بكتيريا طبية medical bacteriology

Introduction of microbiology

The term of microbiology compose three word.

1-    Micro mean – so small can't seen by neked eye.

2-    Bio mean – living.

3-    Logy mean – science.

So micro biology is defined as the study of microbes or living microorganisms of microscopical size.

Microorganisms were first seen about 1675 by Layven hook. He found many microorganisms in material such as water, saliva and intestinal content of healthy subject.

The term (microbe) was introduced by Louis paster (1857 – 1860) whose demonstration that fermentation was caused by the bacterial and yeast growth.

The term microbe was used by Sedillat in 1878 but now is replaced by microorganism.

Robert Koch 1877 described methods for microscopic examination of bacteria in dried fixed films stained dyes and in 1881 devised the simple method for isolating pure culture of bacteria by plating out mixed of single bacteria grow in separate colonies.

Prokaryotes and Eukaryotes

All microorganisms that are capable of self multiplication can be differentiated by their cell type into one of two groups.

1. Prokaryotic     2. Eukaryotic

	Prokaryotic	Eukaryotes
Cell structure	Very simple	Complex
Nuclear membrane	Absent	Present
Genetic material	Lies in cytoplasm	Contained in nuclear membrane
Mitochondria	Absent	Present
Enzymes	Contain simple enzyme	Contain complex enzyme
Type of multiplication	By binary fission	By mitosis
Examples	This group in include bacteria rikettesia, chiomydia and mycoplasma	This group includes protozoa and fungi, moulds& algae

The algae, protozoa, moulds and fungi their cell have the some general type of structure and organization, they are described as eukaryotic.

Viruses

1.     Viruses are the smallest intracellular microorganism that containing only one kind of nucleic acid (DNA or RNA) as their genome.

2.     Viruses can pass through bacteria stopping filter. All species are strictly parasites.

3.     Can grow only in living cells, non can grow on an inanimate nutrient media.

4.     Viruses are distinguished from other many bacteria by having an entirely different method of growth and reproduction.

The Bacteria are a group of single-cell microorganisms with procaryotic cellular configuration. The genetic material (DNA) of procaryotic cells is not contained within a nucleus, which is the definitive characteristic of eukaryotic cells, such as those that make up plants and animals.

Bacterial cells structure

Structurally, a procaryotic cell (Figure 1 below) has three architectural regions: appendages (attachments to the cell surface) in the form of flagella and pili (or fimbriae); a cell envelope consisting of a capsule, cell wall and plasma membrane; and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions. 

Table 2. Summary: Characteristics of typical bacterial cell structures
Structure	Function(s)	Predominant chemical composition
Flagella	Swimming movement	Protein
Pili
Sex pilus	Mediates DNA transfer during conjugation	Protein
Common pili or fimbriae	Attachment to surfaces; protection	Protein against phagotrophic engulfment
Capsules (includes "slime layers" and glycocalyx)	Attachment to surfaces; protection against phagocytic engulfment, occasionally killing or digestion; reserve of nutrients or protection against desiccation	Usually polysaccharide; possible polypeptide
Cell wall
Gram-positive bacteria	Prevents osmotic lysis of cell protoplast and confers rigidity and shape on cell	Peptidoglycan (murein) complexed with teichoic acids
Gram-negative bacteria	Peptidoglycan prevents osmotic lysis and confers rigidity and shape; outer membrane is permeability barrier; associated LPS and proteins have various functions	Peptidoglycan (murein) surrounded by phospholipid protein-lipopolysaccharide "outer membrane"
Plasma membrane	Permeability barrier; transport of solutes; energy generation; location of numerous enzyme systems	Phospholipid and protein
Ribosomes	Sites of translation (protein synthesis)	RNA and protein
Inclusions	Often reserves of nutrients; additional specialized functions	Highly variable; carbohydrate, lipid, protein or inorganic
Chromosome	Genetic material of cell	DNA
Plasmid	Extrachromosomal genetic material	DNA

 

The cell wall

The layers of the cell envelope lying between the cytoplasmic membrane and capsule.

