Clinical Microbiology Preface Clinical microbiology studies the molecular bases and the pathogenic agents of infectious diseases for the development of

Clinical Microbiology

Preface

Clinical microbiology studies the molecular bases and the pathogenic agents of infectious diseases for the development of new diagnostic technologies andnew 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 diseaseand 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 bacterialidentification.Direct examination is a rapid diagnostic method.Visible microrganisms may indicate the possible etiologic agent orcan guide the laboratory in selection of the appropriate isolationmedia 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 locatedbelow 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 thespecimen.

Body tube contains an ocular lens 10xand nosepiece with several objective lenses (10x, 40x, 100x)The image is brought into focus by thecoarse 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 becomevisible.

The electron microscope

The electron microscope uses electrons instead of light. Someelectrons pass through the specimen and are focused by anelectromagnetic objective lens, which presents an image to the projectorlens system for enlargement.

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 possibleto observe the detailed structures of prokaryotic and eukaryotic cells

The image can be recorded on photographic film.

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 electronsprojected from an electron gun and focused by an electromagnetic condenser lens.

The SEM has a lower resolving power than the TEM, but it produces three-dimensional 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, enhancesthe 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

Blue stain is considered as a simple stain, in contrastwith the Gram and Acid-Fast stains, which require a counterstaining step

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)

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 artefacts and may disrupt the normal morphology of bacteria.

The Methylene blue stainMaterials: 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 stainFlagella 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) thatsurround bacterial cells

Capsules are usually demonstrated by the negative staining procedure

The cells are treatedwith a suspensionof 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.

MethodSpread the culture on glass slideAir dry complitelyStain with crystal violet (1 min)Wash with copper sulfate 10% Air dryObserve the smear using oil immersion

Gram stain technique

Gram stain procedure was developed by H. Christian Gram to differentiatepneumococci 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 positivecoccus

Gram negativebacillus

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

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

1. Flood slide with crystal violet (30 seconds)

ResulsGram 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 bacteriato 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: methodPrepare 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 anthraxits 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 conditionis often fatal and severe. In inhalation form, spores are carried by macrophagesfrom 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 orpsycrophiles.In addition, most species grow optimally in aerobic condition, but others require CO2 or total removal of O2

Bacteriological media

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)

General

for example Nutrient broth or blood agar

(used for cultivation or isolation of microrganisms: fastidious and non fastidious)

Bacteriological media:other types

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).

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.

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 aureusStaphylococcus epidermidisStaphylococcus 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 inmmunocompromised patients

These infections are linked to biofilm formation

What’s the biofilm?

Biofilm is a community of bacterial cells contained in a self-producedpolymer matrix adherent to biotic or abiotic surface.This structure is very stable and resistant to the physical, chemicalagents 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 epidermidisStaphylococcus lugdunensisStaphylococcus warneriStaphylococcus hominisStaphylococcus simulansand 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 cultureStaphylococci 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 staphylococcion the skin and in the throat (opportunistic pathogen)

As a nosocomial pathogen, S. aureus has been a cause of morbidity andmortality.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 cellularsubstances.

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 nontoxicsoluble 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. Thisbinding actives a large number of T cells with secretions of proinflammatorycytokines

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 in18 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.

positivenegative

Slide coagulase test

negative positive


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 enzymesHeat 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.

positive

negative

Serologic tests

Serologic tests for diagnosis of S. aureus, they have little practical value

Antimicrobial treatmentStaphylococcal isolates should be tested for antimicrobial susceptibilitybecause S. aureus has developed resistance to all antibiotic classesavailable for clinical use.

Resistance to Penicillin

The most common mechanism of S. aureus resistance to β-lactam involvespenicillinase 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 Methicilllinwas created.

Hospital acquired methicillin- resistant 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 firstMRSA was described at aboud the same time.The prevalence of MRSA in hospital has increased rapidly in the lastperiods (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 wallsynthesis.

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

theirbactericidal activity.

Quinupristin-Dalfopristin is a combination of a streptogramin B and AThis association is sinergic and shows a large activity against MRSA strains.

