Transcript Clinical Microbiology

Clinical Microbiology
Preface
Clinical microbiology studies the molecular bases and the pathogenic agents
of infectious diseases for the development of new diagnostic technologies and
new terapeutic treatment.
Bacteria are the oldest and the most adaptable forms of life on Earth.
They have existed for some 3.5 billion years.
For the first 2 billion years prokaryotes alone colonized every accessible ecological
niche.
We are surrounded and exposed to bacteria including those that cause disease
and death. Diseases caused by bacteria include some of the most common infections
in the world.
Therefore, the knowledge of pathogenic bacteria, diseases and
current therapeutic strategies, is critical for all scientists, especially for
clinical microbiologist.
Laboratory procedures for the identification
of microrganisms in clinical samples
In clinical cases, to prescribe a correct antibiotic treatment,
identification of pathogenic organisms in samples, is determinant.
The identification techniques must be as much rapid and accurate as
possible, to discover the origin of the community-acquired or nosocomial
infections, to block the trasmission of diseases between individuals, the
emergence of a new hypervirulent or drug-resistant strains.
Microbial identification: optical methods
Microscopic examination of specimen is the first step for bacterial
identification.
Direct examination is a rapid diagnostic method.
Visible microrganisms may indicate the possible etiologic agent or
can guide the laboratory in selection of the appropriate isolation
media and can guide clinical microbiologist in selection of the
empirical antibiotic therapy.
Microbial identification: optical methods
The light microscope
Microscope is an instrument of primary importance for clinical microbiology
Microscopes are used for viewing some objects that are too small to see,
without magnification.
Parts of the light microscope
The stage is a platform that holds
the slide containing the specimen to be
viewed (mechanism for moving the slide).
The light source this is usually located
below the stage.
The diaphragm located below the
stage is used to regulate the light.
The condenser contains two groups of
lenses. Light, from the light source,
passes through the diaphragm and
condenser, continuing up through the
specimen.
Body tube contains an ocular lens 10x
and nosepiece with several objective
lenses (10x, 40x, 100x)
The image is brought into focus by the
coarse and fine focus knobs.
Oil immersion
The 100x objective (1000x total magnification) requires that a drop of
immersion oil must be placed between the slide and the lens.
After viewing with oil, the lens must be cleaned with fluid for this purpose
Resolving power
The resolving power is the smallest distance between two points
required to distinguish as separate two different points.
With yellow light and with objective 100x, the smallest separable
diameters are about 0,2 µm.
Particles 0,2 μm in diameter are magnified to about 0,2 mm and so become
visible.
The electron microscope
The electron microscope uses electrons instead of light. Some
electrons pass through the specimen and are focused by an
electromagnetic objective lens, which presents an image to the projector
lens system for enlargement.
The image can be recorded on photographic film.
The superior resolution of the electron microscope is due to the fact
that electrons have a shorter wavelength than the photons of white
light.
With the high resolving power of the electron microscope is possible
to observe the detailed structures of prokaryotic and eukaryotic cells
Types of electron microscopes
Different types of electron microscopes:
the trasmission electron microscope TEM
the scanning electron microscope
SEM
the confocal microscope
The TEM was the first to be developed, it employs a beam of electrons
projected from an electron gun and focused by an electromagnetic condenser
lens.
The SEM has a lower resolving power than the TEM, but it produces threedimensional images of the surface of microscopic objects.
Confocal microscope uses laser light beams, it produces three-dimensional images
Microscopical observation of
samples: techniques
Simple wet mounts
Clinical samples can be placed directly under the microscope. However,
many samples look better when placed in a drop of water on the
microscope slide. This is Known as a “wet mount”.
Simple wet mounts, consisting of clinical material in a drop of saline
solution, allow determination of cellular composition, morphology and
motility of bacteria.
The specimens can be examined by light, phase-contrast, or dark-field
microscopy.
The wet mounts do not involve fixation of the clinical materials. They
are viewed immediately upon preparation.
Simple wet mounts
This method is used for direct clinical examination of stool,
vaginal discharge, urine sediment, aspirate.
Is used to detect organism motility and morphology
Is vary rapid, requires few minutes
It requires experience
Wet mounts
Tools and materials
 Microscope
 Flat slides
 Cover slides
 Eyedropper or pipette
 Saline solution, water or broth
 Toothpick
 Paper towels
Wet mounts
Method
 using an eyedropper put a drop of water on the sample
 put a drop on the slide
 place one end of the cover slip on the slide and lower the other end
using a toothpick (this will help to prevent air bubbles)
Single stain method
This method, with a single stain such as methylene blue or iodine, enhances
the visualization of microrganisms by increasing the contrast of structures
The smears must be fixed (in contrast with the wet mount)
All organisms and cellular components have a similar color
Single stain method
The dyes are usually salts, two important chemical groups:
chromophore and auxochrome, complete the dye compound.
The auxochrome group determines whether a dye is classified as cation
(basic) or anion (acid)
Blue stain is considered as a simple stain, in contrast
with the Gram and Acid-Fast stains, which require a counterstaining step
Preparation of the smear
Correct preparation of the smear:
Make a thin film of the material on a clean glass slide
using a sterile loop or swab
 Air
 Fix
dry
the slide by passing it several times through a
flame.
The slide very hot, may cause staining artefacts and
may disrupt the normal morphology of bacteria.
The Methylene blue stain
Materials:
1% Methylene blue stain solution
Glass microscope slides
Culture
Method:
Make a thin film of the material on a clean glass slide,
using a sterile loop or swab
Air dry
Fix the slide by passing it several times through a flame
Flood with methylene blue solution
Leave stain in contact with the smear for 30 sec-1 min
Rinse well, blot dry and examine under oil immersion
The flagella stain
Flagella are appendages, composed by proteins. They are organs of locomotion and
are too fine (12-30 nm in diameter) to be visible using the light microscope. Three
types of arrangement are known: monotrichous, lophotricous and peritrichous.
However their presence can be demonstrated by treating the cells with a colloidal
suspension of tannic acid salts causing a heavy precipitate around the cell wall and
flagella. The treatment increases the diameter of flagella.
Subsequent staining with basic fuchsin, makes the flagella visible in the light
microscope
Flagilla observation methods
• Hanging drop
methods
In this method a drop of culture is
placed on a cover-slip and then
this cover-slip is placed on a slide
with a cavity in the middle.
The slide is focused to see the
bacteria vitality
The capsule stain
Many bacteria synthetize a large amount of extracellular
polymers when growing in their natural environment.
Capsules are composed of polymers (sugars and proteins) that
surround bacterial cells
Capsules are usually demonstrated by the negative staining procedure
The cells are treated
with a suspension
of indian ink.
Capsules are colourless
The capsule stain (Welch method)
This method involves treatment with hot crystal violet solution followed by a rinsing
with copper sulfate solution; the latter solution is used to remove excess stain
because the conventional washing with water would dissolve the capsule.
The copper salt gives color to the background. The cell and background appear
dark blue, the capsule as much paler blue.
Method
Spread the culture on glass slide
Air dry complitely
Stain with crystal violet (1 min)
Wash with copper sulfate 10%
Air dry
Observe the smear using oil immersion
Gram stain technique
Gram stain procedure was developed by H. Christian Gram to differentiate
pneumococci from klebsiella pneumoniae
The procedure involves the application of a solution of iodine
(potassium iodide) to cells previously stained with crystal violet.
This procedure produces “purple colored iodine-dye complexes” in
the cytoplasm of bacteria
The cells that are previously stained with crystal violet and iodine are next
treated with a decolorizing agent such 95% ethanol or a mixture of acetone
and alcohol
The difference between Gram+ and
Gram- bacteria
The difference is in the permeability of the cell wall to “purple iodine-dye
complexes” when treated with decolorizing solvent.
Gram positive bacteria retain purple iodine-dye complexes after the
treatment with decolorizing solvent
Gram negative bacteria do not retain complexes when decolorized, so we
use a red counterstain with safranin to observe Gram negative bacteria
Gram stain procedure
Gram positive
coccus
Gram negative
bacillus
Preparation of the smear
Correct preparation of the smear:
 Make a thin film of the material on a clean glass slide, using a sterile loop
or swab
 Air dry
 Fix the slide by passing it several times through a flame
( the slide very hot, may cause staining artifacts and disrupt the normal
morphology of bacteria)
Staining procedure
1. Flood slide with crystal violet (30 seconds)
2. Wash with running tap water
3. Flood with Gram’s iodine (30 seconds)
4. Wash with water
5. Decolorize with 95% ethanol until the smear are colorless
(this step is very critical and affected by variation in timing and
reagents)
6. Wash with water
7. Flood with safranin (30 seconds)
8. Wash with water, air dry or with absorbent paper
Resuls
Gram positive bacteria are stained purple because retain the violet-iodine complexes
Gram negative bacteria do not retain violet-iodine complexes after washing in
ethanol, but stain red from the safranin counterstain.
The acid fast stain (Zielh Neelsen)
The acid fast stain is primarily of clinical application to detect members of the
genus Mycobacterium.
Mycobacterium tubercolosis, the etiologic agent of tuberculosis, is the most
common pathogen of this group.
Other microoganisms, particularly the Nocardia, can be identified by their
acid-fast characteristic.
The term acid-fast is derived from the resistance displayed by acid-fast bacteria
to decolorization by acid once they have been stained by another dye.
(Cell wall conteins fatty acids and phospholipids responsible for acid fast stain)
Matherials:
Mycobacterium culture
carbol fuchsine
acid-alcohol solution (70% ethanol 0,5% hydrochloric acid)
methylene blue counterstain
The acid-fast stain: method
Prepare a bacterial smear in the conventional method
(as for Gram-staining) air-dried and heat fixed
Flood the smear with carbol fuchsin reagent heated and allow to
remain in contact for 5 min
Rinse the excess stain with deionized water
Decolorize the smear with acid alcohol until the color runs from
the smear
Wash the smear with deionized water
Counterstain for 30 sec with 1% methylene blue
Rinse, blot dry, and examine under oil immersion.
Zielh Neelsen stain (Mycobacterium)
Acid fast bacteria will appear an intense red (retaining the carbol fuchsine)
Other material will be blue from the counterstain
The spore stain:
(Schaeffer-Fulton method)
Some bacteria are able to spores forming. The most common genera are Gram
positive rods ( aerobic Bacillus genus and anaerobic Clostridium genus )
The spore wall is relatively impermeable, but dyes can be made to
penetrate it by heating the preparation.
Spores are commonly stained with malachite green or carbolfuchsin
Bacillus anthracis is the best-known microorganism in Bacillus group, and anthrax
its clinical condition. It remains a specific agent in biological war and in
bioterroristic attacks.
Anthrax is divided in cutaneous forms and in pulmonary forms, the last condition
is often fatal and severe. In inhalation form, spores are carried by macrophages
from the lungs to lymphatic system. Germination begins inside the macrophages
and vegetative cells Kill the macrophages and are released into the bloodstream.
Staining procedure
Staining procedure
• Suspend a small amount of bacteria in the distilled water
• Air dry the slide, heat fix by passing the slide over a flame
• Flood the slide with malachite green and flame the slide
steaming for 5 min
• Throw the excess
• Flood the slide with eosin
• Rinse, blot dry and examine under oil immersion.
Cultivation of microrganisms:
bacteriological media
Bacteria can be cultivated in laboratory on particular substrates.
Many components optimize the growth of microrganisms on media,
they include:
 Nutrient sources
 Solidifying agents (for solid media)
 Specific pH
 Specific additives (for fastidious bacteria)
Some organisms can utilize a very simple medium, most require specific
nutrient sources including: nitrogen, carbon, inorganic salts, minerals,
vitamins and other substances.
The pH is important because many microrganisms have strict pH
requirements, most species grow in a range of pH neutrality
Bacteriological media: other factors
Other factors allowing the growth include: the incubation temperature and
the gas in growth environment.
Most clinically significant organisms are mesophiles, other are thermophiles or
psycrophiles.
In addition, most species grow optimally in aerobic condition, but others require
CO2 or total removal of O2
Bacteriological media
General
for example Nutrient broth or blood agar
(used for cultivation or isolation of microrganisms: fastidious and non
fastidious)
Selective
are media that contain additives that enhance the growth of
desired organisms by inhibiting other organisms. The selection activity is
obtained with addition of dyes, salts or antibiotics.
Examples include MSA that contains 7,5% NaCl, which inhibits most
organisms, it contains mannitol and pH indicator (phenol red)
Bacteriological media:other types
Differential
they base the identification on the organism’s appearance on
the media.This can be demonstrated by colony colour or by precipitate
around the colony.
Examples include the medium used for the isolation of enteric pathogens
such as Mac Conkey, it contains: lactose bile salts and pH indicator
(neutral red).
TSI for enteric bacteria it contains: lactose, sucrose, glucose and iron salt,
pH indicator.
Enriched
enriched media are media that allow the growth of fastidious organisms
requiring the presence of specific nutrient additives such as hemin, cysteine etc.
(fastidious organisms do not grow on general media).
Bacteriological media:other
types
Specialized
are media containing additives for specific pathogens (legionella
species etc)
Anaerobic media
anaerobic media include: peptones, yest extract, vitamines, and
reducing agents
Other types: transport media
Transport media
are used to transport, from the bedside to inoculation in the clinical laboratory,
fastidious organisms not surviving in environment
They are packaged in a plastic tube with a small amount of liquid medium, a swab
attached to a cap is used for collection of specimen, which is then placed into
the tube.
Staphylococci
Staphylococci are Gram-positive spherical cells, usually arranged in
irregular clusters (grape-like)
They grow on many types of media, are active metabolically, fermenting
carbohydrates and producing pigments that vary from white to deep yellow
Some are members of normal flora of skin and mucous membranes
of humans, others cause suppuration, abscess formation, toxin mediated
diseases, and fatal septicemia
Classification
Staphylococcus genus has about 30 species
The main species of clinical importance are:
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
Staphylococcus aureus is coagulase-positive, which
differentiates it from the other species
Coagulase-negative staphylococci
Coagulase-negative staphylococci sometimes cause
infections often associated to implanted medical devices especially in
mmunocompromised patients
These infections are linked to biofilm formation
What’s the biofilm?