The cell wall provides protection and imports shape to the cell.

The cell wall of gram positive (G+ve) bacteria differ in its structure and composition form that of gram negative (G-ve)

In the Gram-positive Bacteria (those that retain the purple crystal violet dye when subjected to the Gram-staining procedure) the cell wall is thick, consisting of several layers of peptidoglycan as well as teichoic acids.

In the Gram negative Bacteria (which do not retain the crystal violet) the cell wall is relatively thin and is composed of a single layer of peptidoglycan (no teichoic acid) surrounded by a membranous structure called the outer membrane. The outer membrane of Gram-negative bacteria invariably contains a unique component, lipopolysaccharide (LPS or endotoxin), which is toxic to animals.

The Plasma Membrane or Cytoplasmic membrane (Cell membrane or plasma membrane).Its main function is a selective permeability barrier that regulates the passage of substances into and out of the cell. It is barrier between interior and exterior of the bacterial cell.

The Cytoplasm

The cytoplasm of most bacteria contain DNA, ribosomes RNA and storage granule.

Storage granule

Temporarily hold excess metabolites storage granule known as volutine and lipid granules (metachromatic granules).

DNA, double – stranded circular molecule.

Plasmid is extra chromosomal circular and smaller then DNA. Plasmids carry genes involved in antibiotic resistance called.

Ribosomes: composed 2 subunit. One with a sedimentation coefficient of 50 Sved berg units (50s) and other 30s = 70s.

RNA: The function of RNA is translation of Genetic code tram DNA for protein synthesis.

The capsule or glycocolyx.

Many bacteria secret around themselves polysaccharide substance, often referred to as a slime layer. This may become sufficiently thick to form a definite capsule around the organism.

1.     protects the cell from phagocytosis.

2.     adherence of bacterium to surface of the cells (tissue).

3.     Virulence factor

Flagella     

-         Arrangement basis for classification

-         Monotrichous; 1 flagella

-         Lophotrichous (polar flagella); tuft at one end

-         Amphitrichous; both ends

-         Peritrichous; all around bacteria

Flagella: present in many bacteria, it is responsible for motility.

-         Peritrichous flagella: many flagella distributed over the bacterial surface.

-         Monotrichous flagella: bacteria have a single flagellum.

-         Polar flagella: the bacteria have small bundle of flagella located atone end.

Pili or Fimbriae:

1.     Protein fibers that cover the entire surface of G-ve bacteria

2.     Ploy a major in bacterial adherence to the cell surface.

3.     Sex pili involved in bacterial conjugation (and gene transfer).

Classification of microorganisms

The majority of microorganisms maybe classified in the following biological groups. 1. Algae. 2. Protozoa. 3. Mould.   4. Fungi 5. Bacteria. 6. Spirochaetes. 7. Mycoplasmas. 8. Chlamydiaceae. 9. Rickettsieae. 10. Viruses

Morphological classification of bacteria

Morphologically bacteria car resemble.

Cocci                    (singular: coccus)

Coccobacilli          (singular: coccobacillus)

Rods (bacilli)        (singular: rod, bacillus)

Vibrios                 (singular: vibrio)

Spirilla                 (singular: spirillum)

Spirochetes                  (singular: spirochete)

Staphylococcus aureus

Staphylococcus: are cluster forming Gram positive cocci.

The main species of medical importance is: staphylococcus aureus. Other species may also cause disease include S.epidermidis and S.saprophyicus.

Habitat: widely distributed in the environment. They form part of the normal microbial flora of the skin, upper respiratory tract and intestinal tract. S. aureus is carried in the nose of 40% or more of healthy people.

Pathogenicity

S. aureus causes boils, styes, pustules, impetigo, infections of wounds (cross-infections), ulcers and burns, osteomyelitis, mastitis, septicaemia, meningitis, pneumonia and pleural empyema.