Linezolid belongs to a new oxazolidinone family of molecules, it isactive 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 characteristicsStreptococci 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 changein blood agar (γ or nonhemolytic). Other reduce hemoglobin and cause agreenish discolouration of agar.(α hemolytic)

β

γα

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 lysedbut colonies are surrounded by greenish discolouration of agar due tostreptococcal 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-acetylglucosaminefor group B, it is rhamnose-glucosamine polysaccharide etc.

Characteristics of clinically important streptoc.

Piogenic infection

Small colonies<0,5 mm

Throat, colonFemale gen. tract

alpha, beta

F, A, C, GStreptoc.anginosusgroup

Dental cariesEndocarditisabscess

Optochin resist. colonies not solub. in bile

Mouth throat, female gen. tract

alphaUsually not typed

Viridans streptoc.

Pneumonia, meningitis,endocarditis

Susceptib. optochin, soluble in bile

throat alpha -Strept. pneumon.

Neonatal sepsis

Hippurate hydrolysis

Female geni tal tract

Β γ BStrep.agalactiae

PharyngitisImpetigo rheumatic fever

Colonies >0,5 mm

Throat,skin

beta AStreptoc.pyogenes

DiseasesLabor. criteria

habitatHemolysis

Group spec.sub.

Name

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

CultivationGroup A streptococci are nutritionally fastidious and usually cultivatedin complex media often supplemented with blood or serum.When cultured on blood agar plates S. pyogenes appears as white to graycolonies 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


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. Thisbinding actives a large number of T cells with secretions of proinflammatorycytokines

Invasive streptococcal infections of skin and soft tissues

Erysipelas

is a superficial cutaneous processrestricted to the dermis with lymphaticinvolvement.This disorder is more common in

infants,young children and older adults. In

olderreports erysipelas was described asinvolving the butterfly area of the

face.At the present the lower extremities

aremore frequently involved, in thiscase, the lesions break the cutaneousbarrier 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 in group A is a polymer of rhamnose and N-acetylglucosamine

M protein 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…..


Samples 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

Smears 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 platesMedia 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 °CMany streptococci require 5% CO2 concentration (pneumococci and some viridansstrains)

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, GPositive controlExtraction enzyme (should be reconstituted with distilled water)Disposable reaction cards

Preparation 1) Dispense 0,4 ml of extraction enzyme to test tube2) Select 2-5 colonies with a bacteriological loop and shake in enzyme preparation3) 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 Astreptococcal antigens from throat swabs.These kits use enzymatic or chemical methods to extract antigensand use EIA or agglutination tests to demonstrate presence of antigens.

Rapid tests are 60-90% sensitive and 98-99% specific when comparedto culture methods.

Antistreptolysin O titre (ASO)

ASO titre is used to demonstrate the body’s reaction to an infection causedby 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 producingantistreptolysin 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 butit indicates if a streptococcal infection is or has been present.

Normal resultsAntistreptolysin 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, isa presence of beta-lactamase producing bacteria at side of infectionor poor patient compliance with dosing regiments.Doses of penicillin for 10 days prevent poststreptococcal diseases

Most strains are sensitive to erythromycinAn increase in erythromycin resistance in S. pyogenes was noted duringthe 1990 in many sides in worldwide. High rates of resistance(20-40% of isolates tested) have been documented in different geographicareas 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 agarare 3 to 4 mm in diameter,

grayor white in color.Colonies are often surroundedby a zone of β-hemolysis,

aboud2% 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 serotypesdesigned I, II, III.Later distinct differences in serotypes I strains have beenreported, 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

womencolonized with group B streptococci at the time of delivery isapproximately 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 mostlikely 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 infectionsin 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


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.


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 oflysis 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.


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 itselfthe 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).


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 pneumococcaldiseases

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 with 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


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 adhereto cerebral capillaries, after in cerebrospinal fluid a variety of pneumococcalcomponents, 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


Gram positive staining


Hemolytic activityS. pneumoniae is α hemolyticorganism.