Biofilm is a community of bacterial cells contained in a self-produced
polymer matrix adherent to biotic or abiotic surface.
This structure is very stable and resistant to the physical, chemical
agents used in medicine.
Recently, biofilm producing organisms were described in different
infections such as in reactivated chronic bronchitis, in cystic fibrosis,
in chronic prostatitis, in musculoskeletal infections
Coagulase negative staphylococci
Infections caused by coagulase-negative staphylococci are due to:
Staphylococcus epidermidis
Staphylococcus lugdunensis
Staphylococcus warneri
Staphylococcus hominis
Staphylococcus simulans
and other less common species
Staphylococcus saprophyticus is the cause of urinary tract infection
in young women
Staphylococci: morphology
Staphylococci are spherical cells about 1µm in diameter arranged in
irregular clusters.
Single cocci, pairs, tetrads and chains are seen especially in liquid cultures.
Staphylococci are:
Gram positive, (are stained purple)
capsulate
nonmobile
and do not form spores.
Their colonies can be white, yellow or orange.
Staphylococci culture
Staphylococci grow on most bacteriologic media under aerobic or
microaerophilic condition at 37° C.
The colonies on solid media are round, smooth, raised, glistening.
Staphylococcus aureus form gray to deep golden yellow colonies
Staphylococcus epidermidis form gray to white colonies
Staphylococci: characteristics
Staphylococci: produce catalase (which differentiates them from the
streptococci)
ferment many carbohydrates
are resistant to drying, heat and 7.5% sodium chloride
are sensitive to many antimicrobial drugs
Staplylococcus aureus
Staphylococcus aureus is a major pathogen for humans.
Many people are asymptomatic carriers; they have staphylococci
on the skin and in the throat (opportunistic pathogen)
As a nosocomial pathogen, S. aureus has been a cause of morbidity and
mortality.
In hospitals, the areas at risk for severe staphylococcal infections are
the newborns nursery, intensive care units, operating rooms and cancer
chemotherapy wards.
Diseases caused by S. aureus
1) Skin and soft tissues:
a) Abscesses, furuncles
b) Wound infections
c) Cellulitis
d) Impetigo
2) Blood and cardiovascular system:
a) Bacteremia
b) endocarditis
3) Muscoloskeletal:
a) Osteomyelitis
b) Arthritis
4) Toxin mediated diseases:
a) Toxic shock syndrome
b) Food poisoning
c) Scalded skin syndrome
5) Metastatic abscesses (brain)
6) Pulmonary
Antigenic structure
Staphylococcus aureus contains polysaccharides, proteins
and other substances on cellular surface.
Capsular component: glucuronic acid
capsule inhibits phagocytosis by polymorphonuclear
leukocytes
Teichoic acids: they are polymers of glycerol or ribitol phosphate linked
to peptidoglycan
Protein A is a component that binds to FC portion of IgG molecules
Virulence factors
Staphylococci can produce diseases through their ability to multiply in
tissues and through production of many extracellular and cellular
substances.
Toxins
Hemolysins (cytolytic)
α it is potent hemolysin, degrades red blood cells of rabbits
β it degrades sphingomyelin and is toxic for red blood cells of
sheep
γ it lyses red blood cells of humans
δ it disrupts biologic membranes and may have a detergent role
Poreforming α hemolysin
α hemolysin is secreted in nontoxic
soluble form, it multimerizes on eukaryotic
membranes to form lytic pores
Toxins
Leukocidin (Panton-Valentine toxin)
this toxin has two components. It can kill white blood cells
of humans and rabbits; two components act together to
form pores and they increase cation permeability.
Exfoliative toxins are two distinct proteins with same molecular
weight, they cause generalized desquamation on
staphylococcal scalded skin syndrome. SSSS is typical in
newborns and in infants younger than 1 year (superantigen)
Toxic Shock Syndrome toxin TSST-1 (superantigen). It binds
to MHC class II molecules yielding T cell stimulation which
promotes the manifestations of toxic shock syndrome.
This syndrome is associated with fever, shock and
multisystem involvement including desquamative skin rash.
Superantigens
Superantigens are potent immunostimulators able to simultaneously bind to
major histocompatibility complex class II molecules and T-cell receptor. This
binding actives a large number of T cells with secretions of proinflammatory
cytokines
Toxins
Enterotoxins there are multiple enterotoxins; like TSST-1 they are
superantigens. Enterotoxins are heat-stable and
resistant to action of enzymes. Enterotoxins cause
food poisoning, they are produced when S. aureus
grows in fatty foods; ingestion results in vomiting
and diarrhea. Emetic effect probably is the result
of central nervous system stimulation (vomiting centre)
when the toxin acts on neuron receptors in intestinal tract.
Enzymes
Coagulase this enzyme clots oxalated or citrated plasma. Coagulase
binds to prothrombin; together they become enzymatically
active and initiate fibrin polymerization. Coagulase may
deposit fibrin on surface of staphylococci altering their
ingestion by phagocytic cells.
Catalase catalase converts hydrogen peroxide into water and oxygen.
Catalase test differentiates staphylococci (positive)
from streptococci (negative).
Hyaluronidase it is spreading factor
Staphylokinase fibrinolitic enzyme, but acting much more slowly than
streptokinase
Proteinases and lipases they act on proteinic and lipidic components
Diagnostic laboratory tests
Samples:
a throut swab, a samples of pus, blood, tracheal aspirate,
or spinal fluid may be used (in according with the different
localization of process).
Direct examination with a direct microscopic examination is not
possible to distinguish saprophyticus from pathogenic
organisms; culture and appropriate identification techniques
must confirm this report.
Isolation
Specimens planted on blood agar
plates develop the typical colonies in
18 hours at 37° C, but hemolysis
and
pigment production are optimal at
room temperature.
Identification
Isolated colonies on blood agar
medium are planted on Mannitol
salt agar.
This medium conteins: mannitol,
7,5% of Na Cl and a pH indicator.
S. aureus ferments mannitol. The salt
(7,5% NaCl) inhibits the most
other normal flora but not
staphylococci. Mannitol salt agar is
medium used to screen S. aureus
from S. epidermidis
Biochemical identification
Coagulate test two different coagulase tests can be performed:
a tube test for free coagulase, and a slide test for bound
coagulase or clumbing factor.
Free coagulase: the tube coagulase test is best performed
by mixing 0.1 ml of an overnight culture in brain heart infusion
broth with 0.5 ml of citrated rabbit plasma, incubating the
mixture at 37°C in a water bath, and observing tube for
clot formation. If clots form in 1-4 hours the test is positive.
The slide coagulase test is performed by making uniform
suspension of growth, adding 1 drop of plasma and observing
for clumping within 10s (this test may be used as rapid
screening technique to identify S. aureus).
Tube coagulase test: free coag.
negative
positive
Slide coagulase test
negative
positive
Diagnostic laboratory tests
Catalase staphylococci produce catalase, which forms water and oxygen
from hydrogen peroxide. The test differentiates
staphylococci, which are positive, from streptococci,
which are negative.
positive
Catalase test reaction
Staphylococci are positive when tested with 3% hydrogen peroxide
To perform the test, a loopful of growth is transferred from agar
plate culture to glass microscope slide.
Reaction (evolution of bubbles) on additional of drop of 3% H2O2 ,
positive result is considered.
Other enzymes
Heat stable nuclease can cleave DNA or RNA it is produced by most
strains of S. aureus. TNase can be detected by using a
metachromatic agar diffusion method and Dna-toluidine
blue agar.
negative
positive
Serologic tests
Serologic tests
for diagnosis of S. aureus, they have little practical value
Antimicrobial treatment
Staphylococcal isolates should be tested for antimicrobial susceptibility
because S. aureus has developed resistance to all antibiotic classes
available for clinical use.
Resistance to Penicillin
The most common mechanism of S. aureus resistance to β-lactam involves
penicillinase an enzyme that hydrolyzes penicillin into inactive penicilloid
acid.
Penicillinase-producing strains emerged rapidly after penicillin introduction
in the mid 1940s and became prevalent in hospitals and in communities.
(Penicillin G resistant S. aureus strains, producing penicillinase, now
constitute about 90% of isolates in communities).
In the late 1950 a new penicillinase-resistant penicillin called Methicilllin
was created.
Hospital acquired methicillinresistant Staphylococcus aureus
Resistance to Methicillin
The first penicillinase-stable β-lactam such as methicillin and the
isoxazolyl penicillins became available in the late 1950s. The first
MRSA was described at aboud the same time.
The prevalence of MRSA in hospital has increased rapidly in the last
periods (more than 60% in hospital centers with great geographic
variations).
Mechanism of Methicillin resistance
The mechanisms is mediated by a new acquired PBP2A, encoded by
mecA gene.
Because of its low β-lactam affinity, PBP2A can complete cell wall
synthesis.
Alternative treatments for MRSA
Treatment of infections with hospital multiresistant MRSA
appears problematic.
Vancomycin is the first choice in these situation.
Members of aminoglycosides associated with vancomycin increase their
bactericidal activity.
Quinupristin-Dalfopristin is a combination of a streptogramin B
and A
This association is sinergic and shows a large activity against MRSA
strains.
Linezolid belongs to a new oxazolidinone family of molecules, it is
active against all multiresistant Gram positive pathogens
Daptomycin is a cyclic lipopeptide antibiotic active against MRSA
Streptococci
The streptococci are Gram positive spherical bacteria
characteristically arranged in pairs or chains.
Lengths of chains are conditioned by environmental factors
Growth characteristics
Streptococci grow in solid media, supplemented with blood, as discoid
colonies, usually one to two mm in diameter.
Some streptococci (β hemolytic) can lyse blood cells and cause a complete
clearing of blood all around the colonies. Other strains cause no change
in blood agar (γ or nonhemolytic). Other reduce hemoglobin and cause a
greenish discolouration of agar.
(α hemolytic)
β
α
γ
Hemolytic reactions on culture media
In beta hemolytic reaction red blood cells are completely lysed
In alpha hemolytic reaction red blood cells are not completely lysed
but colonies are surrounded by greenish discolouration of agar due to
streptococcal action on hemoglobin
Nonhemolytic or gamma hemolytic streptococci have no effect on blood
agar
Lancefield classification
Lancefield subdivided streptococci based on cell wall antigen
(antigen C).
This carbohydrate forms the basis of serologic grouping,
Lancefield group A-H K-T,
For group A streptococci, antigen C is rhamnose-N-acetylglucosamine
for group B, it is rhamnose-glucosamine polysaccharide etc.
Characteristics of clinically important streptoc.
Name
Hemolysis
Group
spec.sub.
Streptoc.
pyogenes
A
beta
Strep.
agalactiae
B
Β γ
-
alpha
Strept.
pneumon.
habitat
Labor.
criteria
Diseases
Throat,
skin
Colonies
>0,5 mm
Pharyngitis
Impetigo
rheumatic
fever
Female geni
tal tract
Hippurate
hydrolysis
Neonatal
sepsis
throat
Susceptib.
optochin,
soluble in
bile
Pneumonia,
meningitis,
endocarditis
Viridans
streptoc.
Usually not
typed
alpha
Mouth
throat,
female gen.
tract
Optochin
resist.
colonies not
solub. in bile
Dental caries
Endocarditis
abscess
Streptoc.
anginosus
group
F, A, C, G
alpha,
beta
Throat, colon
Female gen.
tract
Small
colonies
<0,5 mm
Piogenic
infection
Streptococcus pyogenes
Streptococcus pyogenes (group A streptococcus) is one of the most
important bacterial pathogens of humans,it is cause of acute pharyngitis,
and also cutaneous and systemic infections.
The organisms are Gram positive
nonmobile
non-spore forming
catalase negative
facultatively anaerobic
Cultivation
Group A streptococci are nutritionally fastidious and usually cultivated
in complex media often supplemented with blood or serum.
When cultured on blood agar plates S. pyogenes appears as white to gray
colonies surrounded by complete β hemolysis.
Some strains appear mucoid because they produce abundant capsular
material
Clinical manifestations
Streptococcal pharyngitis
is characterized by pharyngeal pain and erythema with fever and
anterior cervical adenopathy. This disease occurs primarily among children
5 to 15 years of age with the peak during the first years of school.
Suppurative sequelae (peritonsillar abscesses, meningitis, endocarditis etc.
may ensue to contiguous tissue or by bacteremic dissemination.
(Patients do not receive antibiotic therapy)
Nonsuppurative sequelae include: rheumatic fever (some strains contain
cell membrane antigens that cross-react with human heart tissue
antigens) and acute glomerulonephritis (the disease may be caused by
the deposit of antigen-antibody complexes on the glomerular basement
membrane). Autoimmune diseases they are considered
Clinical manifestations
Scarlet fever this infection results with a streptococcal strain that
elaborates streptococcal pyrogenic exotoxins (SPE). Rush appears
usually on second day. Tongue is covered with yellowish white coat
through which may be seen the red papillae (strawberry tongue)
Other infections
In recent years there has been an increase in number of severe
S. pyogenes infections, including necrotizing fascitiis and infections
associated with toxic shock syndrome.
Ability of streptococcal pyrogenic exotoxins (SPE) to act as
superantigens contributes to production of shock in these infections
Necrotizing fascitiis
SPEs family
Streptococcal pyrogenic exotoxins (SPEs) are a family of bacterial
superantigens associated with streptococcal toxic shock syndrome, necrotizing
fascitiis and other severe infections.
Scarlet fever: SPE A and C
Necrotizing fascitiis: SPE B
Streptococcal toxic shock syndrome: SPE F and streptococcal superantigens
recently identified
Superantigens
Superantigens are potent immunostimulators able to simultaneously bind to
major histocompatibility complex class II molecules and T-cell receptor. This
binding actives a large number of T cells with secretions of proinflammatory
cytokines
Invasive streptococcal infections
of skin and soft tissues
Erysipelas
is a superficial cutaneous process
restricted to the dermis with lymphatic
involvement.
This disorder is more common in infants,
young children and older adults. In older
reports erysipelas was described as
involving the butterfly area of the face.
