Clinical Microbiology

Preface Clinical Microbiology Clinical microbiology studies the molecular bases and the pathogenic agents of infectious diseases for the development of new diagnostic technologies and new terapeutic treatment. Bacteria are the oldest and the most adaptable forms of life on Earth. They have existed for some 3.5 billion years. For the first 2 billion years prokaryotes alone colonized every accessible ecological niche. We are surrounded and exposed to bacteria including those that cause disease and death. Diseases caused by bacteria include some of the most common infections in the world. Therefore, the knowledge of pathogenic bacteria, diseases and current therapeutic strategies, is critical for all scientists, especially for clinical microbiologist.

Laboratory procedures for the identification of microrganisms in clinical samples In clinical cases, to prescribe a correct antibiotic treatment, identification of pathogenic organisms in samples, is determinant. The identification techniques must be as much rapid and accurate as possible, to discover the origin of the community-acquired or nosocomial infections, to block the trasmission of diseases between individuals, the emergence of a new hypervirulent or drug-resistant strains.

Microbial identification: optical methods Microscopic examination of specimen is the first step for bacterial identification. Direct examination is a rapid diagnostic method. Visible microrganisms may indicate the possible etiologic agent or can guide the laboratory in selection of the appropriate isolation media and can guide clinical microbiologist in selection of the empirical antibiotic therapy.

Microbial identification: optical methods The light microscope Microscope is an instrument of primary importance for clinical microbiology Microscopes are used for viewing some objects that are too small to see, without magnification.

Parts of the light microscope The stageis a platform that holds the slide containing the specimen to be viewed (mechanism for moving the slide). The light sourcethis is usually located below the stage. The diaphragmlocated below the stage is used to regulate the light. The condensercontains two groups of lenses. Light, from the light source, passes through the diaphragm and condenser, continuing up through the specimen. Body tubecontains anocular lens10x and nosepiece with severalobjective lenses(10x, 40x, 100x) The image is brought into focus by the coarseand fine focusknobs.

Oil immersion The 100x objective (1000x total magnification) requires that a drop of immersion oil must be placed between the slide and the lens. After viewing with oil, the lens must be cleaned with fluid for this purpose

Resolving power The resolving power is the smallest distance between two points required to distinguish as separate two different points. With yellow light and with objective 100x, the smallest separable diameters are about 0,2 µm. Particles 0,2 μm in diameter are magnified to about 0,2 mm and so become visible.

The electron microscope The electron microscope uses electronsinstead of light. Some electrons pass through the specimen and are focused by an electromagnetic objective lens, which presents an image to the projector lens system for enlargement. The image can be recorded on photographic film. The superior resolution of the electron microscope is due to the fact that electrons have a shorter wavelength than the photons of white light. With the high resolving power of the electron microscope is possible to observe the detailed structures of prokaryotic and eukaryotic cells

Types of electron microscopes Different types of electron microscopes: the trasmission electron microscopeTEM the scanning electron microscopeSEM the confocal microscope The TEM was the first to be developed, it employs a beam of electrons projected from an electron gun and focused by an electromagnetic condenser lens. The SEM has a lower resolving power than the TEM, but it produces 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, enhances the visualization of microrganisms by increasing the contrast of structures The smears must be fixed (in contrast with the wet mount) All organisms and cellular components have a similar color

Single stain method Blue stain is considered as a simple stain, in contrast with 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 stain Materials: 1% Methylene blue stain solution Glass microscope slides Culture Method: Make a thin film of the material on a clean glass slide, using a sterile loop or swab Air dry Fix the slide by passing it several times through a flame Flood with methylene blue solution Leave stain in contact with the smear for 30 sec-1 min Rinse well, blot dry and examine under oil immersion

The flagella stain Flagella are appendages, composed by proteins. They are organs of locomotion and are too fine (12-30 nm in diameter) to be visible using the light microscope. Three types of arrangement are known:monotrichous, lophotricous and peritrichous. However their presence can be demonstrated by treating the cells with a colloidal suspension of tannic acid salts causing a heavy precipitate around the cell wall and flagella. The treatment increases the diameter of flagella. Subsequent staining with basic fuchsin, makes the flagella visible in the light microscope

Flagilla observation methods • Hanging drop methods In this method a drop of culture is placed on a cover-slip and then this cover-slip is placed on a slide with a cavity in the middle. The slide is focused to see the bacteria vitality

The capsule stain Many bacteria synthetize a large amount of extracellular polymers when growing in their natural environment. Capsules are composed of polymers (sugars and proteins) that surround bacterial cells Capsules are usually demonstrated by the negative staining procedure The cells are treated with a suspension of indian ink. Capsules are colourless