Optochin sensitivityPneumococci form a 16 mm zoneof inhibition around a 5 mg optochindisc.Viridans streptococci are not inhibited by optochin


Bile sensitivity

Addition of a few drops of 10% deoxycholate to liquid culture at 37°Clyses 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 antibioticsare 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 conjugatedto carrier proteins such as tetanus or diphtheria toxoid.The resulting antigens are recognized as T-cell dependent; they stimulategood 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 immunocompromizedpatients. Some studies suggest that an initial dose of conjugate vaccinetogether 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

EnterococcusGeneral 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. gallinarumE. faecium E. hiraeE. durans E. mundtiiE. avium E. raffinosusE. casseliflavus E. solitariusE. malodoratus E. pseudoavium

Enterococci: characteristics

Enterococci are facultative anaerobes that are able togrow 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 inclinical microbiology laboratoryE. faecium accounted for 5% to 10% of isolated.More recent evidence suggests that the prevalence of E. faecium, especialymultiresistant 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 gastrointestinaltract of humans and of other animals (significant part of the normalintestinal 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 patientswith 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 respira-tory 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 infectionsUrinary 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 acquiredbacteremia 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, becauseenterococcal bacteremia commonly occurs in debilitated patients

Meningitis and wound infection

Enterococci, rarely cause meningitis in normal adults. Most causes of enterococcalmeningitis 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 immuno-deficiecies including acquired immunodeficiency syndrome

Meningitis

Wound infectionsEnterococci are rarely implicated in tissue infections. They are frequently isolatedfrom mixed cultures with Gram-negative bacilli and anaerobes in surgical woundinfections, 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 withbacteremia 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 resistanceAminoglycosidic 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 mechanismsof resistance to a wide variety of antimicrobial agents.Most of these resistance mechanisms are mediated by genes encoded on plasmidsor transposons. Enterococci have evolved a number of efficient methods of transferring resistance genes, and this greatly facilitates the acquisition of new resistance determinants.

Acquired resistanceAminoglycosidic aminocyclitols (high level)Penicillin and ampicillin (β-lactamase)β-Lactams (altered PBPs)FluorochinolonesLincosamides (high level)MacrolidesTetracyclinesVancomycin (a number of phenotypes of vancomycin resistant have been discovered)

Therapeutic approachesTreatment 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 penicillinor for organisms with high-level penicillin resistance.

Fluoroquinolones such as ciprofloxacin and ofloxacin have in vitro activity againstenterococci (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 glycylglycynesshow 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) andNeisseria meningitidis (meningococci) arepathogenic for humans and tipically are foundassociated with or inside polymorphonuclearcells.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, nonmotilediplococci, approximately 0,8 µm in diameter

In 48 hours, on enriched media: Mueller Hinton,modified Thayer Martin, gonococci andmeningococci form convex, glistening, mucoidcolonies 1-5 mm in diameter.Colonies are transparent or opaque, nonpigmented and nonhemolytic.Neisseria flavescens and subflava have yellowpigmentation.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 gonorrhoeaeGonococci 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 coloniesoften occur; the opaque colonies are associated with the presence of a surface-exposed protein, Opa.

Neisseria gonorrhoeae: antigenic structure

Cell envelope

peptidoglycan

Outer membrane

Cytoplasmaticmembrane

piluspilus

PiliPili 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.


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

POR


OPA (opacity associated protein)

The OPA proteins are usually found on colonies withan opaque fenotype.These proteins are implicated in adhesion of gonococciand 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

RMPThis protein is antigenically conserved in all gonococci. It is associated with Por to form pores in cell surface.

Lipooligosaccharide LOSGonococcal LPS does not have long O-antigen chains. Toxicity in gonococcal infections is due to endotoxic effects of Lipooligosaccharide LOS.

IgA1 proteaseInactivates 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. Theprocess 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


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.


Specimens

Pus and secretions are collected from the urethra, cervix, rectum, conjunctivaand throat for culture and smear

Smears

Gram-stained smears of urethral or endocervical exudate show many diplococcimixed to cells.


Immediately after collection, pus or mucus arestreaked on enriched selective medium such asmodified 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 organismscan be identified by their Gram negativeaspect, by oxidase test or other laboratory tests.A liquid culture may be prepared to detectthe different fermentation of sugars.

Different fermentation of sugars

Glucose Maltose lactose saccharose

N. meningitidis

+

+

-

-

N.gonorrhoeae + - - -

M.catarrhalis - - - -


Serology

In infected people, antibodies to gonococcal pili and outer membrane proteinscan 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

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.

Antigenic structure


Bacteremia.