At the present the lower extremities are
more frequently involved, in this
case, the lesions break the cutaneous
barrier causing local infections
Virulence factors: somatic constituents
Capsule
the organism is enveloped in a hyaluronic acid capsule that serves such
as a virulence factor in retarding phagocytosis by polymorphonuclear
leukocytes
Cell wall
Group-specific carbohydrate C
and N-acetylglucosamine
M protein
in group A is a polymer of rhamnose
is a major virulence factor. GAS may be divided into serotypes on
basis of antigenic differences in M-protein molecules and into
genotypes on basis of differences in emm gene encoding molecules.
More than 120 serotypes are recognized. The M protein is anchored
to cell membrane protruding such as fibril from cell surface.
Virulence factors: somatic constituents
Opacity factor
this antigen, associated with M-protein, is expressed by
40% of strains. It contains 2 domains, one domain, able to serum
opacify, disrupts the structure of high density lipoproteins and serum
becomes cloudy, other domain binds fibronectin. OF is so-called for
its ability to horse serum opacify.
Additional surface proteins (T,U,W,X,Y)are related to M protein.
Genes encoding these proteins have been designated as members
of emm gene superfamily.
C5A peptidase cleaves the
complement component C5A
Virulence factors: extracellular products (toxins)
Streptolysin O
all strains of GAS produce the toxin. Streptolysin is toxic
to a variety of cells including: erythrocytes, leukocytes and tissue
culture cells, can be inactivated by oxygen. Streptolysin O is an
immunogenic single chain protein; measurement of antistreptolysin O
antibodies in humans is used as an indicator of recent streptococcal
infection
Streptolysin S
most strains of GAS produce streptolysin S, this toxin is
produced by the organism in the presence of serum and is
nonantigenic. Streptolysin S consists of a polypeptide that has a
lytic effects for red and white blood cells and is responsable for the
hemolysis on culture plates.
Pyrogenic exotoxins
GAS produce different types of exotoxins (A,B,C,F…)
they are responsible for causing scarlet fever rash, endotoxic shock; they
can stimulate T cells proliferation and as superantigens are referred .
Virulence factors: extracellular products
(enzymes)
GAS release a large number of proteins in environment.
Streptokinase
catalyzes the conversion of plasminogen to plasmin leading
the digestion of fibrin
Hyaluronidase
hydrolyses hyaluronic acid found in connective tissues
Nucleases
serve to facilitate liquefaction of pus and invasion of
streptococci through tissues (cellulitis and necrotizing fascitiis)
Other enzymes:
proteinase, NADse, lipoproteinase…..
Diagnostic laboratory tests
Samples
Smears
are different in according with the different localization of
streptococcal infection. A throat swab, pus or blood or spinal fluid
are used for cultural methods.
Serum is used for antibody determinations
obtained from pus or blood show, to direct osservation, Gram positive
cocci, arranged in single cells, in pairs or in chains .
Smears obtained from throat swabs are rarely useful because other
streptococci i.e. viridans are always present and have the same aspect
as group A streptococci on stained smears.
Isolation procedures
Suspected specimens are planted on blood agar plates
Media additionated with blood, are preferred to determine the hemolytic reaction.
Columbia agar with sheep blood and gentamicin has been described as superior
for isolation of group A and B streptococci from sites containing normal flora
Cultures for isolation of streptococci should be incubated at 35 to 37 °C
Many streptococci require 5% CO2 concentration (pneumococci and some viridans
strains)
Streptococcal colonies vary in color from gray to whitish and are usually
glistening. Some strains of S. pyogenes may form mucoid colonies.
IDENTIFICATION
Microscopical observation
of isolated colonies with Gram stain, shows
Gram positive cocci arranged in chains
Hemolytic reactions on culture media
beta-hemolysis appears as
complete clearing (lysis of red blood cells) of the medium. This reaction
may be blocked by the inhibition of streptolysin O by oxigen; so
anaerobic incubation or observation of hemolysis in areas around
colonies is optimal for accurate determination of beta hemolytic
reaction.
Group A streptococci may be presumptively identified by inhibition
of growth by bacitracin, but this method should be used only when
more definitive test are not available.
A latex agglutination test for the identification of
streptococcal groups from isolated colonies
Streptococcal grouping kit
Reagents:
Latex grouping reagents A, B, C, D, F, G
Positive control
Extraction enzyme (should be reconstituted with distilled water)
Disposable reaction cards
Preparation
1) Dispense 0,4 ml of extraction enzyme to test tube
2) Select 2-5 colonies with a bacteriological loop and shake in enzyme
preparation
3) Incubate for 10 minutes at 37 °C (after 5 min remove each tube)
A latex agglutination test for the identification of
streptococcal groups from isolated colonies
Test method
1) Dispense 1 drop from each latex reagent (at room temperature
warming bottles by hands) on the reaction cards
2) Using a Pasteur pipette add 1 drop of extract to each of 6 rings
3) With mixing sticks spread the mixure
4) Gently rock the card. Agglutination will normaly take place within 30’’
5) Dispose the reaction card into a suitable disinfectant
Rapid Antigens Detection test
Several commercial kits are available for a rapid detection of group A
streptococcal antigens from throat swabs.
These kits use enzymatic or chemical methods to extract antigens
and use EIA or agglutination tests to demonstrate presence of
antigens.
Rapid tests are 60-90% sensitive and 98-99% specific when compared
to culture methods.
Antistreptolysin O titre (ASO)
ASO titre is used to demonstrate the body’s reaction to an infection caused
by group A beta-hemolytic streptococci.
GAS produce the enzyme streptolysin A, which can lyse red blood cells.
Because streptolysin O is antigenic (foreign protein) the body reacts by producing
antistreptolysin O (neutralizing antibody).
ASO appears in blood serum one week to one month after infection.
A high titre of ASO is not specific for any type of poststreptococcal disease but
it indicates if a streptococcal infection is or has been present.
Normal results
Antistreptolysin O titre adult: 160 units/ml
child six months to two years: 50 units/ml
child two to four years: 160 units/ml
child five to 12 years 179 units/ml
newborn: similar to the mother’s value
Treatment
All beta-hemolytic group A streptococci are sensitive to penicillin G.
A possible explanation for treatment failure especially in pharyngitis, is
a presence of beta-lactamase producing bacteria at side of infection
or poor patient compliance with dosing regiments.
Doses of penicillin for 10 days prevent poststreptococcal diseases
Most strains are sensitive to erythromycin
An increase in erythromycin resistance in S. pyogenes was noted during
the 1990 in many sides in worldwide. High rates of resistance
(20-40% of isolates tested) have been documented in different geographic
areas also in Italy.
Some are resistant to tetracyclines
Other streptococci
Streptococcus agalactiae (Group B Streptococcus)
History
Descovered by Frey in 1938 as etiological agent of three cases of fatal puerperal
sepsis and identified later by Lancefield in vaginal cultures from asyntomatic women
Human group B streptococcal infections were reported infrequently until the early
1960s.
By the 1970s, group B streptococci have become the predominant pathogens causing
septicemia and meningitis in neonates and infants younger than 3 months.
In the past two decades, has been recognized as etiological agent of infections also
in adults.
Streptococcus agalactiae:
morfological characteristics
Group B streptococci are
facultative anaerobic, Gram
positive streptococci,
growing on a variety of
bacteriological media.
Isolated colonies on blood agar
are 3 to 4 mm in diameter, gray
or white in color.
Colonies are often surrounded
by a zone of β-hemolysis, aboud
2% of strains are nonhemolytic
Streptococcus agalactiae:
classification and typing
Group B streptococci are serologically classified by capsular
polysaccharide type and by cell-surface expressed proteins.
Lancefield classified group B streptococci into 3 serotypes
designed I, II, III.
Later distinct differences in serotypes I strains have been
reported, and in 1970 these were designated: Ia, Ib, Ic.
Additional capsular polysaccharides types: IV, V, VI, VII, VIII
have been defined in the last years.
Streptococcus agalactiae:
epidemiology
Group B streptococci have been isolated from genital or lower
gastrointestinal tract cultures of pregnant and nonpregnant women
(from 10 to 40%).
Colonization more frequently occurs in case of diabetes mellitus or
in particular ethnic group.
Sexual activity is an important risk factor for vaginal acquisition.
Also multiple partners is associated with increased risk for vaginal
group B colonization.
Streptococcus agalactiae
Trasmission to neonates
Mucous membrane colonization of newborns results from vertical
trasmission of the organism from the mother, either in utero, or at
the time of delivery.
The rate of vertical trasmission among neonates born to women
colonized with group B streptococci at the time of delivery is
approximately 50%.
In addition to maternal intrapartum exposure, nosocomial
colonization of the neonate occasionally may occur.
Infant-to-infant spread by the hands of personnel is the most
likely acquisition system.
Group C and G streptococci
Group C and G streptococci
these microrganisms sometimes in the
nasopharynx occur and may cause sinusitis,
bacteremia, endocarditis and pharyngeal infections.
The clinical symptoms are similar to those of S.
pyogenes, except for the absence of nonsuppurative
sequelae.
Other streptococci of particular medical
interest
Viridans Group streptococci
so-called because they form a green pigmentation that surrounds
colonies grown on blood agar (alpha haemolysis). Viridans group
streptococci are normal inhabitants of the oral cavity, gastrointestinal
tract, and female genital tract. However, their presence may be
associated whith subacute bacterial endocarditis (S. sanguis, mitis,
gordonii). Members of mutans group are associated with dental
caries, dental plaque and with endocarditis. S. anginosus may be
isolated from oral abscesses and from female genital infections
The viridans group streptococci are assuming an increasing role in infections
in immunocompromised patients. The complications include: endocarditis,
acute respiratory syndrome, and shock
Streptococcus pneumoniae
History
S. pneumoniae is a very important organism in history of microbiology.
Identified in 1881 it has a central role in the discovery of DNA.
Experiments done by Griffith in the 1920s showed that intraperitoneal
injection of live, unencapsulated pneumococci together with heat-killed
encapsulated pneumococci into mice led to emergence of viable, encapsulated
bacteria; he called this process trasformation
Streptococcus pneumoniae
Morphology
Streptococcus pneumoniae are Gram-positive, lancet cocci. Usually they
are seen as pairs of cocci (diplococci), but they may also occur singly and
in short chains.
Streptococcus pneumoniae
When S. pneumoniae is cultured on blood agar is alpha hemolytic.
Pneumococci produce pneumolysin (called a-hemolysin) which breaks
hemoglobin into a green pigment, so pneumococcal colonies are surrounded
by a green zone on blood agar plates, but pneumolysin is not responsible of
lysis of red blood cells, in fact the greenish color appears around the
colonies of S. pneumoniae during growth also on chocolate agar: medium in
which all red blood cells have been lysed during preparation.
Streptococcus pneumoniae
Cultivation
Streptococcus pneumoniae is fastidious bacterium, growing in 5% carbon
dioxide, requires a source of catalase (e.g. blood) to neutralize the hydrogen
peroxide produced by this bacterium. Pneumococci initialy form a small
round, glistening colonies and later develop a central plateau with an elevated
rim.
Streptococcus pneumoniae is a very fragile bacterium and contains itself
the enzymatic ability to disrup the cells. The enzyme is called autolysin.
This enzyme kills the entire culture when grown to stationary phase.
All clinical isolates produce autolysin and a lysis begins between
18-24 hours after initiation of growth under optimal conditions.
Autolysis consists in changes of colony morphology. Colonies appear with a
plateau-type morphology when autolysis begins (piece colony).
Streptococcus pneumoniae
Characteristics
Gram positive
lanced diplococci
do not form spores
nonmotile
catalase negativity
susceptibility to optochin (ethyl hydrocupreine)
solubility in bile salts
ferment glucose to lactic acid
Pneumococcal antigens
Capsular polysaccharide
immunologically distinct in more than 90 types, capsular polysaccharide
is covalently bound to peptidoglycan and to cell wall polysaccharides
M protein
superantigen
C polysaccharide
it precipitates, in the presence of Ca++ , with a serum protein called
(C-reactive protein).
CPR is a serum protein that increases in response to infection, or
inflammation. CPR reacts with cell wall C-polysaccharide, and this reaction
can lead to complement activation.
Virulence factors
Capsule
a capsule composed by polysaccharides envelops the pneumococcal cells.
Capsule is an essential determinant of virulence in invasion.
Capsule interferes with phagocytosis by preventing C3b opsonization
of bacterial cells and causes inactivation of complement. 90 different
capsule types of pneumococci have been identified and form the basis of
antigenic serotyping of the organism.
In fact, anti-pneumococcal vaccines are produced by capsular antigens derived
from highly-prevalent strains.
Noncapsular virulence factors
Noncapsular constituents including: pneumolysin, surface proteins,
neuraminidase, autolysin . They contribute to pathogenesis of pneumococcal
diseases
Pneumolysin
all serotypes of S. pneumoniae produce pneumolysin, a thiol-activated toxin
that inserts ifself into the lipid bilayer of cell membranes via its interaction wit
cholesterol. Pneumolysin is cytotoxic for phagocytic and respiratory epithelial
cells and causes inflammation by activating complement, and inducing
production of tumor necrosis factor.
Surface proteins
are located on pneumococcal surface, mediate the invasion of
mammalian cells. They block the complement cascate.
Noncapsular virulence factors
Autolysin
disrupts the bacterial wall, in nature this enzyme contributes to cell wall
remodeling. In infection it contributes to disease by releasing peptodoglycan
components (toxic) and other substances such as pneumolysin (intracellular)
Neuraminidase
may contribute to bacterial adherence and colonization by cleaving sialic acid
on mucous membrane surfaces
Hyaluronidase
the role in pathogenesis has not been demonstrated
Clinical manifestations
Pneumococcal pneumonia
Pathogenesis
S. pneumoniae is a normal inhabitant of the human respiratory tract, (nasopharyngeal colonization occurs in approximately 80% of the population). This
bacterium can cause pneumonia, sinusitis, otitis media, or meningitidis.
Pneumonia and otitis media are the most common infections, meningitidis much
more variable
Colonization
pneumococci adhere to the nasopharyngeal epithelium by multiple mechanisms,
in particolar conditions, invasion of the lungs or middle ear occurs (pneumolysin
is cytotoxic on ciliated cells of the cochlea or respiratory tract).
