant increase in antibody titer by indirect
immunofluorescence assay. Since there are at least 6
serotypes of L. pneumophila, a pool of antigens is
used.
Growing this fastidious organism on medium
supplemented with iron and systein. However, this is
rarely successful with sputum culture, because other
organisms present in sputum usually overgrow the
agar.
Demonstrating Legionella antigens in infected lung
tissue by using fluorescent-antibody staining.
Treatment
Erythromycin
It is also effective against Mycoplasma pneumoniae
and Streptococcus pneumoniae.
Prevention
Reducing cigarette and alcohol consumption
Eliminating aerosols from water sources
Reducing Legionella in hospital water supplies by using
high temperatures and hyperchlorination.
Pasteurella
Pasteurella multocida
 - A short encapsulated gram negative rod exhibiting
bipolar staining.
- Facultative anaerobic, Oxidase positive, catalase
positive.
- Wound infections associated with cat and dog
bites.
Virulence Factors
Capsule
Endotoxin
Pathogenesis & Epidemiology
The organism is part of the normal flora in the
mouths of many animals, particularly domestic cats
and dogs.
Transmitted by biting.
About 25% of animals bites become infected with
the organism., with sutures acting as a predisposing
factor to infection.
Pathogenesis
Most bite infections are polymicrobial, with a
variety of facultative anaerobes and anaerobic
organisms present in addition to P. multocida.
The rapid onset of cellulitis at the site of an animal
bite
Clinical Findings
Osteomyelitis can complicate cat bites in particular,
because cats’ sharp, pointed teeth can implant the
organism under the periosteum.
Laboratory diagnosis
Bipolar staining microscopy
Culturing a sample from the wound site
(on common lab media) and then Biochemical tests
Treatment & Prevention
Penicillin G
The use of Ampicillin as prophylaxis, especially for
cat bites
Francisella tularensis
A pathogenic species of gram-negative bacteria and
the causative agent of tularemia or rabbit fever.
It is a facultative intracellular bacterium.
Due to its ease of spread by aerosol and its high
virulence, F. tularensis is classified as a Class A
agent.
Nonmotile
Encapsulated
survive up to weeks at low temperatures in water,
soil, and animal carcasses
mortality rate 30 to 60% of the cases.
Virulence factors
Strains produce different hemolytic agents,
which may facilitate degradation of the
phagosome.
Pili
Rabbit fever
Tularemia, also known as “rabbit fever,”
Tularemia is typically found in animals, especially
rodents, rabbits, and hares.
Tularemia is usually a rural disease
Transmission
F. tularensis is capable of infecting a number of small
mammals such as voles, rabbits, and muskrats, as
well as humans.
Tularemia
No case of initiated by human-to-human
transmission.
Cases are caused by contact with infected animals or
vectors such as ticks, mosquitos, and deer flies.
Pathogenesis
Facultative intracellular bacterium that is capable
of infecting most cell types but primarily infects
macrophages.
After isolation in phagosome. F. tularensis then
breaks out of this phagosome into the cytosol and
rapidly proliferates.
Eventually the infected cell undergoes apoptosis,
and the progeny bacteria are released to initiate new
rounds of infection.
Treatment
Streptomycin is the drug of choice.
Gentamicin
Prophylaxis
Isolation is not recommended for tularemia patients,
given the lack of person-to-person transmission.
In hospitals, standard precautions are recommended.
Streptomycin, doxycycline, or ciprofloxacin in the
incubation period of tularemia and continuing
treatment daily for 14 days
Brucella
Microbiology characteristics
Small bacilli
Gram negative
Aerobic
Capsule
Nonmotile
Co2 needed
Fastidious
Brucella
Most clinical isolates come from blood cultures
(Castenada’s media which has both a solid and a
liquid phase)
Requires enriched media containing meat infusion
or tryptone
Will grow on Columbia blood agar (CBA) or
chocolate agar
Growth is slow and may take 72 hours
Colonies start as tiny pinpoint, translucent colonies
that become gray with age.