Neisseria meningitidis is a normal inhabitant of nasopharynx tract. In case of lowedhost defences, in presence of other infections, the microrganisms become invasiveand may reach the bloostream, producing bacteremia.Meningococcemia is more severe, with high fever and hemorrhagic rash


Meningitis

Meningitis is the most complication of meningococcemia. It usually beginssuddenly, with intense headache, vomiting and progresses to coma within few hours.In meningitis, the meninges are acutely inflammed, with thombosis and exudationof polymorphonuclear leukocytes, so the surface of the brain is covered with purulent exudate.


specimens

Specimens of blood are used for culture; specimens of spinal fluid are usedfor 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.


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 asceftriaxone or cefotaxime can be used

Moraxella catarrhalis

Moraxella catarrhalis was previously namedBranhamella 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 otherNeisseriae because does not ferment carbohydrates butit produces Dnase.

Micobacterium: general characteristics

Many species within the genus Mycobacterium are prominent pathogens such asMycobacterium tubercolosis and Mycobacterium leprae.

In addition numerous species of environmental mycobacteria called nontuberculousMycobacteria (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 includedthe advent of the AIDS epidemic, immigration from other countries, trasmissionin high-risk settings (hospital and prison) and the increase in the number of casesof 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 decreasethe 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 thespecies, ranging from nonpigmented to pigmented. Colonies are yellow, orange, or rarely pink, usually due tocarotenoid pigments

Mycobacterium: cell wall

The cell wall conteins meso-diaminopimelicacid, alanine, glutamic acid and mycolic acids(number of carbon atoms ranging from 60 to 90), togheter with free lipids (trehalose-dimycolate), provide a hydrophobic permeabi-lity barrier.Other important fatty acids are waxes andphospholipids.The high content of complex lipids of the cell wall prevents access by common anilinedyes. Mycobacteria are usually consideredGram positive but not are stained by Gram’smethod. Once stained with special proce-durs, 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 acidsas 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°CCompared to other bacteria, growth of mostmycobacterial species is slow.Visible colonies appear after few days to 6weeks of incubation under optimum conditions.Mycobacteria require less than 7 days whensubcultured on Löwenstein-Jensen, but mayalso take several weeks on primary culturefrom clinical specimens

Mycobacterium tuberculosisMorphology

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 containing3% 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-fastbacteria

Constituents of tubercle bacilli

Lipids

Mycobacteria are rich in lipids. These include mycolic acids, waxes and phosphatidesIn 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” inwhich 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 alveoliWhere moltiplication beginsThe disease results from establishment and proliferation of virulent organismsand 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 eliminatesmall numbers of bacteria

Pathogenesis of tuberculosis: immunology

Tuberculosis is the prototype of infections that require a cellular immune responsefor their control.During infection, abundant antibodies are produced, but they play no apparent rolein 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 oftubercle bacilli. In 1934 S.iebert made a simple protein precipitate (purifiedprotein 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 skintest 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 multiplication2) 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 caseationnecrosis. 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 lymphaticchannels 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, whichdistributes bacilli to all organs (miliary distribution).When caseating lesion discharges its contents into a bronchus, they are aspiratedand 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 theprimary 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 testsA 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, biopsymaterial, 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, nowcan 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.


Smears

Sputum exudates, or other material is examinated for acid-fast bacilli byZiehl-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


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 to8 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 clinicallyimportant mycobacteria from several weeks to as little as 1 day.


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, whichvary 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 smearspositive for acid-fast bacilli; the PCR test is approved for this use on sputumspecimens that are acid-fast stain-positive.

Enzyme immunoassays have been used to detect mycobacterial antigens, but thesensitivity 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 resistantto 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 usedin 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 RNApolymerase

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 riskfor such infections, should be treated according to susceptibility test results forthe infecting strain

Therapy should include a minimum of three or more than three drugs to whichthe 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 infectionwith virulent tubercle bacilli without a danger. The available vaccines are anadequate,so in the United States, the use is suggested only for tubercolin-negative personswho are exposed (members of tuberculous families, medical personnel)

Other mycobacteria

In addition to tubercle bacilli (M. tuberculosis and bovis), other mycobacteriaof varying degrees of pathogenicity have been grown from human samples in pastdecades.These “atypical” mycobacteria were initial grouped according to speed of growthat various temperatures and production of pigments