Pneumococcal pneumonia
Invasion
Invasion is due to bacterial resistance to host phagocytis response.
Cell wall components directly activate multiple inflammatory cascades
including the alternative pathway of complement cascate.
After pneumococci begin to lyse in response to host defenses and to antimicrobial
agents, they release cell wall components, pneumolysin and other substances
with cytotoxic effects
If bacteriemia occurs the risk of meningitis increases. Pneumococci can adhere
to cerebral capillaries, after in cerebrospinal fluid a variety of pneumococcal
components, particularly cell wall components, are released causing inflammation.
Streptococcus pneumoniae: other
clinical syndromes
S. pneumoniae causes infection of the middle ear, sinuses, trachea, bronchi
and to the lungs by direct spread of organisms from the nasopharyngeal site
of colonization.
S. pneumoniae causes infection of the central nervous system, heart
valves, bones and peritoneal cavity by hemotogenous spread
Otitis media
Many studies have shown S. pneumoniae to be the most common isolate of acute
otitis media ( second only to Haemophilus influenzae, Moraxella catarrhalis is
usually third).
S. pneumoniae is implicated in about 40% to 50% of cases in which an etiologic
agent is isolated or in 30% to 40% of all cases.
Pneumococcus is the most prevalent pathogen in otitis media in adults
Streptococcus pneumoniae: other
clinical syndromes
Sinusitis
acute infection is caused by the same organisms as acute otitis media.
S. pneumoniae dominates or is second to H. influenzae. Important in
pathogenesis of this infection is congestion of mucosal membranes. The
accumulation of fluid in paranasal sinus cavities provides a medium for
bacterial proliferation and subsequent acute sinus infection.
Meningitis
S. pneumoniae is the most common cause of bacterial meningitis in adults
(except during an epidemic meningococcal infection). In countries that have
implemented a vaccination programs for H. influenzae type b; pneumococcus
has become the most common cause of meningitis in children.
Pathogenesis of meningitis, by direct dissemination from sinuses or the
middle ear can occur or may result by bacteremia
Streptococcus pneumoniae:
identification
A laboratory criteria for identification and differentation of pneumococci
from other streptococci are:
Gram stain
Hemolytic activity
Optochin sensitivity
Bile sensitivity
Streptococcus pneumoniae:
identification
Gram positive staining
Streptococcus pneumoniae:
identification
Hemolytic activity
S. pneumoniae is α hemolytic
organism.
Optochin sensitivity
Pneumococci form a 16 mm zone
of inhibition around a 5 mg optochin
disc.
Viridans streptococci are not
inhibited by optochin
Streptococcus pneumoniae:
identification
Bile sensitivity
Addition of a few drops of 10%
deoxycholate to liquid culture at 37°C
lyses the entire culture in minutes.
The ability of sodium deoxicholate to
dissolve the cell wall, depends by the
production of autolytic enzyme.
Streptococcus pneumoniae: treatment
Antibiotic generally used is penicillin.
Strains manifesting increased resistance to this drug and to other antibiotics
are isolated with significant frequency.
Penicillin inhibits replication of S. pneumoniae by binding one or more enzymes
required to synthesize peptidoglycan including higher-molecular-weight
transpeptidases (PBP).
Resistant isolates strains, produce PBPs with decreased affinity for penicillin.
Erytrhomycin or clindamycin may be used in allergic patients to penicillin,
but susceptibility to antibiotics should be tested before the treatment.
Tetracyclines may be used, but tetracycline-resistant strains are frequent
Chloramphenicol should be used only in treatment of pneumococcal meningitis in
allergic patients to penicillin
Vaccines
A vaccination program to protect against pneumococcal infection
has been reviewed extensively in recent years.
Two pneumococcal vaccines are available:
 pneumococcal capsular polysaccharide vaccine (Pneumovax), it contains
25 mg of capsular polysaccharides from each of 23 common infecting
serotypes of Streptococcus pneumoniae.
 protein-conjugated pneumococcal vaccine, it contains lesser amounts of
capsular material from seven pneumococcal serotypes that are most
implicated in disease of children (this vaccine is released only for
pediatric use).
Protein conjugate vaccines
Pneumococcal capsular polysaccharides have been covalently conjugated
to carrier proteins such as tetanus or diphtheria toxoid.
The resulting antigens are recognized as T-cell dependent; they stimulate
good antibody responses in children younger than 2 years of age
(who do not respond to polisaccharides antigens) and they induce
immunologic memory.
In adults conjugate vaccine can induce a response in immunocompromized
patients. Some studies suggest that an initial dose of conjugate vaccine
together a second dose of nonconjugate vaccine stimulates a better
response in patients with Hodgkin’s disease.
Other surface proteins are currently under study for use
in a vaccine
Enterococcus
General clinical microbiology
enterococci are Gram-positive cocci that occur in singles, pairs and short chains.
Because they are difficult to distinguish morphologically from true streptococci
until recently were classified as streptococci (in the Lancefield classification
scheme they were included among the group D streptococci). In the 1980s,
it was shown that enterococci differ sufficiently from streptococci and they
have been classified in their own genus. Enterococcus contains about 12 species.
Enterococcus species
E. faecalis
E. faecium
E. durans
E. avium
E. casseliflavus
E. malodoratus
E. gallinarum
E. hirae
E. mundtii
E. raffinosus
E. solitarius
E. pseudoavium
Enterococci: characteristics
Enterococci are facultative anaerobes that are able to
grow under rather extreme conditions.
They are able to grow in 6,5% NaCL, at pH 9,6 and at a
temperature ranging from 10° C to 45° C.
Many can survive 30 min at 60° C, and they grow in the
presence of 40% of bile salts.
They hydrolyze esculin to esculetin
Enterococci: classification
Most clinical isolates of enterococci are Enterococcus faecalis, which
until recently accounted for 80% to 90% of the enterococci isolated in
clinical microbiology laboratory
E. faecium accounted for 5% to 10% of isolated.
More recent evidence suggests that the prevalence of E. faecium, especialy
multiresistant strains is increasing in a number of hospital centers
Enterococci occur in environment because they are able to grow and
survive under harsh conditions (opportunistic pathogens)
They can be found in soil, food, water and a wide variety of living animals.
The major habitat of these organisms appears to be the gastrointestinal
tract of humans and of other animals (significant part of the normal
intestinal flora).
Pathogenetic mechanisms and
virulence
Until recently little was known about a factors that contribute to enterococci
ability to cause infections in humans.
Most enterococci do not have classic virulence factors, (do not secrete exotoxins
or produce superantigens) so a resistance of enterococci to multiple antimicrobial
agents allows them to survive and proliferate in patients receiving antimicrobial
chemotherapy (enterococci are recognized for their ability to cause superinfections
in patients receiving a number of different broad-spectrum antimicrobial agents).
Some studies have documented significant mortality rates (42% to 68%) in patients
with enterococcal bacteremia. Because most of this patients have been
debilitated, enterococcal bacteremia were a marker of this state and not a cause
of death. In this case enterococci were part of polymicrobial bacteremia and their
contribution to mortality, difficult to assess
Enterococci are not virulent organisms such as Staphylococcus aureus
or Streptococcus pyogenes
Pathogenetic mechanisms and
virulence
Several extracellular molecules play an important role in colonization and in
adherence.
Enterococci are able to adhere to the heart valves and to the renal epithelial
cells, this properties contribute to cause endocarditis and urinary tract infections.
Enterococci are able to colonize the oropharynx, but rarely they cause respiratory tract infections.
A second extracellular surface protein, designated EPS appears to play an
important role in colonization and in infection of humans with enterococci
Biofilm production contributes to ability of these organisms to colonize and
infect urinary and vascular catheters and to colonize heart valves.
Several investigators suggest that plasmid-mediated hemolysins secreted by
some strains may contribute to virulence, but their role remains to be determined
Clinical infections
Urinary tract infections
urinary tract infections are the most common clinical disease produced
by enterococci, and urine cultures are the most frequent sources of
enterococci in the clinical microbiology laboratory.
Most enterococcal urinary tract infections are nosocomial and they are
associated with medical implanted devices (catheters, instruments etc)
Several evidences suggest that a prevalence of nosocomial enterococcal
urinary tract infections is increasing in hospitals.
In contrast, enterococci only rarely cause infections such as
uncomplicated cystitis in nonhospitalized women.
Bacteremia and endocarditis
Most cases of enterococcal bacteremia are not associated with endocarditis.
Endocarditis is much more common in patients with a community acquired
bacteremia that in patients with nosocomial enterococcal bacteremia.
Nosocomial enterococcal bacteremia are commonly polymicrobial.
The most important causes of enterococcal bacteremia include:
the urinary tract infections, intra-abdominal or pelvic sepsis, wounds
especially thermal burns, decubitus ulcers, or diabetic foot infections,
intravenous catheters.
The mortality rate in patiens with enterococcal bacteremia is high, because
enterococcal bacteremia commonly occurs in debilitated patients
Meningitis and wound infection
Meningitis
Enterococci, rarely cause meningitis in normal adults. Most causes of enterococcal
meningitis in patients with anatomic defects of the central nervous system occur.
Meningitis is a rare complication of bacteremia in patients with enterococcal
endocarditis.
Meningitis can complicate enterococcal bacteremia in patients with severe immunodeficiecies including acquired immunodeficiency syndrome
Wound infections
Enterococci are rarely implicated in tissue infections. They are frequently isolated
from mixed cultures with Gram-negative bacilli and anaerobes in surgical wound
infections, decubitus ulcers and diabetic foot infections.
Enterococci have also been found in burn patients.
Neonatal sepsis
Enterococci have been documented to cause neonatal sepsis
characterized by fever, lethargy and respiratory difficulty with
bacteremia or meningitis or both.
Several nosocomial manifestations due to Enterococcus faecium or
Enterococcus faecalis have been described in premature or
low-birth-weigth neonates
Antimicrobial susceptibility and
resistance
The most known characteristic of enterococci is the relative and absolute
resistance of these organisms to a variety of antimicrobial agents used for
treatment of infections caused by Gram-positive organisms.
Not only these organisms are intrinsically resistant to a large number of
antimicrobial agents, but they show a remarkable ability to acquire a new
mechanisms of resistance
Intrinsic resistance
Aminoglycosidic aminocyclitols (low level)
β-Lactams (relatively high MICs)
Lincosamides (low level)
Trimethoprim-sulfamethoxazole (in vivo only)
Quinupristin/Dalfopristin (Enterococcus faecalis-resistant)
Antimicrobial susceptibility and
resistance
In addition to their intrinsic resistance, enterococci have acquired new mechanisms
of resistance to a wide variety of antimicrobial agents.
Most of these resistance mechanisms are mediated by genes encoded on plasmids
or transposons. Enterococci have evolved a number of efficient methods of
transferring resistance genes, and this greatly facilitates the acquisition of new
resistance determinants.
Acquired resistance
Aminoglycosidic aminocyclitols (high level)
Penicillin and ampicillin (β-lactamase)
β-Lactams (altered PBPs)
Fluorochinolones
Lincosamides (high level)
Macrolides
Tetracyclines
Vancomycin (a number of phenotypes of vancomycin resistant have been discovered)
Therapeutic approaches
Treatment of enterococcal infections is complicated by their unusual patterns
of susceptibility or resistance (it is necessary to use specific techniques to
demonstrate their susceptibility in clinical microbiology laboratory)
Penicillin or ampicillin remain the antibiotics of choice for treatmen of enterococcal
infections such as urinary tract infections, peritonitis, and wound infections that do
not require bactericidal treatment.
Vancomycin or teicoplanin are the alternative agent in patients allergic to penicillin
or for organisms with high-level penicillin resistance.
Fluoroquinolones such as ciprofloxacin and ofloxacin have in vitro activity against
enterococci (levofloxacin, gatifloxacin and moxifloxacin are more active than
ciprofloxacin)
Quinupristin/dalfopristin (a combination of streptogramins A and B) has a broad
spectrum of activity against enterococci, also novel oxazolidinones and glycylglycynes
show potent activity against enterococci.
Neisseriae
Neisseria family includes Neisseria species and Moraxella catarrhalis
Members of Neisseria genus are Gram negative
cocci usually occuring in pairs.
Neisseria gonorrhoeae (gonococci) and
Neisseria meningitidis (meningococci) are
pathogenic for humans and tipically are found
associated with or inside polymorphonuclear
cells.
Neisseria sicca, N. subflava,
N. flavescens, N. cinerea are normal
inhabitants of the human respiratory tract,
and rarely cause diseases.
Morphology and culture
Neisseriae are Gram negative, nonmotile
diplococci, approximately 0,8 µm in diameter
In 48 hours, on enriched media: Mueller Hinton,
modified Thayer Martin, gonococci and
meningococci form convex, glistening, mucoid
colonies 1-5 mm in diameter.
Colonies are transparent or opaque,
nonpigmented and nonhemolytic.
Neisseria flavescens and subflava have yellow
pigmentation.
Neisseria sicca produces opaque brittle colonies
Growth characteristics
Most Neisseriae grow well under aerobic conditions, some strains grow also in
anaerobic conditions.
Most Neisseriae ferment carbohydrates, producing acid but not gas
(carbohydrate fermentation serve to distinguish them).
The Neisseriae produce oxidase (oxidase positive).
Test: bacteria are spotted on a filter paper,
with the addition of tetramethylparaphenylenediamine
culture rapidly turn dark purple.
Growth characteristics
Meningococci and gonococci grow best on bacteriological media containing complex
organic substances such as heated blood, hemin, and animal proteins and in an
atmosphere containing 5% Co2
This organism are rapidly killed by drying, sunlight, moist heat and many
disinfectants.
They produce autolytic enzymes that result in rapid swelling and lysis in vitro
at 25°C and at an alkaline pH
Neisseria gonorrhoeae
Gonococci ferment only glucose and differ antigenically from other neisseriae.
Gonococci usually produce smaller colonies than those of the other neisseriae.
Gonococci isolated from clinical specimens form typical small colonies containing
piliated bacteria.
On nonselective subculture, larger colonies containing nonpiliated gonococci are
also formed. Opaque and trasparent variants derived by small or large colonies
often occur; the opaque colonies are associated with the presence of a surfaceexposed protein, Opa.