Transmission
A zoonotic organism
From domestic animals:
B. melitensis from goat
B. abortus from cow
B. suis from pig
B. canis from dog
Entering portals: Mouth, conjunctive, respiratory
tract, abraded skin.
Brucella
Brucella
B. abortus requires 10% CO2 for growth, others do not
Biochemistry
Speciated based on the ability to grow in the presence of
the dyes basic fuchsin and thionine
H2S produced by B. abortus and B. suis
Nonfermentative
Urease +\catalase +
Oxidase +
Brucella
Antigenic structure
2 antigens that are part of the LPS are recognized: A and M
• B. melitensis has the highest concentration of M and causes the
most serious infections
Virulence factors
– Endotoxin
Clinical significance
Has a tropism for erythritol
• Animal fetal tissues and placenta, other than those in humans, are
rich in erythritol and, therefore, the organisms often cause abortions
in these animals.
Brucella
Causes Brucellosis or undulent fever in man
following ingestion of contaminated milk or cheese
from:
goats (B. melitensis),
cows (B. abortus),
pigs (B. suis),
canines (B. canis).
Virulence factors
Endotoxin
No exotoxin
The organism is an obligative intracellular parasite.
Some organisms survive within macrophages.
Portals of entry
Oral - most common route
Ingestion of contaminated animal products
(often raw milk or its derivatives)
contact with contaminated fingers
Aerosols
Inhalation of bacteria
Contamination of the conjunctivae
Percutaneous infection through skin abrasions or
by accidental inoculation
Brucella
Can affect any organ or organ system
Pathogenesis
Entering through ingestion / skin / mucosa
Localization in mononuclear phagocytes to the
reticuloendothelial system: lymph nodes, liver,
spleen, and bone marrow
Effective host defense depends primarily on cellmediated immunity.
The host responses by granulomatous along with
lymphocytes and epithelioid giant cells, which can
progress to form focal abscesses and caseation.
Clinical findings
Enlarged lymph nodes, liver and spleen
The onset may be insidious or abrupt.
Undulant (rising and falling )fever
Subclinical infection is common
Sweating, weakness and fatigue
Incubation period: 2-4 weeks
Severe limb and back pains
Influenza like onset
B. melitensis infections tend to be more severe and
prolonged, whereas those caused by B. abortus are
more self-limited.
Osteomyelitis is the most frequent complication.
In untreated cases, symptoms may continue for 2-4
weeks.
Most patients recover entirely within 3-12 months
but some develop complications marked by
involvement of various organs.
Brucella
Man can also acquire the organism via contact with
infected animals.
Clinical manifestations range from subclinical, to
chronic with low grade symptoms of low fever and
muscular stiffness, to acute with fever and chills.
The fever typically spikes each evening and this
coincides with the release of organisms from
phagocytes (hence the name undulent fever).
The patient may also experience malaise, weakness,
enlarged lymph nodes, weight loss, and arthritis.
Control
Pasteurizing milk
Eradicating infection from herds by immunization of
animals and slaughtering of infected animals.
Using safety precautions (protective clothing and
laboratory safety).
Incubation period
1 to 6 weeks (sometimes to 6 or more months)
The length of the incubation period may be
influenced by many factors
virulence of the infecting strain
size of the inoculum
route of infection
resistance of the host
COURSE OF BRUCELLOSIS
If the disease is not treated, the symptoms may
continue for 2 to 4 weeks
Many patients will then recover
spontaneously
Others may suffer a series of complications
May produce an undulant fever in which the
intensity of fever and symptoms recur and recede.
Brucellosis
Cyprus fever/Gibraltar fever/Malta fever/Rock
fever/Undulant fever
Most affected persons
recover entirely within 3 to
12 months
Some will develop
complications
involvement of various
organs,
a few may enter an illdefined chronic syndrome.
Clinical Manifestations
The presentation of brucellosis is characteristically
variable and can effect any organ.
The onset may be insidious or abrupt
Influenza-like with fever reaching 38 to 40oC
Limb and back pains are unusually severe, night
sweating and fatigue are marked.