Mycobacterium avium complex

The Mycobacterium avium complex is often calledMAC 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 causedisease in immunocompetent humans; however dissemina-ted MAC infection is one of the most common opportu-nistic infections of bacterial origin in AIDS patients.Environmental exposure can led to MAC colonization ofeither the respiratory or gastrointestinal tract.Any organ can be involved: in the lungs, nodules, diffuseinfiltrates, cavities, and endobronchial lesions are common.Other manifestations include pericarditis, soft tissue abscesses,skin lesions, lymph node involvement. The patients presentnonspecific symptoms of fever, abdominal pain, diarrhea.The diagnosis is made by culturing MAC organisms from blood ortissue. For treatment may be used. Rifabutin, fluoroquinolonesand amikacin.

Mycobacterium kansasii

Mycobacterium kansasii is a photochromogen thatrequires complex media for growth at 37 °C.It can produce pulmonary and systemic diseaseindistinguishable from tuberculosis.Sensitive to rifampin, it is often treated with thecombination of rifampin, ethambutol and isoniazidwith 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, especiallyin Asia

Mycobacterium leprae: morphology

Typical acid-fast bacilli, single in parallelbundles or in globular masses, are regularly found in scraping from skin ormucous 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 anestheticmacular lesions (1-10 cm in diameter), diffuseerythematous infiltrated nodules 1-5 cm in diameter.Neurologic disturbances are manifested withresultant anesthesia, paresthesia, trophic ulcers,bone resorption and shortening of digits.In untreated cases, this condition may be extreme.

Leprosy: two typesThe 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 characterizedby 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 skinlesions 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 smearedon a slide and stained by the Ziehl Neelsen technique.No serologic tests are of value

For treatment, sulfones such as dapsone are first-linetherapy for both tubercoloid and lepromatous leprosy.Rifampin or clofazimine generally is included in the initialtreatment regiments.Other drug active include: minocycline, clarithromycin andsome fluorochinolones.World Health Organization recommends several years oftherapy

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 exploredfor 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 thebook of Exodus can be described.In 1881 Pasteur descovered the first heat-attenuated anthraxvaccine able to protect the animals.Anthrax has been a disease of animals primarily, with a long historyof animals-associated disease in humans.Most recently, in 2001 human cutaneous and inhalational forms ofanthrax were acquired a new importance when B. anthracis sporeswere sent in contaminated letters as an act of bioterrorism in theUnited 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

contrastsmears 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-lysismediated by cytokines release, particularly tumor necrosis factor-and 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 calmodulin-dependent adenylate cyclase enzyme that converts ATP to cyclicadenosine monophosphate (cAMP).Intracellular increase in cAMP results in dysregulation of waterand ions.

Anthrax spores

Anthrax spores can survive for months or decades depending on pH,temperature and nutrients in soil. Spores ingested from the soil bycattle or other herbivores then germinate into the vegetative form in thespleen or lymph nodes resulting in bacteremia and hemorrhage as aterminal event.Vegetative forms are deposited in soil and sporulation occurscontinuing 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 theexposed area of the neck, head or upper extremity. The papule

progresses andthe central lesion becomes necrotic and hemorrhagic and maydevelop peripheral vesicles.Finally the classic central black eschar appereas, often accompaned byedema. The progression of cutaneous anthrax lesions from papule to

blackeschar surrounded by edema is mediated by toxin and occurs also in

case ofappropriate antibiotic treatment. Mortality more likely in patients

developingbacteremia occurs.

Gastrointestinal anthraxGastrointestinal anthrax is a rare disease accounting for less than 5% of

allcases of anthrax in humans. When anthrax is ingested in food or liquid it

cancause two syndromes: oropharyngeal and/or intestinal anthrax. The

symptomsof oropharyngeal form are: a painful swelling of the neck caused by

cervicaladenophaty and soft tissue edema. Oral lesions and edema were seen on

thetonsils, hard palate. After one week ulceration and central necrosis occurforming a pseudomembrane covering the ulcers. The intestinal form is

morecommon characterized by abdominal pain, nausea, vomiting and signs of

ascites.