The capsule stain (Welch method) This method involves treatment with hot crystal violet solution followed by a rinsing with copper sulfate solution; the latter solution is used to remove excess stain because the conventional washing with water would dissolve the capsule. The copper salt gives color to the background. The cell and background appear dark blue, the capsule as much paler blue. Method Spread the culture on glass slide Air dry complitely Stain with crystal violet (1 min) Wash with copper sulfate 10% Air dry Observe the smear using oil immersion

Gram stain technique Gram stain procedure was developed by H. Christian Gram to differentiate pneumococci from klebsiella pneumoniae The procedure involves the application of a solution of iodine (potassium iodide) to cells previously stained with crystal violet. This procedure produces “purple colored iodine-dye complexes” in the cytoplasm of bacteria The cells that are previously stained with crystal violet and iodine are next treated with a decolorizing agent such 95% ethanol or a mixture of acetone and alcohol

The difference between Gram+ and Gram- bacteria The difference is in the permeability of the cell wall to “purple iodine-dye complexes” when treated with decolorizing solvent. Gram positive bacteriaretainpurple iodine-dye complexes after the treatment with decolorizing solvent Gram negative bacteria do not retaincomplexes when decolorized, so we use a red counterstain with safranin to observe Gram negative bacteria

Gram stain procedure Gram negative bacillus Gram positive coccus

Preparation of the smear Correct preparation of the smear: • Make a thin film of the material on a clean glass slide, using a sterile loop or swab • Air dry • Fix the slide by passing it several times through a flame ( the slide very hot, may cause staining artifacts and disrupt the normal morphology of bacteria)

Staining procedure 1.Flood slide with crystal violet (30 seconds) 2.Wash with running tap water 3.Flood with Gram’s iodine (30 seconds) 4.Wash with water 5.Decolorize with 95% ethanol until the smear are colorless (this step is very critical and affected by variation in timing and reagents) 6.Wash with water 7.Flood with safranin (30 seconds) 8.Wash with water, air dry or with absorbent paper

Resuls Gram positive bacteria are stained purple because retain the violet-iodine complexes Gram negative bacteria do not retain violet-iodine complexes after washing in ethanol, but stain red from the safranin counterstain.

The acid fast stain (Zielh Neelsen) The acid fast stain is primarily of clinical application to detect members of the genus Mycobacterium. Mycobacterium tubercolosis, the etiologic agent of tuberculosis, is the most common pathogen of this group. Other microoganisms, particularly the Nocardia, can be identified by their acid-fast characteristic. The term acid-fast is derived from the resistance displayed by acid-fast bacteria to decolorization by acid once they have been stained by another dye. (Cell wall conteins fatty acids and phospholipids responsible for acid fast stain) Matherials: Mycobacterium culture carbol fuchsine acid-alcohol solution (70% ethanol 0,5% hydrochloric acid) methylene blue counterstain

The acid-fast stain: method • Prepare a bacterial smear in the conventional method (as for Gram-staining) air-dried and heat fixed • Flood the smear with carbol fuchsin reagent heated and allow to remain in contact for 5 min • Rinse the excess stain with deionized water • Decolorize the smear with acid alcohol until the color runs from the smear • Wash the smear with deionized water • Counterstain for 30 sec with 1% methylene blue • Rinse, blot dry, and examine under oil immersion.

Zielh Neelsen stain (Mycobacterium) Acid fast bacteria will appear an intense red (retaining the carbol fuchsine) Other material will be blue from the counterstain

The spore stain: (Schaeffer-Fulton method) Some bacteria are able to spores forming. The most common genera are Gram positive rods ( aerobic Bacillus genus and anaerobic Clostridium genus ) The spore wall is relatively impermeable, but dyes can be made to penetrate it by heating the preparation. Spores are commonly stained with malachite green or carbolfuchsin Bacillus anthracis is the best-known microorganism in Bacillus group, and anthrax its clinical condition. It remains a specific agent in biological war and in bioterroristic attacks. Anthrax is divided in cutaneous forms and in pulmonary forms, the last condition is often fatal and severe. In inhalation form, spores are carried by macrophages from the lungs to lymphatic system. Germination begins inside the macrophages and vegetative cells Kill the macrophages and are released into the bloodstream.

Staining procedure Staining procedure • Suspend a small amount of bacteria in the distilled water • Air dry the slide, heat fix by passing the slide over a flame • Flood the slide with malachite green and flame the slide steaming for 5 min • Throw the excess • Flood the slide with eosin • Rinse, blot dry and examine under oil immersion.