Neisseria gonorrhoeae: antigenic structure
Pili
Cell envelope
Pili are hair-like appendages that extend up to
several micrometers from the gonococcal
surface. They promote the attachment to host
cells and resistance to phagocytosis.
The pili of different strains of N. gonorrhoeae
are antigenically different.
peptidoglycan
Outer membrane
Cytoplasmatic
membrane
pilus
pilus
Neisseria gonorrhoeae: antigenic structure
POR
Por proteins (PorA and PorB) are heat-stable integral proteins.
They occur in trimers to form pores on outer membrane. The molecular
weight of por varies from 34,000 to 37,000. Each strain of gonococcus
expresses only one type of Por. Por of different strains are antigenically
different.
Por proteins may influence intracellular killing of organisms in
polymorphonuclear leukocytes by preventing phagosome-lysosome fusion and
by diminishing oxidative burst
Neisseria gonorrhoeae: antigenic structure
OPA (opacity associated protein)
The OPA proteins are usually found on colonies with
an opaque fenotype.
These proteins are implicated in adhesion of gonococci
and in attachment to host cells.
They are heat-labile, the molecular weight of OPA
ranges from 24.000 to 32.000.
Each strain of gonococcus can express
one, two, or occasionally three types of OPA.
Other proteins
RMP
This protein is antigenically conserved in all gonococci. It is associated
with Por to form pores in cell surface.
Lipooligosaccharide LOS
Gonococcal LPS does not have long O-antigen chains. Toxicity in gonococcal
infections is due to endotoxic effects of Lipooligosaccharide LOS.
IgA1 protease
Inactivates IgA1, a major mucosal immunoglobulin of humans, (also other bacteria
elaborate similar IgA1 proteases)
Pathogenesis, pathology and clinical findings
Gonococci attack and colonize the mucous membranes of the genitourinary tract,
eye, rectum, and throat producing acute suppuration.
In males, they produce usually urethritis with yellow pus and painful urination. The
process may extend to epididymis.
In females expecially the endocervix is infected. The process extends to the
urethra and vagina.
Infertility occurs in 20% of women with gonococcal salpingitis.
Gonococcal bacteremia leads to skin lesions on the hands,
feet and legs.
Gonococci sometimes cause meningitis and endocarditis
Pathogenesis, pathology and clinical
findings
Gonococcal ophthalmia neanatorum, is an infection of the eye acquired
by newborns during the passage through an infected birth canal.
To prevent gonococcal ophthalmia, instillation of tetracycline,
erythromycin or silver nitrate into the conjunctival sac of the
newborns is used.
Diagnostic laboratory tests
Specimens
Pus and secretions are collected from the urethra, cervix, rectum, conjunctiva
and throat for culture and smear
Smears
Gram-stained smears of urethral or endocervical exudate show many diplococci
mixed to cells.
Diagnostic laboratory tests
Immediately after collection, pus or mucus are
streaked on enriched selective medium such as
modified Thayer Martin medium and incubated
in an atmosphere containing 5% CO2. This
selective medium contains antimicrobial drugs
(vancomycin, colistin, amphotericin B, and
trimethoprim).
Forty-eight hours after culture, the organisms
can be identified by their Gram negative
aspect, by oxidase test or other laboratory tests.
A liquid culture may be prepared to detect
the different fermentation of sugars.
Different fermentation of sugars
Glucose Maltose
lactose
saccharose
N.
meningitidis
+
+
-
-
+
-
-
-
-
-
-
-
N.
gonorrhoeae
M.
catarrhalis
Diagnostic laboratory tests
Serology
In infected people, antibodies to gonococcal pili and outer membrane proteins
can be detected by immunoblotting, radioimmunoassay, and ELISA tests.
Treatment
Gonococcal resistance to penicillin is increasing in this period; many strains now
require high concentration of penicillin G, and penicillinase-producing
strains are isolated.
High resistance levels against Tetracycline, Spectinomycin and other antimicrobials
have been described.
Uncomplicated genital or rectal infections are treated with ceftriaxone.
Ceftriaxone associated with doxicycline twice a day for 7 day is recommended in
presence of possible concomitant chlamydial infection.
Erithromycin is used instead of doxycycline in pregnant women.
Neisseria meningitidis
Antigenic structure
Capsule
13 serogroups of meningococci have been identified on the basis of
immunological differences in capsular polysaccharides.
The most important serogroups associated with disease in humans are:
A.B,C, Y and W135. Meningococcal antigens are found in blood and in
cerebrospinal fluid of patients with active disease.
Outer membrane proteins
are classified on the basis of molecular weight.
Pili
LPS
is responsable for most of the toxic effects.
Pathogenesis, pathology and
clinical findings
Bacteremia.
Neisseria meningitidis is a normal inhabitant of nasopharynx tract. In case of lowed
host defences, in presence of other infections, the microrganisms become invasive
and may reach the bloostream, producing bacteremia.
Meningococcemia is more severe, with high fever and hemorrhagic rash
Pathogenesis, pathology and
clinical findings
Meningitis
Meningitis is the most complication of meningococcemia. It usually begins
suddenly, with intense headache, vomiting and progresses to coma within few
hours.
In meningitis, the meninges are acutely inflammed, with thombosis and exudation
of polymorphonuclear leukocytes, so the surface of the brain is covered with
purulent exudate.
Diagnostic laboratory tests
specimens
Specimens of blood are used for culture; specimens of spinal fluid are used
for smears, culture and chemical determination.
Nasopharyngeal swab cultures are used for carrier screening.
smears
Spinal fluid must be centrifugated. Collected sediment, observed to microscope
with Gram stain method, shows typical neisseriae within polymorphonuclear
leukocytes.
Diagnostic laboratory tests
Culture
Cerebrospinal fluid specimens are plated on heated blood agar (chocolate agar)
and incubated at 37°C in an atmosphere of 5% CO2.
A modified Thayer-Martin medium with antibiotics (vancomycin, colistin,
amphotericin) is used for nasopharyngeal cultures because inhibits many other
bacteria
Suspected colonies can be identified by Gram stain and oxidase test
Spinal fluid and blood generally produce pure cultures that can be identified by
carbohydrate fermentation reactions and agglutination with type-specific serum.
Treatment
Amoxicillin is the drug of choice for treatment of meningococcal disease.
Also chloramphenicol or a third-generation cephalosporin such as
ceftriaxone or cefotaxime can be used
Moraxella catarrhalis
Moraxella catarrhalis was previously named
Branhamella catarrhalis and before Neisseria catarrhalis.
It is a member of the normal flora in 40-50% of normal
school children. M. catarrhalis causes bronchitis,
pneumonia, sinusitis, otitis media and conjunctivitis,
it causes infections also in immunocompromized patients.
Most strains of M. catarrhalis produce β-lactamase.
M. catarrhalis can be differentiates from the other
Neisseriae because does not ferment carbohydrates but
it produces Dnase.
Micobacterium: general
characteristics
Many species within the genus Mycobacterium are prominent pathogens such as
Mycobacterium tubercolosis and Mycobacterium leprae.
In addition numerous species of environmental mycobacteria called nontuberculous
Mycobacteria (atypical mycobacteria or mycobacteria other than tubercle bacilli)
are responsable for various kinds of mycobacterioses.
Tubercolosis remains a major global public health problem.
In 1997 there were 8 million estimated new cases including 3,5 million cases of
smear-positive pulmonary tubercolosis.
In the future the World Health Organization estimates that between 2000 and
2020, 35 million people will die from tubercolosis.
Mycobacterium: general
characteristics
Factors facilitating the resurgence of tuberculosis in recent years included
the advent of the AIDS epidemic, immigration from other countries, trasmission
in high-risk settings (hospital and prison) and the increase in the number of cases
of multidrug-resistant tubercolosis
Prevention strategies and control measures implemented by the health authorities,
including the use of more rapid and efficient laboratory methods, serve to decrease
the number of reported cases.
In this context, the clinical mycobacteriology laboratory plays an
important role
Mycobacterium: general characteristics
Mycobacteria are aerobic (some species are able to grow under a reduced O2
atmosphere)
Non spore-forming
Nonmotile
Straight or curved rods (filamentous or mycelium-like growth sometimes occurs)
Mycobacterium colonies
Colony morphology varies among the
species, ranging from nonpigmented
to pigmented. Colonies are yellow,
orange, or rarely pink, usually due to
carotenoid pigments
Mycobacterium: cell wall
The cell wall conteins meso-diaminopimelic
acid, alanine, glutamic acid and mycolic acids
(number of carbon atoms ranging from 60
to 90), togheter with free lipids (trehalosedimycolate), provide a hydrophobic permeability barrier.
Other important fatty acids are waxes and
phospholipids.
The high content of complex lipids of the
cell wall prevents access by common aniline
dyes. Mycobacteria are usually considered
Gram positive but not are stained by Gram’s
method. Once stained with special procedurs, they are not easily decolorized, even
with acid-alcohol, in fact they are acid-fast.
Nutritional requirements and growth
Most species adapt to growth on simple substrates using ammonia or amino acids
as nitrogen sources and glycerol as a carbon source.
Growth of Mycobacteria is stimulated by fatty acids, which may provide in the form
of cell wall compounds.
Optimum temperature vary from 30 to 45°C
Compared to other bacteria, growth of most
mycobacterial species is slow.
Visible colonies appear after few days to 6
weeks of incubation under optimum conditions.
Mycobacteria require less than 7 days when
subcultured on Löwenstein-Jensen, but may
also take several weeks on primary culture
from clinical specimens
Mycobacterium tuberculosis
Morphology
In tissue, tubercle bacilli are straight rods measuring about 0,4 x 3 μm.
On artificial media, coccoid and filamentous forms are seen with variable
morphology.
Tubercle bacilli are characterized by “acid fastness” (95% ethyl alcohol containing
3% hydrochloric acid decolorizes all bacteria except the mycobacteria).
Acid-fastness depends on the integrity of the waxy envelope
The Ziehl-Neelsen technique
of staining is employed for
identification of acid-fast
bacteria
Constituents of tubercle bacilli
Lipids
Mycobacteria are rich in lipids. These include mycolic acids, waxes and phosphatides
In the cell wall the lipids are largely bound to proteins and polysaccharides.
Muramyl dipeptide complexed with mycolic acids can cause granuloma formation;
phospholipids induce caseous necrosis in tuberculosis.
Virulent strains of tubercle bacilli form in liquid medium, “serpentine cords” in
which acid-fast bacilli are arranged in parallel chains.
Cord formation is correlated with virulence.
A “cord factor” (trehalose-6,6-dimycolate) has been extracted from virulence
bacilli with petroleum ether. It inhibits migration of leukocytes, causes chronic
granulomas and can serve as an immunologic “adjuvant”.
Pathogenesis of tuberculosis
Mycobacteria in droplets (1-5 µm in diameter) are inhaled and arrive in the alveoli
Where moltiplication begins
The disease results from establishment and proliferation of virulent organisms
and interactions with the host.
Injected bacilli survive for months or years in the normal host.
Resistance and hypersensitivity of the host can influence the development of the
disease
The initial focus is usually in the midlung zone where airflow favours deposition
of bacilli.
The bacteria are ingested by alveolar macrophages, which may be able to eliminate
small numbers of bacteria
Pathogenesis of tuberculosis:
immunology
Tuberculosis is the prototype of infections that require a cellular immune response
for their control.
During infection, abundant antibodies are produced, but they play no apparent role
in host defence mechanisms.
Small inhaled inocula multiply in alveolar spaces or in alveolar macrophages. Entry
into macrophages involves interactions with complement receptors.
Replication process proceeds for weeks.
The development of tissue hypersensitivity and cellular immunity ultimately
Supervenes.
All persons have a population of lymphocytes able to recognize mycobacterial
antigens that have been processed and presented by macrophages in a major
histocompatibily complex class II context.
When lymphocyte encounters antigen, it is activated and proliferate producing a
clone of T cells.
T cells produce secretory proteins, which attract and activate macrophages with
accumulation of lytic enzymes that increase their mycobactericidal competence
Reactions to tubercolin
When the population of lymphocytes is activated, cutaneous delayed reactivity to
tmubercolin or tissue hypersensitivity, become manifest
Tubercolin test
Material
Koch’ tubercolin (old tubercolin) was an extract of a boiled culture of
tubercle bacilli. In 1934 S.iebert made a simple protein precipitate (purified
protein derivative PPD) of old tubercolin, which became preferred reagent.
Reactions to tubercolin
In an individual who has not had contact with mycobacteria, there is no reaction
to PPD.
An individual who has had a primary infection with tubercole bacilli, develops
induration, edema, erythema in 24-48 hours with vary intense reactions. The skin
test should be read in 48 or 72 hours. Positive tests tend to persist for several
days
Interpretation of tubercolin test
A positive tubercolin test indicates that an individual has been infected in the past
and continues to carry viable mycobacteria in some tissue.
Tubercolin-positive persons are at risk of developing disease from reactivation of
the primary infection.
Tubercolin-negative persons are not subject to that risk, but may become infected
from an external source
Interpretation of tubercolin test
A positive tubercolin test indicates that
an individual has been infected in the
past and continues to carry viable
mycobacteria in some tissue.
Tubercolin-positive persons are at
risk of developing disease from
reactivation of the primary infection.
Tubercolin-negative persons are not
subject to that risk, but may become
infected from an external source
Tuberculosis: pathology
The production and development of lesions in tubercolosis are determined by:
1) The number of mycobacteria in the inoculum and their subsequent multiplication
2) The resistance and hypersensitivity of the host
Two principal lesions
1.Exudative type This consists of an acute inflammatory reaction with edema fluid,
polymorphonuclear leukocytes, and, later monocytes around the tubercle
bacilli. This type is seen particularly in lung tissue. It may heal by resolution
(entire exudate becames absorbed) or it may develop into the second
productive type of lesion. The tubercolin test becomes positive
2. Productive type In this phase appears a chronic granuloma, which consists of
three zones
Tuberculosis pathology
Two principal lesions
1.Exudative type This consists of an acute inflammatory reaction with
edema fluid, polymorphonuclear leukocytes, and, later monocytes around the
tubercle bacilli. This type is seen particularly in lung tissue. It may heal by
resolution (entire exudate becames absorbed) or it may develop into the
second productive type of lesion. The tubercolin test becomes positive
2. Productive type in this phase a chronic granuloma appears. It consists of
three zones: 1) a central area of large, multinucleated cells 2) a mid zone of
epithelioid cells 3) a peripheral zone of fibroblast, lymphocytes and monocytes.