Also, toxic food-poisoning (rapid onset, no fever), toxic shock syndrome and toxic skin exfoliation. S. aureus is carried in the nose and on the skin of many healthy people. It is easily spread in hospitals, particularly on surgical wards.

Extracellular enzymes and toxins produced by strains of S. aureus that contribute to its invasiveness and pathogenicity

● Coagulase: Clots plasma, interferes with phagocytosis, facilitates spread in the tissues.

● Haemolysins: Lyze red cells.

● Leukocidin: Kills leucocytes.

● Fibrinolysin: Digests fibrin.

● Lipase: Breaks down fat.

● Hyaluronidase: Facilitates spread in tissues by destroying hyaluronic acid (component of connective tissue).

● Protein A: Antiphagocytic (prevents complement activa-tion).

● Enterotoxins (heat stable): Cause food-poisoning (particularly vomiting).

● Toxic shock syndrome toxin-1: Shock, rash, desquamation of skin.

● Epidermolytic toxins A and B: Generalized peeling of the skin.

● Chemotaxis inhibitory protein: Inhibits migration and activation of neutrophils.

LABORATORY FEATURES

Specimens: Pus and swabs from infected sites, sputum, cerebrospinal fluid, blood for culture. Faeces, vomit and the remains of food when foodpoisoning is suspected.

Morphology

Staphylococci are Gram positive cocci of uniform size, occurring characteristically in groups but also singly and in pairs. They are non-motile and non capsulate.

Culture

Staphylococci grow well aerobically and in a carbon dioxide enriched atmosphere. Most strains also grow anaerobically, but less well. Temperature range for growth is 10–42 ^oC, with an optimum of 35–37^oC .

Blood agar, chocolate (heated blood) agar: S. aureus produces yellow (golden yellow) to cream or occasionally white 1–2 mm in diameter colonies. Pigment is less pronounced in young colonies. Some strains are  betahaemolytic when grown aerobically. Colonies are slightly raised and easily emulsified.

MacConkey agar: Smaller (0.1–0.5 mm) colonies. Most strains are lactose fermenting.

On Mannitol salt agar:selective and differential media for S.aureus isolation from faecal specimens when investigating staphylococcal food-poisoning and nasal carrier screening. S.aureus ferment manitol and the colonies surrounded by yellow zones due to acid production.

 S. aureus ferments mannitol and is able to grow on agar containing 70–100 g/l sodium chloride. Mannitol salt agar containing 75g/l sodium chloride (plus 4 mg/l methicillin) is recommended, particularly for isolating MRSA strains.

Biochemical tests: S. aureus is:

● Coagulase positive.

● DNA-ase positive.

● Catalase positive.

latex agglutination test kits are available to identify S. aureus based on the detection of clumping factor and, or, protein A

Pastorex Staph Plus test

It detects all strains of S. aureus, including up to 95% MRSA strains (reagent contains antibodies to the capsular polysaccharides found in MRSA as well as fibrinogen and protein A).

Dryspot Staphytect Plus

It detects up to 97% of S. aureus strains, including most MRSA. Colonies of S. aureus are emulsified in saline and mixed with the dry reagent. Agglutination of the blue latex particles indicates a positive test.

Commercially available test kits to confirm MRSA

Test kits have become available to detect penicillin binding protein 2 (PBP2) for the rapid detection of MRSA. An example of a PBP2 latex agglutination test is Mastalex MRSA. The test has been shown to be 97% specific and  sensitive for the detection of MRSA. PBP2-based tests are expensive.

Antimicrobial susceptibility

Antibiotics with activity against S. aureus include: Penicillins* Vancomycin, Macrolides, Cephalosporins and Fusidic acid *Most strains of S. aureus (particularly hospital strains) are resistant to penicillin due to the production of plasmid-coded beta-lactamase.

MRSA (methicillin resistant S. aureus): These strains are resistant to methicillin and related penicillins and are particularly difficult to treat because they are also resistant to most other common antibiotics. Vancomycin is often needed to treat MRSA infections.

Other pathogenic Staphylococcus species

_ Staphylococcus saprophyticus: Causes urinary tract infections in sexually active women.