Anorexia, weakness, severe fatigue and loss of
weight, depression
Headache
Relative leukopenia with a relative lymphocytosis
Splenomegaly
Human congenitally infected infants
Low birth weight
Failure to thrive
Jaundice
Hepatomegaly
Splenomegaly
Respiratory difficulty
General signs of sepsis (fever, vomiting)
Asymptomatic
Prognosis
May last days, months, or years
Recovery is common
Disability is often pronounced
About 5% of treated cases relapse
Case-fatality rate: <2% ( untreated)
Endocarditis caused by B. melitensis
CLINICAL and LAB
DIAGNOSIS
Laboratory diagnosis
History of exposure.
Blood sample: Enriched culture media and
incubation in 10% Co2.
Blood cultures are positive in early disease, but
serology is the mainstay of diagnosis.
Interpretation is complicated by subclinical infections
and persistent antibodies.
Laboratory diagnosis
Isolation of organism
Blood, bone marrow, other tissues
Serum agglutination test
Four-fold or greater rise in titer (Rose Bengal,
Wright, coombs wright, 2-ME)
Samples 2 weeks apart
Immunofluorescence
Organism in clinical specimens
PCR
Treatment
Doxycycline some times combined with streptomycin
for 21 days
CNS cases treat 6-9 months
Psuedomonaceae
Gram Negative rods
Nonfermentative (Strict aerobic)
Oxidation of sugars
Cytochrome C oxidase
Motile
Pseudomonas aeroginosa (Piocianic bacillus)
Growth in soil and water containing only traces of
nutrients.
A remarkable ability to withstand disinfectants
(found in soap solutions, in antiseptics and in
detergents).
Persistent in the hospital environment
An important role in hospital-acquired infections
P.a. Producing 2 pigments:
Pyocyanin (colors the pus in a wound blue-green)
Pyoverdin /Fluorescein (a yellow-green pigment that
fluoresces under ultraviolet light
In the lab, these pigments diffuse into the agar,
imparting a blue-green color that is useful in
identification of the species.
In cystic fibrosis patients, P. aeroginosa has a slime
layer (glycocalyx):
very mucoidal colonies. The slime layer mediates
adherence of the organism to mucous membranes
of the respiratory tract and prevents antibody from
binding to the organism.
Pathogenesis
Virulance factors:
Endotoxin
Exotoxin A (inhibits eukaryotic protein synthesis
by the same mechanism as diphteria
exotoxin)
An opportunistic pathogen when
neutrophil counts is below 500/uL
In those with extensive burns (skin host defenses are
destroyed)
In those with chronic respiratory disease (such as
cystic fibrosis)
10-20% of hospital-acquired infections.
In immunosuppressed
In those with catheters
Clinical finding
Can cause infections virtually anywhere in the
body, but more frequent in:
Urinary tract infections (UTIs)
Pneumonia
External otitis
Wound infections (especially burns).
Sepsis with mortality rate of over 50%.
Epidemiology
10% of people carry it in the normal flora of the
colon and on the skin in moist areas.
It can colonize the upper respiratory tract of
hospitalized patients.
Its ability to grow in simple aqueous solutions has
resulted in contamination of respiratory therapy
and anesthesia equipments, and even distilled
water.
Lab diagnosis
Non-lactose-fermenting (colorless) colonies on
MacConkey or EMB agar.
Blue-green pigment on nutrient agar
Catalaze and gelatinase positive
In TSI: Alkalin/Alkalin
Gram negative rods
Oxidase-positive
Fruity aroma
Oxidase Test
Detecting cytochrome C oxidase enzyme
Indicator: 1% tetra methyl-para-phenylene diamine
dihydrochloride
OF (Oxidation Fermentation) Test
Indicator: Bromothymol blue
PH: 7.1
Treatment
Resistant to many antibiotics
Antibiogram test is essential
Usually is chosen from penicillins or cephalosporins
along with an aminoglycoside.
Prevention
Keeping neutrophil counts above 500/uL
Removing indwelling catheters promptly
Taking special care of burned skin