Inhalation anthraxInhalation 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 producewater-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, measuringabout 0,6x2 µm

It is Gram-negative and occurs as single bacteria, in pairs and occasionaly in short chains

Pseudomonas aeruginosa: cultureP. aeruginosa is an obligate aerobe that grows onmany type of culture media, producing a sweet orgrape-like odor.Some strains hemolyze the blood

P. aeruginosa forms smooth round colonies witha fluorescent greenish color.It often produces the nonfluorescens bluishpigment pyocyanin which diffuses into the agar.Other strains produce the fluorescens pigmentpyoverdin which gives a greenish color to the agar

Pseudomonas aeruginosa: culture

Pseudomonas aeruginosa causes superinfections inpatients affected by cystic fibrosis Cultures from patients with cystic fibrosisoften form mucoid colonies as a result of overproduction of alginate, an exopolysaccharide.In cystic fibrosis patients, the exopolysaccharidesappears to provide the matrix for the organisms to live in a biofilm

P. aeruginosa: growth characteristic

Pseudomonas aeruginosa grows well at37-42°C; its growth at 42°C helps todifferentiate it from other Pseudomonasspecies in the fluorescens group.It is oxidase-positive.It does not ferment carbohydrates, butmany 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:

elastatesproteases

Most Pseudomonas aeruginosa isolates produce several toxins such as:

phospholipase C a heat-labile hemolysinglycolipid a heat-stable hemolysinexotoxin A 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 hospitalizedpatients 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, invadeslocally and produces systemic diseaseThese 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 moresusceptible 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 bycatheters and instruments or in irrigating solutions.

Involvement of the respiratory tract, results innecrotizing pneumonia. (the lungs are damaged bycavities)

Clinical findings

P. aeruginosa is often found in mild otitis externa inswimmers

Infection of the eye, which may lead torapid destruction of the eye, occurs mostcommonly after surgical procedures

Clinical findings

In infants or debilitated people, P. aeruginosa may invade the bloodstreamand result in fatal sepsis.This occurs in patients with leukemia or lymphoma who have receivedantineoplastic drugs, radiation therapy and in patients whith severe burns.

Culture observed by ultravioletfluorescence

Occasionally fluorescens pigment can be detected inwounds, burns, or urine by ultraviolet fluorescence


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 ente- rics

Pseudomonas aeruginosa does not fermentlactose and so is easily differentiated fromthe lactose-fermenting bacteria (E. coli).

Identification

Selective mediumPseudomonas 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 growthat 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 controlof infection are similar to those of other nosocomial pathogens

Should be paid attention to sinks, water baths, showers, hot tubs and other wetareas

Vaccine is reserved to high-risk patients, it provides protection against pseudomonas sepsis

Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an aerobic,nonfermentative Gram negative rod, initiallyclassified as Pseudomonas maltophilia and also grouped in the genus Xanthomonas. In 1993 wasclassified 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 befound 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 hospital-acquired infections in patients receiving antimicrobial therapy and in immunocompromized patients.In hospital environment it colonizes breathing tubes such as endotracheal ortracheostomy 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 infectionIn 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 playsan 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 stationaryphase. They are generally encapsulated non mobile with a tendencyto retain crystal violet and so incorrectly identified as Gram positive.They are widely distributed in soil and water and the ability toutilize a variety of carbon and energy sources allows them tosurvive in nature and to grow on many laboratory mediaMay be isolated from skin, mucous membranes, secretions,and in the hospital environment.The most commonly isolated species are:

Acinetobacter baumaniiAcinetobacter johnsoniiAcinetobacter haemolyticusAcinetobacter lwoffli

Acinetobacter: morphology characteristics and cultivation

Acinetobacters are usually coccobacillary or coccal in appearance.They resemble Neisseriae on smears, because diplococcalforms predominate, rod-shaped forms also occur.The bacteria appear to be Gram positive when staineddirectly from specimens.

Acinetobacter grows well on most types ofmedia. Colonies are 1-2 mm, nonpigmented, mucoidwith smooth surface.The inability to reduce nitrate or to grow anaerobicallydistinguished 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


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


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 rodsliving 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 rodsisolated in the clinical laboratory and along with staphylococci and streptococciare the most common bacteria that cause disease.