_ Staphylococcus epidermidis: May cause endocarditis and bacteraemia following infection of cannulae, indwelling catheters, shunts or other appliances positioned in the body. Infections are difficult to treat due to the resistance of S. epidermidis to many antimicrobials.

Microscopically: S. saprophyticus and S. epidermidis resemble S. aureus.

Culturally the colonies of S. epidermidis are white and usually non-haemolytic. The colonies of S. saprophyticus may be white or yellow. They are non-haemolytic. Growth may not occur on MacConkey agar. S. saprophyticus and S. epidermidis are coagulase negative.

Biochemical reactions that differentiate S. epidermidis and S. saprophyticus from S. aureus

Test                      S. aureus              S. epidermidis       S. saprophyticus

Coagulase             +                 ––                         –       

DNA-ase              +                 +Weak                  –

Mannitol*             +                 -                            +

Trehalose*           +                 -                            +      

Sucrose*               +                 +                           +

Novobiocin (5µg)S                  S                           R

Notes: *Fermentation tests: __Sugar fermented with acid production (sugar tablet tests are available from Rosco Diagnostics) S _ Susceptible, R _ Resistant

Streptococcus pyogenes

Classification of streptococci and enterococci

These organisms are broadly classified by their haemolytic activity on blood agar (alpha, beta, non-haemolytic) and by Lancefield group antigens in their cell wall (envelope). Streptococci, formerly classified as Group D streptococci, are now included in the genus Enterococcus e.g. S. faecalis has been reclassified E. faecalis.

Pathogenicity

S. pyogenes (Lancefield Group A) causes sore throat (tonsillitis, pharyngitis), peritonsillar abscess (quinsy), scarlet fever, otitis media, cellulitis, impetigo, necrotizing fasciitis, rysipelas, puerperal sepsis, septicaemia, and occasionally toxic shock syndrome. Also immune mediated post-streptococcal rheumatic fever (following throat infections) and glomerulonephritis (after skin or throat infections).

Note: S. pyogenes can be found as a commensal in the upper respiratory tract, particularly of children.

Extracellular enzymes and toxins produced by strains of S. pyogenes that contribute to its invasiveness and pathogenicity

●Streptolysins (toxins that haemolyze red cells):

– Streptolysin S that is active aerobically (beta-haemolysis on blood agar). It is non-antigenic. Streptolysin O that haemolyzes red cells under anaerobic conditions, e.g. sub-surface agar stabs. It is antigenic, stimulating the production of antistreptolysin O antibody (ASO), see later text.

● Streptokinase, a protease that lyzes fibrin.

● Hyaluronidase: Facilitates spread in the tissues by destroying hyaluronic acid.

● Leukocidin: Destroys leucocytes.

● Lipoteichoic acid: Facilitates adherence to pharyngeal epithelial cells.

● M-proteins (antigens): Anti-phagocytic virulence factors.

● NADase (nicotinamide adenine dinucleotidase): Kills leukocytes. Antibody formed after infection.

● DNA-ases (deoxyribonuclease) A, B, C, D that break down DNA and stimulate an antibody response, particularly against DNA-ase B. Anti-DNA-ase B tests are available.

● Erythrogenic toxin: Responsible for the rash seen in scarlet fever and is also associated with streptococcal toxic shock syndrome..

LABORATORY FEATURES

Specimens: Throat swab (avoiding saliva contamination) or swabs of pus and serous fluid depending on the site of infection, and blood for culture.

Culture media should be inoculated as soon as possible or a swab placed in a tube of silica gel.

Testing for ASO antibody in serum is helpful in diagnosing rheumatic fever.

Morphology

Streptococci are Gram positive cocci, occurring characteristically in short chains, but also in pairs and singly. Long chains are formed in fluid cultures. The organisms are non-motile. Some strains are capsulated.