Later peripheral fibrous tissue develops and the central area sustains a caseation
necrosis. This lesion is called: “tubercle”.
A caseous tubercle may break into a bronchus, empty its contents there, and form
a cavity. It may heal by fibrosis or calcification.
Tuberculosis: productive type
A caseous tubercle may
break into a bronchus,
empty its contents there,
and form a cavity in the
lung tissue.
Spread of organisms in the host
Tubercle bacilli spread in the host by direct extension through the lymphatic
channels and bloodstream, through bronchi and gastrointestinal tract.
In the first infection, tubercle bacilli always spread from the initial site to the
regional lymph nodes. Bacilli may spread farther and reach the bloodstream, which
distributes bacilli to all organs (miliary distribution).
When caseating lesion discharges its contents into a bronchus, they are aspirated
and distributed to other parts of the lungs or are swallowed and passed into the
stomach and intestines.
Primary infection and reactivation
types of tuberculosis
When a host has first contact with tubercle bacilli, this cases are usually observed:
1) An acute exudative lesion develops and rapidly spreads to the lymphatics and
regionanal lymph nodes. The exudative lesion often heals rapidly.
1) The lymph nodes undergoes massive caseation, which usually calcifies.
3) The tubercolin test becomes positive
This primary infection type occurred in the past, especially in children, but now
frequently in adults remained free from infection and so tubercolin negative
The reactivation type is usually caused by tubercle bacilli that have survived in the
primary lesion. Reactivation tuberculosis is characterized by chronic tissue lesions,
the formation of tubercle, caseation and fibrosis.
Primary infection and reactivation
types of tuberculosis
Differences between primary infection and reactivation are attributed to:
1) Resistance (capacity to localize tubercle bacilli, retard their moltiplication,
limit their spread lymphatic dissemination
2) Hypersensitivity induced by the first infection of the host with tubercle bacilli
Diagnostic laboratory tests
A positive tubercolin test does not prove the presence of active disease due to
tubercle bacilli. Isolation of tubercle bacilli provides such proof.
Specimens
Specimens consist of fresh sputum, gastric washings, urine, pleural fluid, biopsy
material, blood, or other suspected material.
Decontamination and concentration of specimens
Specimens from sputum and other nonsterile sites should be liquefied with
N-acetyl-l-cysteine, decontamineted with NaOH (kills many other bacteria and
fungi), neutralized with buffer and concentrated by centrifugation. Specimens, now
can be used for acid-fast stains and for culture.
Specimens from sterile sites, such as cerebrospinal fluid, do not need the
decontamination procedure but can be directly centrifugated, examined and cultured.
Diagnostic laboratory tests
Smears
Sputum exudates, or other material is examinated for acid-fast bacilli by
Ziehl-Neelsen staining. Stains of gastric washings and urine generally are not
Racommended, because saprophytic mycobacteria may be present.
Fluorescence microscopy with auramine-rhodamine stain is more sensitive than
acid-fast stain.
If acid-fast organisms are found in an appropriate specimen, this is presuntive
evidence of mycobacterial infection
Diagnostic laboratory tests
Culture
Specimens derived from nonsterile or sterile sites can be cultured into selective
media. A selective agar media (eg. Löwenstein-Jensen should be inoculated in
parallel with broth media cultures. Incubation is at 37°C in 5-10% CO2 for up to
8 weeks
Identification
Conventional methods for identification of mycobacteria include observation of
rate of growth, colony morphology, pigmentation and biochemical profiles.
The conventional methods for classifying mycobacteria are rapidly becoming of
historical interest, because molecular probe methods are much faster and easier.
The probes can be used on mycobacterial growth from solid media or from broth
cultures.
The use of these probes has shortened the time to identification of clinically
important mycobacteria from several weeks to as little as 1 day.
Diagnostic laboratory tests
Identification: other methods
High-performance liquid chromatography (HPLC) has been applied to mycobacteria
identify. The methods is based on development of profiles of mycolic acids, which
vary from one species to another
The polymerase chain reaction holds great promise for the rapid and direct
detection of Mycobacterium tuberculosis in clinical specimens.
The test has the highest sensitivity when applied to specimens that have smears
positive for acid-fast bacilli; the PCR test is approved for this use on sputum
specimens that are acid-fast stain-positive.
Enzyme immunoassays have been used to detect mycobacterial antigens, but the
sensitivity and specificity are less than with other methods.
Treatment
The primary treatment for mycobacterial infection is specific chemotherapy.
Between one in 106 and one in 108 tubercle bacilli are spontaneous mutants resistant
to first-line antituberculosis drugs. When the drugs are singly, the resistant
tubercle bacilli emerge rapidly and multiply. Therefore, treatment regimens use
drugs in combination.
The two major drugs used to treat tuberculosis are: isoniazid and rifampicin.
The other first-line drugs are pyrazinamide, ethambutol and streptomycin.
Second-line drugs are more toxic or less effective (or both), and they should be used
in therapy only under particular circumstances (treatment failure, multiple drug
resistance).
Second-line drugs include: Kanamycin, capreomycin, ethionamide,
cycloserine, ofloxacin and ciprofloxacin
Drug resistance in M. tuberculosis
Drug resistance in Mycobacterium tuberculosis is a wordwide problem.
Mechanisms explaining the resistance phenomenon for many but not all of the
resistant strains have been defined.
Isoniazid resistance has been associated with deletions or mutations in the
catalase-peroxidase gene (these isolated become catalase-negative.
Streptomycin resistance has been associated with mutations in genes encoging
the ribosomal S12 protein and 16S rRNA respectively.
Rifampicin resistance has been associated with alterations in the b subunit of RNA
polymerase
A four-drug regimens of isoniazid, rifampicin, pyrazinamide and ethambutol is
recommended for persons who have a risk for infection with drug-resistant
tubercle bacilli. The risk factors include recent emigration from Latin America or
Asia, persons with HIV infections or living in an area with a low prevalence of
multidrug-resistant tubercle bacilli.
Multidrug-resistant Mycobacterium
tuberculosis
Multidrug-resistant M. tuberculosis (resistant to both isoniazid and rifampicin)
is a major and increasing problem in tuberculosis treatment and control.
These strains are prevalent in certain geografic areas and in certain populations
(hospital and prison). There have been many outbreaks of tuberculosis with
multidrug resistant strains.
Persons infected with multidrug-resistant organisms or who are are at high risk
for such infections, should be treated according to susceptibility test results for
the infecting strain
Therapy should include a minimum of three or more than three drugs to which
the organisms have demonstrated susceptibility.
Immunization
Immunization
Various living avirulent tubercle bacilli, particularly BCG (bacillus Calmette-Guerin,
an attenuate bovine organism), have been used to induce resistance in exposed to
infection subjects.
Vaccination with this organisms can be considered a sostitute for primary infection
with virulent tubercle bacilli without a danger. The available vaccines are anadequate,
so in the United States, the use is suggested only for tubercolin-negative persons
who are exposed (members of tuberculous families, medical personnel)
Other mycobacteria
In addition to tubercle bacilli (M. tuberculosis and bovis), other mycobacteria
of varying degrees of pathogenicity have been grown from human samples in past
decades.
These “atypical” mycobacteria were initial grouped according to speed of growth
at various temperatures and production of pigments
Mycobacterium avium complex
The Mycobacterium avium complex is often called
MAC or MAI (M. avium intracellulare) complex.
These organisms grow optimally at 41° C and produce
smooth, soft, nonpigmented colonies.
They are ubiquitous in the environment. They can cause
disease in immunocompetent humans; however disseminated MAC infection is one of the most common opportunistic infections of bacterial origin in AIDS patients.
Environmental exposure can led to MAC colonization of
either the respiratory or gastrointestinal tract.
Any organ can be involved: in the lungs, nodules, diffuse
infiltrates, cavities, and endobronchial lesions are common.
Other manifestations include pericarditis, soft tissue abscesses,
skin lesions, lymph node involvement. The patients present
nonspecific symptoms of fever, abdominal pain, diarrhea.
The diagnosis is made by culturing MAC organisms from blood or
tissue. For treatment may be used. Rifabutin, fluoroquinolones
and amikacin.
Mycobacterium kansasii
Mycobacterium kansasii is a photochromogen that
requires complex media for growth at 37 °C.
It can produce pulmonary and systemic disease
indistinguishable from tuberculosis.
Sensitive to rifampin, it is often treated with the
combination of rifampin, ethambutol and isoniazid
with good clinical response.
Mycobacterium leprae
This organism was described by Hansen in 1873.
It has not been cultivated on nonliving bacteriologic media,
(but naturally occurring infections in armadillo have been
documented in Texas and Louisiana).
It causes leprosy.
There are more than 10 million cases of leprosy, especially
in Asia
Mycobacterium leprae: morphology
Typical acid-fast bacilli, single in parallel
bundles or in globular masses, are
regularly found in scraping from skin or
mucous membranes
Clinical findings
The lesions involve the cooler tissue of the body:
skin, superficial nerves, nose, pharynx, eyes, hands.
The skin lesions may occur such as anesthetic
macular lesions (1-10 cm in diameter), diffuse
erythematous infiltrated nodules 1-5 cm in diameter.
Neurologic disturbances are manifested with
resultant anesthesia, paresthesia, trophic ulcers,
bone resorption and shortening of digits.
In untreated cases, this condition may be extreme.
Leprosy: two types
The disease is divided into two major types: lepromatous
and tuberculoid with several intermediate stages.
In the lepromatous state , the course is progressive
and malign, it is characterized
by nodular skin lesion with abundant acid-fast bacilli,
bacteremia and a negative lepromin ( extract of
lepromatous tissue) skin test, because in lepromatous
leprosy cell-mediated immunity is deficient.
In the tuberculoid type, the course is benign and
nonprogressive, with macular skin
lesions and a positive lepromin skin test.
In fact in tuberculoid leprosy, cell-mediated
immunity is intact.
Systemic manifestations and anemia may also
occur. Eye involvement is common.
Diagnosis and treatment
For diagnosis, scrapings with a scapel blade from skin or nasal mucosa are smeared
on a slide and stained by the Ziehl Neelsen technique.
No serologic tests are of value
For treatment, sulfones such as dapsone are first-line
therapy for both tubercoloid and lepromatous leprosy.
Rifampin or clofazimine generally is included in the initial
treatment regiments.
Other drug active include: minocycline, clarithromycin and
some fluorochinolones.
World Health Organization recommends several years of
therapy
Prevention and control
Identification and treatment of patients with leprosy are the keys to control.
Experimental BCG vaccination and an M. leprae vaccine are also being explored
for family contacts and possibly for community contacts in endemic areas.
Bacillus anthracis (Anthrax)
History
The word “anthrax” derives from the Greek, applicable to the black
eschar that forms in cutaneous anthrax.
Anthrax, the fifth plague that killed the Egyptians described in the
book of Exodus can be described.
In 1881 Pasteur descovered the first heat-attenuated anthrax
vaccine able to protect the animals.
Anthrax has been a disease of animals primarily, with a long history
of animals-associated disease in humans.
Most recently, in 2001 human cutaneous and inhalational forms of
anthrax were acquired a new importance when B. anthracis spores
were sent in contaminated letters as an act of bioterrorism in the
United States
Bacillus anthracis microbiology
B. anthracis is a Gram positive,
spore-forming rod, aerobic or
facultatively anaerobic.
Smears prepared by cultures show
“cigar-shaped” chains; in contrast
smears obtained from tissues,
blood or fluids, show only short
chains or single cells.
The organism is nonmobile,
nonhemolytic, catalase positive.
Bacillus anthracis: microbiology
B. anthracis grows on sheep blood agar.
Colonies appearence is tipically white, characterized
by a “Medusa’s head” aspect
(elements are similar to a mass of hairs).
Under anaerobic conditions, a polypeptidic
capsule is secreted consisting of poly-D-glutamic
acid. Capsule can be visualized by Indian ink method
Bacillis anthracis: virulence
factors
Capsule is one of the major virulence factors and is responsable
for inhibition of phagocytosis
Toxin
is formed by two parts called “edema factor” (EF) and
“lethal factor” (LF). Both of these factors must bind
the third toxin component called “protective
antigen” (PA), finally, they can penetrate a target cell,
such as a macrophage or dendridic cell.
Bacillus anthracis: pathogenesis
Pathogenesis of anthrax has been attributed to macrophage-lysis
mediated by cytokines release, particularly tumor necrosis factorand IL-1, and to septic shock resulting in death.
Macrophage lysis was not required for anthrax-induced death,
instead tissue hypoxia and liver necrosis were observed (the exact
mechanism for this damage was not defined).
Edema factor (responsible for cutaneous forms) is a calmodulindependent adenylate cyclase enzyme that converts ATP to cyclic
adenosine monophosphate (cAMP).
Intracellular increase in cAMP results in dysregulation of water
and ions.
Anthrax spores
Anthrax spores can survive for months or
decades depending on pH,
temperature and nutrients in soil.
Spores ingested from the soil by
cattle or other herbivores then germinate
into the vegetative form in the
spleen or lymph nodes resulting in
bacteremia and hemorrhage as a
terminal event.
Vegetative forms are deposited in
soil and sporulation occurs
continuing the cycle of infection
Infection of humans occurs most frequently by exposure to B. anthracis
spores or when the vegetative forms are ingested in the meat of
infected animals
Clinical manifestations: Cutaneous
anthrax
Approximately 95% of all human anthrax is cutaneous.
The incubation period is 1 to 12 days. The initial skin lesion is a papule on the
exposed area of the neck, head or upper extremity. The papule progresses and
the central lesion becomes necrotic and hemorrhagic and may
develop peripheral vesicles.
Finally the classic central black eschar appereas, often accompaned by
edema. The progression of cutaneous anthrax lesions from papule to black
eschar surrounded by edema is mediated by toxin and occurs also in case of
appropriate antibiotic treatment. Mortality more likely in patients developing
bacteremia occurs.
