Non-Spore-Forming
Gram-Positive
Bacilli
Non-Spore-Forming Gram-Positive
Bacilli
• Corynebacterium
C. diphtheriae
Disease
Diphteria
Opportunistic infections by other
Corynebacterium species (diphteroids)
Properties
• Club-shaped also V- or L-shaped
• Methachromatic granules (Albert staining): Beaded
appearance
• Nonmotile, no capsule
• Facultative anaerobic
• Classified in CNM (Corynebacterium, Nocardia,
Mycobacterium) group.
Biotypes
(based on colony shape, biochemical properties and virulence)
Gravis
Mitis
Intermedius
Belfanti
Culture
•
Sheep blood agar: It supports the growth
of C.diphtheriae: Granular, gray, irregular
edges, alpha hemolysis (sometimes)
•
Loeffler serum slope: It is not a selective
medium but gives abundant growth and
morphology of C.diphtheriae. This medium
can also be used as a transport medium.
•
Culture
• Blood tellurite agar:
- Tellurite reduction
- It enhances the chances of isolation of
C.diphtheriae from clinical specimen in which
commensal bacterial flora may also be present
- It differentiate different vars.
Growing colonies of
Corynebacterium diphtheria on
Telorit potassium
Blood agar
Clinical finding
• Common diphtheria (Nasopharyngitis)
Incubation period of 2–5 days.
Fibrinous exudate “pseudomembrane”
- Sore throat
- Fever
- Enlargement of neck lymph nodes and neck edema
- Irregulatory of cardiac rhythm and myocarditis
- Difficulties with vision, speech and swallowing
This micrograph reveals an intranuclear
inclusion body in a heart section from a
patient with diphtheria-related
myocarditis.
Clinical findings
• Corrosion of myelin sheaths in the central and
peripheral nervous system leading to
degenerating motor control.
Cutanous diphtheria
(a secondary infection)
Transmission
• Humans are the only natural host
• C. diphtheriae reside in the upper respiratory
tract
• Transmitted by airborn droplet
• Infection at the site of a pre-existing skin lesion
Pathogenesis
• Invasivness
- Cord factor
A glycolipid inhibits eukaryotic cell oxidation.
- Nuraminidase
Removes N-acetyl nuraminic acid from musine
membranes.
• Exotoxin
Exotoxin
(Encoded by gen tox from a temperate phage)
Fragment B. Binding of the toxin
Fragment A. Enzymatic activity
A
B
Exotoxin mechanism
Nicotinamide adenine dinucleotide phosphate (NAD)
Exotoxin
(A fragment)
Nicotinamide
ADP
Reaction with EF2
Protein synthesis
inhibition
ADP-EF2
Testing immunity
(Schick’s test)
• Toxin Intradermal injection (0.1 mL):
I. Cause inflammation (4-7 days later): No
antitoxin in patient
II. No inflammation: Antitoxin is present
(Immune person)
Important points about diagnosis of
diphtheria
• The diagnosis is a laboratory
emergency.
• Mild cases resemble pharyngitis and
pseudomembrane may be lacking.
• Diphtheria is not diagnosed easily on
clinical grounds: Streptococcal pharyngitis
& Vincent's pharyngitis
Important points about diagnosis
of diphtheria
• Non-toxigenic strains of C.diphtheriae have
the potential to cause invasive disease and to
undergo lysogenic conversion to toxin
producers in vivo leading to disease.
• Specimen should be transported (e.i. by
Amies medium)and processed as soon as
possible.
Laboratory diagnosis
• Swabs should be taken both from the throat
and the nose.
• Preferably two swabs should be collected
from the site most affected.
• Microscopic observation (differentiation from
streptococcal and Vincent's pharyngitis)
Laboratory diagnosis
• Isolating the organism
• Demonstrating toxin production
- Animal inoculation
- Eleck test
- ELISA (Enzyme-linked immunosorbent assay)
• PCR to detect tox gene
Elek test
Treatment
• Tracheostomy in children (to prevent
croup)
• Antitoxin
(Intra muscular)
• Penicillin or erythromycin
Prevention
• Vaccination
DPT vaccine: A combination of diphtheria toxoid,
tetanus toxoid, and killed pertusis organism.
Given at 2, 4 an 6 months of age, with a booster at
1 and 6 years of age and then each 10 years
afterward. (DPT or DT)
The toxoid is prepared by treating the exotoxin
with 0.3% formaldehyde.
Listeria monocytogenes
• Small rod (Coccobacillus: 1-2 micron) like
“chinese character”.
• In old cultures 6-20 micrones.
• No capsule, Facultative aerobic.
• Tumbling movement in 25 c
• Growing in 4c
• Resistant to cold, heat, salt, pH extremes, and
bile
In 37 centigrade
In 20 – 25 centigrade
Listeria monocytogenes
• Small, gray and smooth colonies
with a narrow zone of beta
hemolysis.
• Growth in low O2 and 5-10% CO2
• Fermentater, Catalase +, Oxidase +
Transmission
• The organism is distributed worldwide in
animals, plants, soil, stream water,
sewage, unpasteurized milk, vegetables.
• Transmission to human by contact with
animals or their feces.
• Endogenously from gasterointestinal
tract.
Listeriosis
• Rarely the cause of human disease
• Potentially lethal food-borne infection
case fatality rate:
For those with a severe form of infection
is 25%. (Salmonella, in comparison, has a
mortality rate estimated at less than 1%)
Virulence Factors
• Soluble components:
- Hemolysins
- Soluble antigens :
Lypolytic Ags
- Endotoxins (LPS):
Cold Aglotinin: Abs react with RBC at low temp
(28-31 c): Lysis of RBC
Epidemiology
• Primary reservoir is soil and water; animal
intestines. It contaminates foods and grow
during refrigeration
• Listeriosis – most cases associated with dairy
products, poultry, and meat
• Often mild or subclinical in normal adults.