Culture

Blood agar: S. pyogenes produces beta- haemolytic colonies(the colonies are surrounded by a zone of complete haemolysis). Colonies are usually small (0.5–1 mm), colourless, dry, shiny or mucoid. Haemolysis is more marked under anaerobic conditions as seen in colonies growing below the agar surface (following stabs made in the culture medium).

Choice of blood

To isolate beta-haemolytic streptococci, use sheep blood (1st choice), horse, rabbit or goat blood to prepare blood agar plates. Do not use human blood because this may contain unwanted substances such as citrate (e.g. donor blood), antibiotics, or antibodies such as ASO or anti-M protein that could interfere with the growth or haemolytic activity of S. pyogenes.

Sensitivity to bacitracin

Adding a bacitracin disc (0.04 or 0.05 IU, not higher), to a plate of blood agar or preferably a selective medium, is a useful method of screening for S. pyogenes. Most strains are sensitive to bacitracin, but it is not possible to rely completely on sensitivity to bacitracin to identify S. pyogenes. Other non-Group A beta-haemolytic streptococci (e.g. Groups B, C and G) may occasionally also show sensitivity to bacitracin. Serological grouping is required.

Note: S. pyogenes is always sensitive to benzylpenicillin and therefore placing a 1 µg disc of the antibiotic on a primary culture plate (well area) can also help topresumptively identify S. pyogenes.

Crystal violet (1 in 50 000) blood agar: This is a useful inexpensive selective medium for isolating S. pyogenes from patients with impetigo where S. aureus may be present with S. pyogenes. Crystal violet will inhibit the growth of S. aureus. Alternatively, use a 30 µg neomycin disc on the heavy part of the inoculum.

MacConkey agar: S. pyogenes does not grow on this medium.

Biochemical tests: S. pyogenes is:

● Catalase negative (staphylococci are positive)

● PYR positive*

*PYR (pyrrolidonyl) test: This detects pyrrolidonyl peptidase enzyme activity. Besides S. pyogenes, Enterococcus species and occasionally streptococci belonging to groups C and G are also PYR positive. The test can be rapidly and simply performed using PYR impregnated strips.

S. pyogenes belongs to Lancefield Group A can be grouped (identified) using specific Group A antiserum to identify the A antigen extracted from the cell wall of the bacteria  (coagglutination or latex) to Lancefield group betahaemolytic streptococci.

Positive group A test:Indicates that the organism is S. pyogenes (particularly when bacitracin sensitive and PYR positive) but possession of A antigen is not species specific. Very occasionally other beta haemolytic streptococci group as A.

Direct detection of antigen A from throat swab extracts: Several tests can be used to detect antigen A directly extracted from throat swabs without the need to culture the specimen, Most of the rapid direct tests are immunochromatographic (IC) enzyme immunoassays (with built-in control) or latex agglutination techniques.

ASO antibody tests

Measurement of ASO antibody in serum ASO (anti-streptolysin O) antibody is formed in response to infection with S. pyogenes and other streptococci that produce streptolysin 0 (some Group C and G strains).

Rapid, simple to perform latex agglutination to screen for and measure semi-quantitatively raised levels of ASO antibody in serum.

Measurement of ASO antibody titre is important in the investigation of post-streptococcal diseases, particularly rheumatic fever. In rheumatic fever there is a rise in ASO antibody titre in 80–85% of patients. The rise begins early in the course of the disease with highest levels being reached soon after onset of the disease. In the second week, the level begins to fall. Rheumatic fever is a serious post-streptococcal complication because it can lead in later life to chronic valvular disease of the heart.

Measurement of DNA-ase B antibody

Most increases in DNA-ase B (deoxyribonuclease B) antibody titres occur in response to Group A streptococcal infection. The rise in DNA-ase B antibody usually occurs later than the rise in ASO antibody. Measurement of anti-DNA-ase B is of value when investigating acute glomerulonephritis following a streptococcal skin infection (rather than a streptococcal sore throat). This is because the ASO antibody titre is not usually raised following streptococcal skin infections whereas there is a rise in titre of anti-DNA-ase B.