Gastrointestinal anthrax
Gastrointestinal anthrax is a rare disease accounting for less than 5% of all
cases of anthrax in humans. When anthrax is ingested in food or liquid it can
cause two syndromes: oropharyngeal and/or intestinal anthrax. The symptoms
of oropharyngeal form are: a painful swelling of the neck caused by cervical
adenophaty and soft tissue edema. Oral lesions and edema were seen on the
tonsils, hard palate. After one week ulceration and central necrosis occur
forming a pseudomembrane covering the ulcers. The intestinal form is more
common characterized by abdominal pain, nausea, vomiting and signs of ascites.
Inhalation anthrax
Inhalation anthrax is a medical emergency documented by several cases of anthrax
bioterrorism attack. Clinically is present hemorrhagic mediastinal adenopathy,
multilobar pneumonia with hemorrhagic pleural effusion and frequent bacteremia
followed by cardiovascular collapse.
Prevention
Vaccination
At present four nations are producing anthrax vaccines for
humans:
China, Russia, Britain and United States. The FDA has reserved
the use of anthrax vaccine only to US military personnel and to
people working with anthrax in laboratory.
After the anthrax bioterrorism attacks the “Advisory Committee
on Immunization Practices in November 2002 provided
supplemental raccomandations for the use of vaccination.
Treatment
For cutaneous anthrax, prior to the 2001, a 7 to 10 days treatment
using intravenous penicillin and corticosteroids in case of edema
were recommended
After the bioterrorism attacks in 2001 protocol includes:
Ciprofloxacin 400 mg every 12 hours or doxycycline 100 mg every
12 hours and one or two additional antimicrobials (rifampicin,
vancomycin, penicillin, ampicillin, chloramphenicol etc)
The pseudomonad group
The pseudomonad group includes members of pseudomonadaceae family.
They are widely distributed in environment
The pseudomonads are Gram-negative, mobile, aerobic rods, some of which produce
water-soluble pigments.
Pseudomonads in soil, water, plants and animals widely occur
Pseudomonas aeruginosa is frequently present in the normal intestinal flora and
on the skin of humans and is the major pathogen of the group.
Others pseudomonads infrequently cause disease.
Pseudomonas aeruginosa
Pseudomonas aeruginosa is widely distributed in nature and is present in moist
environments in hospitals.
It can colonize normal humans such as saprophyte.
It causes disease in humans with abnormal host defenses.
Pseudomonas aeruginosa:
morphology
P. aeruginosa is motile and rod-shaped, measuring
about 0,6x2 µm
It is Gram-negative and occurs as single
bacteria, in pairs and occasionaly in short
chains
Pseudomonas aeruginosa: culture
P. aeruginosa is an obligate aerobe that grows on
many type of culture media, producing a sweet or
grape-like odor.
Some strains hemolyze the blood
P. aeruginosa forms smooth round colonies with
a fluorescent greenish color.
It often produces the nonfluorescens bluish
pigment pyocyanin which diffuses into the agar.
Other strains produce the fluorescens pigment
pyoverdin which gives a greenish color to the agar
Pseudomonas aeruginosa: culture
Pseudomonas aeruginosa causes superinfections in
patients affected by cystic fibrosis
Cultures from patients with cystic fibrosis
often form mucoid colonies as a result of
overproduction of alginate, an exopolysaccharide.
In cystic fibrosis patients, the exopolysaccharides
appears to provide the matrix for the organisms
to live in a biofilm
P. aeruginosa: growth characteristic
Pseudomonas aeruginosa grows well at
37-42°C; its growth at 42°C helps to
differentiate it from other Pseudomonas
species in the fluorescens group.
It is oxidase-positive.
It does not ferment carbohydrates, but
many strains oxidize glucose.
Identification is usually based on colonial
morphology, oxidase positivity,
characteristic pigments and growth at
42°C
Pseudomonas: antigenic structure
Pili (fimbriae)
they extend from the cell surface and promote attachment to host epithelial
cells.
Exopolysaccharide
it is responsable for the mucoid colonies seen in cultures obtained from
patients with cystic fibrosis
Lipopolysaccharide
it exists in multiple immunotypes. P. aeruginosa can be typed on the basis
of antigenic differences of lipopolysaccharide, and by bacteriocin
susceptibility
Virulence factors: extracellular
enzymes and toxins
In addition to exopolysaccharides most Pseudomonas aeruginosa strains isolates
from clinical infections, produce a number of extracellular enzymes including:
elastates
proteases
Most Pseudomonas aeruginosa isolates produce several toxins such as:
phospholipase C
glycolipid
exotoxin A
a heat-labile hemolysin
a heat-stable hemolysin
it causes tissues necrosis and is lethal for animals when injected in purified form. The toxin blocks proteins synthesis
by mechanism of action identical to that of diphtheria toxin
Lipopolysaccharide causes: fever, shock, leukocytosis, leukopenia, disseminated
intravascular coagulation, and respiratory distress syndrome
Pigments
are toxic and contribute to damage
Pseudomonas aeruginosa: pathogenesis
P. aeruginosa is nosocomial pathogen (rarely causes infections in community)
In immunocompetent subjects is present on the skin and in mucous membranes.
An oppurtunistic pathogen may be considered, it causes diseases in hospitalized
patients when mucous membranes and skin are disrupted by tissue damage,
when intravenous or urinary catheters are used, or when neutropenia is present,
or in cancer chemotherapy
The bacterium attaches to and colonizes the mucous membranes or skin, invades
locally and produces systemic disease
These processes are promoted by the pili, enzymes and toxins described.
Pseudomonas aeruginosa and other pseudomonads are resistant to many
antimicrobial agents and therefore become dominant and important when more
susceptible bacteria of the normal flora are suppressed
Clinical findings
P. aeruginosa produces infection of wounds and burns,
characterized by blue-green pus; causes meningitis,
when introduced by lumbar puncture,
and urinary tract infection, when introduced by
catheters and instruments or in irrigating solutions.
Involvement of the respiratory tract, results in
necrotizing pneumonia. (the lungs are damaged by
cavities)
Clinical findings
P. aeruginosa is often found in mild otitis externa in
swimmers
Infection of the eye, which may lead to
rapid destruction of the eye, occurs most
commonly after surgical procedures
Clinical findings
In infants or debilitated people, P. aeruginosa may invade the bloodstream
and result in fatal sepsis.
This occurs in patients with leukemia or lymphoma who have received
antineoplastic drugs, radiation therapy and in patients whith severe burns.
Occasionally fluorescens pigment can be detected in
wounds, burns, or urine by ultraviolet fluorescence
Culture observed by ultraviolet
fluorescence
Diagnostic laboratory tests
Specimens
specimens obtained from skin lesions, pus, urine, blood, spinal fluid, sputum and
other material should be used for isolation and identification.
Smears
Gram negative rods are often seen in smears
directly observed.
There are no specific morphologic characteristics
that differentiate pseudomonads from enteric or
other Gram negative rods.
Isolation: cultural methods
Culture
specimens are planted on blood agar or
one of the differential media used to
grow enteric gram negative rods.
Pseudomonads grow on these media, but
they may grow more slowly than the enterics
Pseudomonas aeruginosa does not ferment
lactose and so is easily differentiated from
the lactose-fermenting bacteria (E. coli).
Identification
Selective medium
Pseudomonas a. may be isolated in cetrimide agar
(tetradecylmethylammonium bromide)
Final identification may be performed on the base of different
fermentation of carbohydrates, on the base of colonies morphology,
oxidase positivity, pigments formation, characteristic odor and growth
at 42°C
Treatment
Clinically significant infections with Pseudomonas aeruginosa should not be
treated with single-drug therapy, because the bacteria can develop resistance
when single drugs are employed.
A penicillin active against P. aeruginosa (ticarcillin or piperacillin) is used in
combination with an aminoglycoside, usually tobramycin.
Other drugs active include:
aztreonam
imipenem
the newer quinolones
cephalosporins (ceftazidime, cefoperazone)
Epidemiology and control
P. aeruginosa is primarily a nosocomial pathogen, and the methods for control
of infection are similar to those of other nosocomial pathogens
Should be paid attention to sinks, water baths, showers, hot tubs and other wet
areas
Vaccine is reserved to high-risk patients, it provides protection against
pseudomonas sepsis
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia is an aerobic,
nonfermentative Gram negative rod, initially
classified as Pseudomonas maltophilia and also
grouped in the genus Xanthomonas. In 1993 was
classified in Stenotrophomonas genus.
S. maltophilia is smaller than other members of
the genus, is mobile (polar flagella) and grows on
Mac Conkey producing pigmented colonies.
It is catalase positive, oxidase negative.
S. maltophilia is ubiquitous in wet environments, may be
found in water, urine, and respiratory secretions;
it has been used in biotechnology applications
On blood agar, colonies have a gray color
Clinical characteristics
Stenotrophomonas maltophilia is an increasingly important cause of hospitalacquired infections in patients receiving antimicrobial therapy and in
immunocompromized patients.
In hospital environment it colonizes breathing tubes such as endotracheal or
tracheostomy tubes and urinary catheters.
Infection more commonly occurs in presence of prosthetic material
(usually a central venous catheter or similar device)
In immunocompromized patients, S. maltophilia is a source of latent pulmonary
infections; also the number of infections in patients affected by cystic fibrosis
has been increasing.
In immunocompetent individuals, S. maltophilia is an unusual cause of pneumonia,
urinary tract infection or bloodstream infection
In clinical laboratory, it has been isolated from many anatomic sites, including
respiratory tract secretions, urine, skin wounds and blood.
Pathogenetic mechanism
• Contribute to pathogenesis:
•
•
•
•
Biofilm
Toxins (Hemolysin)
Enzymes
Beta-lactamases (L1-L2)
Stenotrophomonas maltophilia:
antimicrobial treatment
Stenotrophomonas is resistant to commonly used antimicrobials including:
cephalosporins, aminoglycosides, imipenem and the quinolones.
Many strains are sensitive to co-trimoxazole and ticarcillin-clavulanate but
resistance has been increasing
The widespread use of the drugs to which S. maltophilia is resistant plays
an important role in increased frequency with which it causes disease.
Acinetobacter
Acinetobacter species are aerobic Gram negative rod shaped
bacteria during rapid growth and coccobacillary in the stationary
phase. They are generally encapsulated non mobile with a tendency
to retain crystal violet and so incorrectly identified as Gram
positive.
They are widely distributed in soil and water and the ability to
utilize a variety of carbon and energy sources allows them to
survive in nature and to grow on many laboratory media
May be isolated from skin, mucous membranes, secretions,
and in the hospital environment.
The most commonly isolated species are:
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
baumanii
johnsonii
haemolyticus
lwoffli
Acinetobacter: morphology
characteristics and cultivation
Acinetobacters are usually coccobacillary or coccal in
appearance.
They resemble Neisseriae on smears, because diplococcal
forms predominate, rod-shaped forms also occur.
The bacteria appear to be Gram positive when stained
directly from specimens.
Acinetobacter grows well on most types of
media. Colonies are 1-2 mm, nonpigmented, mucoid
with smooth surface.
The inability to reduce nitrate or to grow anaerobically
distinguished these organisms to Enterobacteriacee
Acinetobacter: pathogenesis
A limited number of virulence factors reduce this bacterium to the role
of an opportunistic.
No cytotoxins are produced, lipolysaccharide is present in the cell wall, but little
is known of its toxic property.
The possibility to grow in an acidic pH and at lower temperatures may
enhance this microrganism to survive and infect.
Also the bacteriocin production, the presence of capsule and capacity to
live under dry conditions can contribute to pathogenetic mechanism
Acinetobacter:clinical
manifestations
Acinetobacter is an opportunistic pathogen causing infections in
Immunocompromized patients expecially with selective complement
component deficiencies (infections are frequent in summer period)
Respiratory tract
Acinetobacter has been reported to cause communy-acquired
bronchiolitis in children or in immunocompromized adults.
Adult community-acquired pneumonia generally occurs in particular
conditions (alcoholism, tobacco use, diabetes mellitus, renal failure,
other pulmonary disease).
The most severe manifestations of hospital pneumonia has been referred as
ventilator-associated cases.
Predisposing factors include: endotracheal intubation, tracheostomy,
previous antibiotic therapy. Bacteremia and toxic shock are associated with
more severe forms caused by A. baumanii
Acinetobacter:clinical
manifestations
Bacteremia
nosocomial Acinetobacter bacteremia is frequently associated with
respiratory tract infections and use of intravenous catheters, urinary
tract, wound, skin and abdominal infections.
The mortality rate for Acinetobacter bacteremia has been reported
to be 17% to 46% expecially when associated with polymicrobial
bacteremia
Acinetobacter:clinical
manifestations
Genitourinary infections
cases of cystitis and pyelonephritis have been documented in
patients with bladder catheter or nephrolithiasis
Intracranial infections
Acinetobacter meningitis infrequently occurs, it is generally
identified following neurosurgical procedures or head trauma.
A petechial rash has been noted in up to 30% of patients with
Acinetobacter meningitis.
Soft tissue
can cause cellulitis in association with venous catheters introduction
Antimicrobial treatment
Acinetobacter strains are often resistant to antimicrobial agents and
therapy of infection can be difficult.
Susceptibility testing should be recommended in selection of appropriate
antimicrobial therapy.
Acinetobacter strains respond most commonly to gentamicin, amikacin or
tobramycin and to newer penicillin or cephalosporins
Enterobacteriaceae
The Enterobacteriaceae are a large heterogeneous group of Gram-negative rods
living in the intestinal tract of humans and animals
The family includes many genera (Escherichia, Shigella, Salmonella, Enterobacter,
Klebsiella, Serratia, Proteus and others).
Some enteric organisms eg. Escherichia coli are part of the normal flora and
incidentally cause disease, others (Salmonella and Shigella ) are pathogenic for
humans.
The Enterobacteriaceae are facultative anaerobes or aerobes, ferment a wide
range of carbohydrates, possess a complex antigenic structure, and produce a
variety of toxins and other virulence factors.