Cultivation of microrganisms: bacteriological media Bacteria can be cultivated in laboratory on particular substrates. Many components optimize the growth of microrganisms on media, they include: • Nutrient sources • Solidifying agents (for solid media) • Specific pH • Specific additives (for fastidious bacteria) Some organisms can utilize a very simple medium, most require specific nutrient sources including: nitrogen, carbon, inorganic salts, minerals, vitamins and other substances. The pH is important because many microrganisms have strict pH requirements, most species grow in a range of pH neutrality

Bacteriological media: other factors Other factors allowing the growth include: the incubation temperature and the gas in growth environment. Most clinically significant organisms are mesophiles, other are thermophiles or psycrophiles. In addition, most species grow optimally in aerobic condition, but others require CO2 or total removal of O2

Bacteriological media General for example Nutrient broth or blood agar (used for cultivation or isolation of microrganisms: fastidious and non fastidious) Selective are media that contain additives that enhance the growth of desired organisms by inhibiting other organisms. The selection activity is obtained with addition of dyes, salts or antibiotics. Examples include MSA that contains 7,5% NaCl, which inhibits most organisms, it contains mannitol and pH indicator (phenol red)

Bacteriological media:other types Differential they base the identification on the organism’s appearance on the media.This can be demonstrated by colony colour or by precipitate around the colony. Examples include the medium used for the isolation of enteric pathogens such as Mac Conkey, it contains: lactose bile salts and pH indicator (neutral red). TSI for enteric bacteria it contains: lactose, sucrose, glucose and iron salt, pH indicator. Enriched enriched media are media that allow the growth of fastidious organisms requiring the presence of specific nutrient additives such as hemin, cysteine etc. (fastidious organisms do not grow on general media).

Bacteriological media:other types Specialized are media containing additives for specific pathogens (legionella species etc) Anaerobic media anaerobic media include: peptones, yest extract, vitamines, and reducing agents

Other types: transport media Transport media are used to transport, from the bedside to inoculation in the clinical laboratory, fastidious organisms not surviving in environment They are packaged in a plastic tube with a small amount of liquid medium, a swab attached to a cap is used for collection of specimen, which is then placed into the tube.

Staphylococci Staphylococci are Gram-positive spherical cells, usually arranged in irregular clusters (grape-like) They grow on many types of media, are active metabolically, fermenting carbohydrates and producing pigments that vary from white to deep yellow Some are members of normal flora of skin and mucous membranes of humans, others cause suppuration, abscess formation, toxin mediated diseases, and fatal septicemia

Classification Staphylococcus genus has about 30 species The main species of clinical importance are: Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Staphylococcus aureus iscoagulase-positive,which differentiates it from the other species

Coagulase-negative staphylococci Coagulase-negative staphylococcisometimes cause infections often associated to implanted medical devices especially in mmunocompromised patients These infections are linked to biofilm formation What’s the biofilm? Biofilm is a community of bacterial cells contained in a self-produced polymer matrix adherent to biotic or abiotic surface. This structure is very stable and resistant to the physical, chemical agents used in medicine. Recently, biofilm producing organisms were described in different infections such as in reactivated chronic bronchitis, in cystic fibrosis, in chronic prostatitis, in musculoskeletal infections

Coagulase negative staphylococci Infections caused by coagulase-negative staphylococci are due to: Staphylococcus epidermidis Staphylococcus lugdunensis Staphylococcus warneri Staphylococcus hominis Staphylococcus simulans and other less common species Staphylococcus saprophyticusis 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 notform spores. Their colonies can be white, yellow or orange.

Staphylococci culture Staphylococci grow on most bacteriologic media under aerobic or microaerophilic condition at 37° C. The colonies on solid media are round, smooth, raised, glistening. Staphylococcus aureus form gray to deep golden yellow colonies Staphylococcus epidermidis form gray to white colonies

Staphylococci: characteristics Staphylococci: producecatalase(which differentiates them from the streptococci) ferment manycarbohydrates are resistant todrying, heatand7.5% sodium chloride are sensitive to manyantimicrobial drugs

Staplylococcus aureus Staphylococcus aureus is a major pathogen for humans. Many people are asymptomatic carriers; they have staphylococci on the skin and in the throat (opportunistic pathogen) As a nosocomial pathogen, S. aureus has been a cause of morbidity and mortality. In hospitals, the areas at risk for severe staphylococcal infections are the newborns nursery, intensive care units, operating rooms and cancer chemotherapy wards.

Diseases caused by S. aureus 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 capsuleinhibits phagocytosis by polymorphonuclear leukocytes Teichoic acids:they are polymers of glycerol or ribitol phosphate linked to peptidoglycan Protein Ais a component that binds to FC portion of IgG molecules

Virulence factors Staphylococci can produce diseases through their ability to multiply in tissues and through production of many extracellular and cellular substances. Toxins Hemolysins (cytolytic) αitis potent hemolysin, degrades red blood cells of rabbits βit degrades sphingomyelin and is toxic for red blood cells of sheep γitlyses red blood cells of humans δit disrupts biologic membranes and may have a detergent role

Poreforming α hemolysin α hemolysin is secreted in nontoxic soluble form, it multimerizes on eukaryotic membranes to form lytic pores

Clinical Microbiology