Antimicrobial susceptibility

susceptible to penicillin. Erythromycin is usually used to treat patients hypersensitive to penicillin but resistance to erythromycin (and also to tetracyclines) is being increasingly reported.

                                                                   

Streptococcus agalactiae

Pathogenicity

Streptococcus agalactiae (Lancefield Group B) causes septic abortion and puerperal or gynaecological sepsis, and occasionally urinary tract infection. S. agalactiae forms part of the normal microbial flora of the female genital tract. Occasionally it causes neonatal septicaemia and meningitis.

In cattle S. agalactiae is a common cause of bovine mastitis. Human strains are distinct from animal strains.

LABORATORY FEATURES

Specimens: Include cerebrospinal fluid, ear swab, and blood for culture from neonates. High vaginal swab is required from women with suspected sepsis.

Morphology

Group B streptococci are Gram positive cocci, occurring characteristically in short chains but also in pairs and singly. The organisms are non-motile. Most strains are capsulated.

Culture

Blood agar: Most strains produce grey mucoid colonies about 2 mm in diameter, surrounded by a small zone of betahaemolysis (clear area with decolorization of haemoglobin). About 5% of strains are nonhaemolytic. Placing discs of penicillin and gentamicin on the plate can help to identify these strains (penicillin sensitive, gentamicin resistant).

MacConkey agar: Most strains grow on this medium. Neomycin blood agar: A useful selective medium for isolating S. agalactiae from urogenital specimens.

Orange pigment: Produced by S. agalactiae when cultured on serum starch agar anaerobically.

Lancefield grouping

S. agalactiae belongs to Lancefield Group B. Serological identification of the organism can be made by detecting B antigen using Group B antiserum reagent. The technique is similar to that described for grouping Streptococcus Group A.

CAMP (Christie, Atkins, Munch, Peterson) test

This test requires the use of a beta-lysin producing strain of S. aureus to detect the CAMP factor, i.e. extracellular diffusible protein produced by S. agalactiae. This protein interacts with the staphylococcal beta-lysin on sheep (or ox) blood agar producing enhanced haemolysis.

Bile aesculin stope: S. agalactiae does not hydrolyse aesculin. It is able to grow on bile agar. Group A Streptococcus pyogenes gives a variable aesculin hydrolysis reaction and does not grow on bile agar. Group D streptococci hydrolyse aesculin and can grow on bile agar.

Hippurate hydrolysis test

S. agalactiae hydrolyzes hippurate

Direct detection of Group B streptococcal antigen in c.s.f.

When Group B streptococcal meningitis is suspected, a rapid diagnosis can be made by detecting Group B streptococcal antigen directly in c.s.f., serum or urine using a latex or coagglutination slide test. They are particularly useful if antibiotic treatment has been started and it is not possible to isolate S. agalactiae culturally.

Fig . CAMP reaction of Streptococcus agalactiae (Group B).

Antimicrobial susceptibility

S. agalactiae has the same susceptibility profile as S. pyogenes.

Streptococcus pneumoniae

Pathogenicity

S. pneumoniae causes lobar pneumonia, bronchitis, meningitis, bacteraemia, otitis media, sinusitis and conjunctivitis.

Serotypes: Over 80 capsular serotypes of S. pneumoniae have been identified. Less than 15 serotypes are responsible for most infections.

LABORATORY FEATURES

Specimens: Depending on the site of infection, specimens include sputum, exudate, blood for culture, and cerebrospinal fluid.

Morphology

S. pneumoniae is a Gram positive elongated (lanceolate) diplococcus. It also forms short chains, particularly following culture. Pneumococci are nonmotile and capsulated (non-capsulated following culture). In Gram stained smears from specimens, the capsule can often be detected as an unstained empty area around the diplococcus.

Culture

Blood agar: Following overnight incubation. S. pneumoniae forms translucent or mucoid colonies, 1–2 mm in diameter. In young cultures the colonies are raised but later become flattened with raised edges, giving them a ringed appearance (‘draughtsmen’). Strains of some serotypes (e.g. serotype 3) produce large mucoid colonies. Pneumococci show alpha-haemolysis.