Enterobacteriaceae: classification
The Enterobacteriaceae are the most common group of Gram negative rods
isolated in the clinical laboratory and along with staphylococci and streptococci
are the most common bacteria that cause disease.
The taxonomy is complex and rapidly changing (introduction of techniques such as
nucleic acid hybridization and sequencing).
More than 25 genera and 110 species or groups have been defined
Enterobacteriaceae:characteristics
Enterobacteriaceae are Gram negative rods,
motile with peritrichous flagella or nonmotile.
They grow on peptone or meat extract media
without the addition other supplements.
Grow well on Mac Conkey’s agar; grow aerobically
and anaerobically, ferment glucose, often with gas
production, are catalase-positive, oxidase-negative
and reduce nitrate to nitrite.
Different biochemical tests are used to
differentiate the species of Enterobacteriaceae
and commercially prepared kits are used for this
porpose.
Api 20 test
procedure
Enterobacteriaceae:morphology
and culture
The Enterobacteriaceae are short Gram negative rods.
Typical morphology is seen in growth on solid media in vitro, but morphology is
highly variable in clinical specimens.
Capsules are large and regular in Klebsiella and uncommon in the other species.
E. coli forms circular convex, smooth colonies. Enterobacter colonies are similar
but more mucoid. The Salmonella and Shigella produce colonies similar to the
E. coli but do not ferment lactose.
Biochemical characteristics
Enterobacteriacee differ in order their biochemical
characteristics, so carbohydrate fermentation
patterns and the activity of amino acid decarboxylases
and other enzymes are commonly used in biochemical
differentiation.
Some tests ,eg. the production of indole from
tryptophan are commonly used
Voges Proskauer
Other tests eg. the Voges-Proskauer reaction
(production of acetyl-methylcarbinol from dextrose)
are less often used.
Indole production
Different fermentation of
carbohydrates
Culture on differential media that contain special dyes and carbohydrates eg. eosin
methylene blue (EMB), Mac Conkey’s or desoxicholate medium distinguishes
lactose –fermenting (colored) from non-lactose-fermenting colonies (nonpigmented)
and may allow rapid presumptive identification of enteric bacteria.
A medium for identification of enteric bacteria is
triple sugar iron. The medium contains 0,1-%
glucose, 1% sucrose, 1% lactose, ferrous sulfate
and a pH indicator (phenol red).The strain must
be inoculated into a test tube to produce a slant
with a deep butt. If only glucose is fermented,
the slant and the butt initially turn yellow, when
amines formation continues, the slant turns red.
If lactose or sucrose is fermented and so much
acid is produced, slant and butt remain yellow.
Some oganisms can produce acid and gas
(bubbles) in medium.
c
No
ferment.
Only
glucose +
glucose deep prod
H2S
Lactose
+ gas
Lac + suc
+ligh
prod H2S
Antigenic structure
Enterobacteriaceae have a complex antigenic structure
They are classified by more than:
150 different heat-stable somatic O (lipopolysaccharide) antigens
100 heat-labile K (capsular) antigens
50 H flagellar antigens
Virulence factors: Toxins
Many species of Enterobacteriaceae are capsulated, in addition they
possess a complex lipopolysaccharide in the cell walls.
This substance endotoxin, have a variety of pathophysiologic effects.
Many Gram negative enteric bacteria also can produce exotoxins of clinical
importance
Escherichia coli
E. coli, discovered in 1885 by Escherich, is a member of the normal intestinal flora.
The most common species of facultative anaerobe found in human gastrointestinal
tract and the most common pathogen of enterobacterial family is considered.
Other bacteria are found as members of the normal intestinal flora but are less
common than E. coli.
Escherichia coli is an opportunistic pathogen, normally is not cause disease,
but becomes pathogenic only when it reaches tissues outside of normal
intestinal sites.
The most frequent sites of clinically important infections are the urinary tract,
biliary tract, abdominal cavity and respiratory tract.
E. coli: pathogenesis and clinical findings
Urinary tract infection
E. coli is the most common cause of urinary tract
infection and accounts for approximately 90% of
noncomplicated community-acquired cystitis in
young women.
Women are at particular risk to develop urinary
tract infection also older adults are a high risk, on
the contrary cystitis is rare in males.
The symptoms and signs include urinary frequency,
dysuria, hematuria.
Risk of cystitis increases in presence of obstruction
of the bladder, or urethra, insertion of instruments,
diabetes etc.
E. coli: pathogenesis and clinical
findings
Acute pyelonephritis: upper tract
Nephropathogenetic strains of E. coli are
associated with acute pyelonephritis, a
clinical syndrome characterized by
frank pain and fever associated with
dysuria, urgency, frequency and infection of
the kidney but none of these symptoms or
signs are specific for E. coli infection.
Nephropathogenetic E. coli produce a
hemolysin, also K antigens appear to be
important in the pathogenesis of upper tract
infection. Pyelonephritis is caused by specific
strains with a particular type of pilus, P pilus
which binds to P receptors
Enteric-associated
diseases Escherichia coli
E. coli causing diarrhea or dysentery are extremely common in worldwide.
These strains are classified in five groups on the basis of their characteristics
and virulence properties.
Each group causes disease by a different mechanism.
Enteropathogenic E. coli (EPEC)
is an important cause of diarrhea
in infants especially in developing countries. EPEC adhere and invade the mucosal
cells of the small intestine. Characteristic lesions can be seen on electron
micrographs of small intestine biopsy lesions.
Enterotoxigenic E. coli (ETEC)
is a common cause of “traveler’s diarrhea”
and a vary important cause of diarrhea in infants in developing countries. They
adhere to epithelial cells of the small intestine. Some strains produce a heat-labile
exotoxin. It increases the local concentration of cyclic adenosine monophosphate
(cAMP), which results in intense prolunged hypersecretion of water and chlorides.
LT is antigenic and cross-reacts with the enterotoxin of vibrio cholerae.
Some strains produce the heat-stable exotoxin. ST activates guanylyl cyclase in
enteric epitelial cells. Many strains produce both toxins causing a more severe
diarrhea.
Enteric-associated diseases E. coli:
other groups
Enterohemorrhagic E.coli (EHEC) these strains produce verotoxin
named for its cytotoxic effect on vero cells, a line of African green monkey
kidney cells. EHEC has been associated with hemorrhagic colitis, a severe form of
dysentery with hemolytic uremic syndrome, a disease resulting in acute renal
failure, hemolityc anemia and thrombocytopenia. Verotoxin is similar to Shiga toxin
produced by some strains of Shigella dysenteriae type 1
Enteroinvasive E. coli (EIEC) produces a dysentery vary similar to
shigellosis by invading intestinal mucosal epithelial cells
Enteroaggregative E. coli (EAEC) causes acute and chronic diarrhea in
persons in developing countries, they are characterized by their adherence to
human cells. EAEC produce ST-like toxin and a hemolysin.
Additional diseases
Sepsis in immunocompromized patients, E. coli may reach the
bloodstream causing sepsis. Sepsis may occur after an urinary tract infection.
Meningitis E. coli and goup B streptococci cause
meningitis in infants.
Approximately 75% of E. coli strains associated with meningitis possess the K1
antigen.
This antigen cross-reacts with the group B capsular polysaccharide of N. meningitidis
Klebsiella
Klebsiella pneumoniae is a capsulated, no mobile Gram- rod.
Is present in the respiratory tract and faeces of about 5%of normal individuals.
It is responsable for a small number (aboud 1%) of bacterial pneumonia.
K. pneumoniae can produce extensive hemorrhagic necrotizing consolidation of the
lung. It occasionally produces urinary tract infection or bacteremia in debilitated
patients.
K. pneumoniae and K. oxytoca cause hospital-acquired infections
K. ozaenae has been isolated from the nasal mucosa in ozena, it causes a fetid
progressive atrophy of mucous membranes.
K. rhinosleromatis causes a destructive granuloma of the nose and pharynx
Proteus
Proteus species are opportunistic microrganisms. They produce infections in
humans when the bacteria leave the intestinal tract.
Members of Proteus genus produce urinary tract infections, bacteremia and
pneumonia in debilitated patients.
P. mirabilis (indolo negative) causes urinary tract infections and occasionally
other infections.
P. vulgaris (indolo positive) is an important nosocomial pathogen.
Proteus species produce urease, resulting in rapid
hydrolysis of urea with production of ammonia.
In urinary tract infections with Proteus, the urine
becomes alkaline, promoting stone formation.
The rapid motility may contribute to invasion of the
urinary tract.
Strains of Proteus are often resistant to antibiotics
Diagnostic laboratory tests
Specimens used for isolation and identification of E. coli, Klebsiella and Proteus
are: urine, blood, spinal fluid, sputum or other material in according with the
different localization of process.
Culture specimens are planted on blood agar or on differential media.
In this case a rapid preliminary identification is often possible
Identification
identification may be performed on the basis of biochemical
characteristics (commercial kits)
Treatment
No single specific therapy is available.
A vary great variation in susceptibility again penicillins, cephalosporins,
fluorochinolones and aminoglycosides is possible.
Laboratory tests for antibiotic sensitivity are recommended.
Multiple drug resistance is common, under the control of
transmissible plasmids.
Various means have been proposed for the prevention of traveler’s
diarrhea, but none are successful or lacking in adverse effects, it is
recommended caution in regard to food and drink in areas where
environmental hygiene is low.
Shigella
The natural habitat of Shigellae is the intestinal tract, where they produce
bacillary dysentery
Morphology
Shigellae are slender
Gram-negative rods; coccobacillary
forms in young cultures occur.
Classification
dysenteriae
flexneri
sonnei
boydei
Shigella: culture and growth characteristics
Shigellae are facultative anaerobes, but grow best
aerobically. Convex, circular, transparent colonies
reach a diameter of about 2 mm in 24 h.
All shigellae ferment glucose (except S. sonnei),
they do not ferment lactose. The inhability to
ferment lactose, distinguishes shigellae on
differential media. Shigellae form acid from
carbohydrates, but rarely produce gas
Shigella: pathogenesis of bacillary
dysentery
Pathologic process consists in
invasion of the mucosal cells by
inducing phagocytosis. Shigellae
escape from the vacuole,
multiply and spread within the
epitelial cells cytoplasma
and passage to adjacent cells.
Microabcesses in the wall of the
large intestine lead to necrosis of
the mucous membrane, superficial
ulceration, bleeding, and formation
of a “pseudomembrane” on the
ulcerated areas
Shigella: toxins
Endotoxin
upon autolysis, all shigellae release their toxic lipopolysaccharide
Shigella dysenteriae Exotoxin
Shigella dysenteriae type 1 produces a
heat-labile exotoxin (Shiga toxin) active against the intestine and the central
nervous system.
The exotoxin is an antigen lethal for experimental animals.
Acting as an enterotoxin, it produces hemorrhagic colitis (E. coli verotoxin),
acting as a neurotoxin it causes hemolytic uremic syndrome and central nervous
system involvements
Clinical findings of dysentery after a short incubation period (1-2 days) there
is abdominal pain, fever and watery diarrhea. The diarrhea has been attributed to
an exotoxin acting in the small intestine. 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.
Diagnostic laboratory tests
Specimens are streaked on
differential media (Mac Conkey’s or
EMB) and on selective media
(Hektoen or Salmonella-Shigella agar)
Colorless colonies are inoculated into TSI
Shigella produce H2S. Only glucose is
fermented, the slant and the butt initially turn
yellow, when amines formation continues, the
slant turns red.
Neg. Lactose
Pos.glucose
Hydrogen sulphide produced
Salmonella group: carhacteristics and classification
Salmonellae are Gram negative rods, most isolates are mobile with peritrichous
flagella. Salmonellae grow readily on simple media but they never ferment lactose
or sucrose. They usually produce H2S.
Salmonellae are resistant to chemicals (brilliant green, sodium tetrathionate,
sodium deoxycholate. These compounds are therefore useful in media to isolate
Salmonellae from faeces.
Classification
the classification
is complex and in changing.
Salmonella in SS medium
Pathogenesis and clinical findings
Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, S. choleraesuis
are pathogen for humans. They produce three types of disease.
• Enteric fevers this syndrome is produced by Salmonella typhi.
The ingested Salmonellae reach the small intestine from which they penetrate
the lymphatics and then invade the bloodstream. They are carried by the blood
to many organs; the microrganisms multiply in lymphoid tissue, return in intestinal
tract and are excreted in stools.
After an incubation period of 10-14 days, fever, headache, constipation,
bradycardia and myalgia occur. The fever rises to a high plateau, and the spleen
and liver become enlarged.
In pre-antibiotic era, the 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%.
Pathogenesis and clinical findings
Bacteremia with focal lesions this is associated commonly with
S. choleraesuis but may be caused by any Salmonella serotype. After oral infection
there is early invasion of the bloodstream with possible focal lesions in the lungs,
bones and meninges.
Enterocolitis this is the most common manifestation of Salmonella infection.
Eight to 48 hours after ingestion of salmonellae, there is nausea, headache, vomiting
and profuse diarrhea. Low-grade fever is common, but the episode usually resolves
in 2-3 days
Diagnostic laboratory tests
Specimens
Blood for culture must be taken repeatedly (in enteric fevers blood culture are
positive in the first week of disease. Stool specimens in enteric fevers yield
positive from the second or third week
Bacteriologic methods for isolation of Salmonellae
Differential medium: EMB, Mac Conkey’s
or Deoxycholate medium allows to
differentiate lactose fermenting
from lactose-nonfermenting organisms
Diagnostic laboratory tests
Selective medium: salmonella-shigella (SS) agar, Hektoen enteric agar or deoxycholate-citrate agar favour growth of salmonellae and shigellae.
Enrichment cultures the specimen (usually stool) also is put into selenite broth,
which inhibits replication of normal intestinal bacteria and permits moltiplication of
salmonellae
Diagnostic laboratory tests and
treatment
Final identification
Suspect colonies are identified by
biochemical reaction patterns or by
slide agglutination tests with specific sera.
Treatment
Enteric fevers and bacteremias require antimicrobial treatment with ampicillin,
trimethoprim-sulfamethoxazole or a third-generation cephalosporin.
Multiple drug resistance transmitted genetically by plasmids among enteric
bacteria is possible. Susceptibility to antibiotics must be always tested.