The taxonomy is complex and rapidly changing (introduction of techniques such asnucleic 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 aerobicallyand anaerobically, ferment glucose, often with gasproduction, are catalase-positive, oxidase-negativeand reduce nitrate to nitrite.Different biochemical tests are used todifferentiate the species of Enterobacteriaceae and commercially prepared kits are used for thisporpose.

Api 20 testprocedure

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 biochemicalcharacteristics, so carbohydrate fermentation patterns and the activity of amino acid decarboxylasesand other enzymes are commonly used in biochemical differentiation.

Some tests ,eg. the production of indole from tryptophan are commonly used

Other tests eg. the Voges-Proskauer reaction(production of acetyl-methylcarbinol from dextrose)are less often used.

Voges Proskauer

Indole production

Different fermentation of carbohydrates

Culture on differential media that contain special dyes and carbohydrates eg. eosinmethylene 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 istriple sugar iron. The medium contains 0,1-% glucose, 1% sucrose, 1% lactose, ferrous sulfateand 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 muchacid is produced, slant and butt remain yellow.Some oganisms can produce acid and gas (bubbles) in medium.

c Onlyglucose

glucose +deep prod H2S

Lactose + gas

Lac + suc+ligh prod H2S

No ferment.

Antigenic structureEnterobacteriaceae have a complex antigenic structureThey are classified by more than:

150 different heat-stable somatic O (lipopolysaccharide) antigens100 heat-labile K (capsular) antigens50 H flagellar antigens

Virulence factors: Toxins

Many species of Enterobacteriaceae are capsulated, in addition theypossess 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 tractinfection and accounts for approximately 90% of noncomplicated community-acquired cystitis in young women.Women are at particular risk to develop urinarytract infection also older adults are a high risk, onthe contrary cystitis is rare in males.The symptoms and signs include urinary frequency,dysuria, hematuria. Risk of cystitis increases in presence of obstructionof 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, aclinical syndrome characterized byfrank pain and fever associated withdysuria, urgency, frequency and infection of the kidney but none of these symptoms or signs are specific for E. coli infection.Nephropathogenetic E. coli produce ahemolysin, also K antigens appear to beimportant in the pathogenesis of upper tract infection. Pyelonephritis is caused by specificstrains 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 diarrheain 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-labileexotoxin. 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 verotoxinnamed for its cytotoxic effect on vero cells, a line of African green monkeykidney 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 toxinproduced 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 thelung. 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 fetidprogressive 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 andpneumonia 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


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’sdiarrhea, but none are successful or lacking in adverse effects, it is recommended caution in regard to food and drink in areas whereenvironmental 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 bestaerobically. Convex, circular, transparent coloniesreach a diameter of about 2 mm in 24 h.All shigellae ferment glucose (except S. sonnei), they do not ferment lactose. The inhability toferment 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, superficialulceration, bleeding, and formationof a “pseudomembrane” on theulcerated areas

Shigella: toxins

Endotoxin upon autolysis, all shigellae release their toxic lipopolysaccharide

Shigella dysenteriae Exotoxin Shigella dysenteriae type 1 produces aheat-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 nervoussystem 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)

Neg. LactosePos.glucoseHydrogen sulphide produced

Colorless colonies are inoculated into TSIShigella produce H2S. Only glucose is fermented, the slant and the butt initially turn yellow, when amines formation continues, theslant turns red.

Salmonella group: carhacteristics and classification

Salmonellae are Gram negative rods, most isolates are mobile with peritrichousflagella. Salmonellae grow readily on simple media but they never ferment lactoseor 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. choleraesuisare 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 withS. choleraesuis but may be caused by any Salmonella serotype. After oral infectionthere 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, vomitingand profuse diarrhea. Low-grade fever is common, but the episode usually resolves in 2-3 days

Diagnostic laboratory testsSpecimensBlood for culture must be taken repeatedly (in enteric fevers blood culture arepositive in the first week of disease. Stool specimens in enteric fevers yieldpositive 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 fermentingfrom lactose-nonfermenting organisms


Selective medium: salmonella-shigella (SS) agar, Hektoen enteric agar or deoxy-cholate-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 ofsalmonellae

Diagnostic laboratory tests and treatment

Final identificationSuspect colonies are identified bybiochemical reaction patterns or by slide agglutination tests with specific sera.

TreatmentEnteric 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.

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