Note: When cultured anaerobically on blood agar, some strains of S. pneumoniae show betahaemolysis.

Viridans streptococci: These organisms which may be found in sputum are also  alphahaemolytic and require differentiation from S. pneumoniae

Optochin sensitivity

Pneumococci are sensitive to optochin (ethylhydrocupreine hydrochloride). Placing a disc (5µg) on a primary sputum culture and culturing the plate aerobically (not in CO₂ ) can help to provide a rapid presumptive identification of S. pneumoniae. If the zone of inhibition is less than 10 mm (6 mm disc) the colonies should be tested for bile solubility.

Chocolate and lyzed blood agar: S. pneumoniae grows well on chocolate (heated blood) agar and lyzed blood agar. Growth is enhanced when incubated in a carbon dioxide enriched atmosphere (candle jar).

Biochemical tests: S. pneumoniae is

● Catalase negative ● Sensitive to optochin ● Bile soluble

*Bile solubility test

There are several ways of testing pneumococci for bile solubility. Some workers, however, prefer to test suspect alpha-haemolytic colonies directly on a culture plate by touching a colony with a loopful of 2% sodium deoxycholate reagent (pH.7.0), incubating the plate at 35–37 ؛C for 30 minutes, and examining for lysis (disappearance of the colony, indicating S. pneumoniae).

Direct detection of pneumococcal antigen in body fluid

Rapid latex and coagglutination tests are available to detect capsular pneumococcal antigen in c.s.f., pleural fluid, serum and urine.

Antimicrobial susceptibility

Antibiotics with activity against pneumococci include penicillin, erythromycin, and co-trimoxazole. Penicillin-resistant strains are becoming an increasing. When testing for susceptibility to penicillin it is best to use a disc containing 1 µg of oxacillin. A zone size less than 20 mm indicates reduced susceptibility. Isolates should also be tested for susceptibility to tetracycline, erythromycin and chloramphenicol.

Viridans streptococci

Although often alpha-haemolytic on blood agar, the viridans group of streptococci can also be nonhaemolytic and occasionally beta-haemolytic. They form part of the normal microbial flora of the upper respiratory tract (particularly oropharynx) and gastrointestinal tract. They may therefore be found with  S. pneumoniae in sputum (as commensals). A few species are pathogenic (e.g. S. mutans, S. sanguis, S. mitior) causing endocarditis, bacteraemia, and dental caries. The following are the main features which differentiate S. pneumoniae from viridans streptococci:

The S. anginosus group (formerly S. milleri group) is associated with deep abscesses in various sites in the body (abdomen, chest, brain) often in association with other bacteria.

Anaerobic streptococci and cocci

Most of the pathogenic Gram positive anaerobic streptococci and cocci belong to the genus Peptostreptococcus.

Anaerobic streptococci and cocci can be found as commensals on the skin and in the mouth, vagina and gastrointestinal tract. They can cause septicaemia, puerperal sepsis, and bone and joint infections. They are often isolated together with other anaerobes such as Bacteroides fragilis, from abscesses and deep infected wounds and ulcers. Many strains are proteolytic and gas (H₂S)-producing producing unpleasant smell.

Anaerobic streptococci and cocci can be cultured in thioglycollate broth. On subculture to blood agar, the colonies are very small, shiny, and nonhaemolytic. Incubation for up to 72 hours is often required to produce visible growth.  Microscopically: They appear as Gram positive cocci in chains, groups, or singly, variable in size, and catalase negative. Anaerobic cocci are usually susceptible to penicillin, and all are susceptible to metronidazole 5 µg disc).

Enterococcus species

Pathogenicity

E. faecalis (formerly classified Streptococcus. faecalis) is the main pathogen in the genus Enterococcus, causing about 95% of enterococcal infections including infections of the urinary tract, biliary tract, ulcers (e.g. bed sores), wounds (particularly abdominal) and occasionally. Endocarditis or meningitis. It is a normal commensal of the vagina and intestinal tract. A minority of infections are caused by E.faecium.