• In immunocompromised, fetuses, and
neonates affects brain and meninges.
52
Epidemiology
• Pregnant women susceptible
• Infection passed to fetus prenatally
across placenta
• Postnatal neonates acquire infection
in birth canal
53
Disease
• Meningitis and sepsis in:
1. The fetus or newborn
I. Granulomatis Infantiseptica (during pregnancy)
or
II. Late listeriosis (5 days after giving birth).
2. Immunosuppressed adults
(especially renal transplant patients)
3. The infected mother: asymptomatic or influenzalike
illness/ Abortion
Pathogenesis
• Listeria induces macrophage phagocytic uptake:
• Internalins & D-galactose (in their teichoic
acids) bound by the macrophage's
polysaccharide receptors (E-cadherin)
• Once phagocytosed (as Phagosome), then
encapsulated by the host cell's acidic
phagolysosome.
• Listeria, escapes the phagolysosome by lysing
the vacuole‘s membrane with hemolysin
(listeriolysin O).
Pathogenesis
Internalin
E-cadherin
Phagocytosis
Into macrophages
& epithelial cells
Forming filopods
Phagocytiosis
By
macrophages
and
hepatocytes
Inducing actin polymerization in
cytoplasm
Release from phagolysosome
Phagolysosome
formation
(acidic condition)
Lysteriolysin O
secretion
Figure 19.10
Multiplication cycle
of Listeria
monocytogenes
58
Lab. diagnosis
• Microscopic observation: Diphtheroids
• Isolation by culture:
Blood and CSF samples on blood agar
• Cold Enrichment
• Rapid diagnostic tests using ELISA,
immunofluorescence, and DNA analysis
Prevention
• Effective sanitation of food contact surfaces:
Alcohol & Quaternary ammonium
• 4°C discourages bacterial growth.
• pasteurization and well cooking
• Cell-mediated immunity is active (but no immunization)
Treatment
• Penicillin
Resistant are rare
• Ampicillin and trimethoprim/
sulfamethoxazole
Lactobacilli
Lactobacillus
• Methachromatic granules (Albert staining)
• Nonmotile
• no capsule
• Facultative anaerobic, microaerophilic or
aerotolerant anaerobic
• L. casei
L. acidophilus,
L. salivarius,
L. reuteri
Are the representatives of distinct subclades.
Lactobacillus are a major part of
lactic acid bacteria group: mostly
convert lactose and other sugars to
lactic acid.
They are common and usually benign.
In humans they are present in
vagina and gastrointestinal tract
inhibiting the growth of some
harmful bacteria.
Erysipelothrix rhusiopathiae
(causing Erysipeloid)
• Gram-positive rod (sometimes looks like
GNB)
• Single, streptobacilli, sometimes
filamentous.
• Catalase -, Oxidase -, Indol –
• SH2 in TSI
• Microaerophilic needed 5% co2
72
• Small shiny or white colonies
• Alpha hemolytic on BA
• Differentiated from Listeria by Alpha
hemolysis and SH2 & catalase.
Transmission
• in environment and normal flora in sheep,
chickens, and fish, duck (indigenous
zoonotic Infectious disease)
• Transmission from animals or their products
to human
• Primary reservoir – tonsils of healthy sheep
& pigs.
• On the surface of fresh fish
Erysipeloid on hand
75
Pathogenesis & Clinical findings
• Enters through skin abrasion, multiplies to
produce erysipeloid, dark red lesions without
systemic symptoms.
• Latent period: 2-7 days
• Sever pain, inflamation with purple color
lesions on skin.
• No pus (differentiation from Staphyloccocal
and Streptococcal infections)
• Endocardit
76
Control and treatment
•
•
•
•
Penicillin or erythromycin
Treated after 3-4 weeks
Always resistant to vancomycin
Vaccine for sheep and pigs
Lab diagnosis
• Biopsy or aspirated liquids from the
edge of lesions.
• Blood sample in case of endocardit.
Chronic swimming pool
granuloma
80
• In severe cases, they may penetrate the
surrounding bone and muscle to the skin,
where they break open and leak large
amounts of pus. The purulent leakage via the
sinus cavities contains "sulfur granules," not
actually sulfur-containing but resembling such
particles. These granules contain progeny
bacteria.
Diagnosis
• The disease is suggested by a supportive
lesion with Gram-positive filaments in the
exudate.
• Sulfur granules may be present.
• Bacterial isolation and identification
Control
• Good oral hygiene
• Antibiotic trapy: Penicillin
• Drainage of lesion
Actinobacteria
• They include some of the most common soil
life, freshwater life, and marine life, playing an
important role in decomposition of organic
materials, such as cellulose and chitin, and
thereby playing a vital part in organic matter
turnover and carbon cycle.
• Actinobacteria are well known as secondary
metabolite producers and hence of high
pharmacological and commercial interest. In
1940 Selman Waksman discovered that the
soil bacteria he was studying made
actinomycin, a discovery for which he received
a Nobel Prize.
• Since then, hundreds of naturally occurring
antibiotics have been discovered in these
terrestrial microorganisms, especially from the
genus Streptomyces.
• Some Actinobacteria form branching
filaments, which somewhat resemble the
mycelia of the unrelated fungi, among which
they were originally classified under the older
name Actinomycetes. Most members are
aerobic,
• a few, such as Actinomyces israelii, can grow
under anaerobic conditions. Unlike the
Firmicutes, the other main group of Grampositive bacteria, they have DNA with a high
GC-content, and some Actinomycetes species
produce external spores.
• Some types of Actinobacteria are responsible
for the peculiar odor emanating from the soil
after rain, mainly in warmer climates.[7