LABORATORY FEATURES

Morphology

Enterococcus species are Gram positive cocci, occurring in pairs or short chains. They are non-capsulate and the majority are non-motile.

Culture

Enterococci are aerobic organisms capable of growing over a wide temperature range, 10–45 ^OC.

Blood agar: Enterococci are mainly nonhaemolytic but some strains show alpha or beta-haemolysis.

MacConkey and CLED agar: E. faecalis ferments lactose, producing small dark-red colonies on MacConkey agar and small yellow colonies on CLED (cysteine lactose electrolyte-deficient) agar.

Enterococcus species are also able to grow in the presence of 6.5% sodium chloride and 40% bile. Withstand 60 ^OC for 30 minutes and can grow at 45 ^OC. When grown on media containing aesculin, enterococci hydrolyze the aesculin, producing black colonies.

Biochemical tests: Enterococcus species:

● Ferment lactose (also mannitol and other sugars).

● Hydrolyze aesculin.

● Reduce litmus milk.

Lancefield group: Enterococci possess Lancefield Group D antigen (as also some streptococci). E. faecalis, however, is usually identified culturally and biochemically.

Antimicrobial susceptibility: Most are susceptible to ampicillin and resistant to cephalosporins. Resistance to penicillin.

Table show:  Biochemical reaction of Streptococci
CAMP test	Litmus milk reduction	Sensitivity to optichin& bile salt	Bacitracn sensitivity	Catalase	Species
-	-	-	+	-	S-pyogenes(A)
+	-	-	-	-	S-aglactiae (B)
-	+	-	-	-	Entrococci(D)
-	-	-	-	-	Viridans strept
-	-	+	-	-	S-pneumoniae

Bacillus anthracis

Pathogenicity

B. anthracis causes anthrax which is mainly a disease of sheep, cattle, goats and other herbivores with humans becoming infected only after coming into contact with infected animals or their skins.

Anthrax in animals

Animals become infected by ingesting B. anthracis when feeding. Pastureland that has become contaminated with spores from excreted bacilli or from the bodies of dead animals (highly infectious) can remain a source of infection for many years, e.g. 50–60 y.

Sources of anthrax in humans

Human infections (zoonoses) can occur from handling infected animals or coming into contact with skins containing anthrax spores, when using skins as clothing, water-carrying containers, or sleeping mats. Other sources of infection include animal hair, bones, and the bedding of infected animals. Less commonly, infection is caused by eating infected meat.

Depending on the source and site of infection, B. anthracis can cause:

_ Cutaneous anthrax (commonest form): Bacilli enter damaged skin, producing a blister (‘malignant pustule’) which usually ulcerates and eventually forms a dry black scab surrounded by oedema. Fatal septicaemia, toxaemia, and meningoencephalitis may develop, especially in non-immune persons. Ocular anthrax may also occur.

_ Pulmonary anthrax: Caused by inhaling large numbers of B. anthracis spores (‘woolsorters’ disease). Infections are usually fatal.

_ Enteric anthrax: A severe form of gastroenteritis with fever, abdominal pain and bloody diarrhoea, due to ingesting infected meat. Septicaemia often develops.

_ Meningoencephalitis: Usually as a complication of septicaemia and occasionally as primary anthrax meningoencephalitis.

Virulence factors

B. anthracis produces a polypeptide capsule which is antiphagocytic and a toxin which affects the central nervous system leading to respiratory distress, shock, cardiac collapse and death.

LABORATORY FEATURES

Specimens: Include fluid aspirated from cutaneous lesions and when indicated, sputum, cerebrospinal fluid, and blood for culture.

Caution: B. anthracis is a high risk infectious pathogen, therefore handle specimens and infected material with care.

Morphology

B. anthracis is a large, 5–8X1.5 µm, Gram positive (or Gram variable) non-motile bacillus, often appearing joined end to end in chains.

In smears from specimens: Bacilli are capsulated. The capsular material often appears irregular and fragmented. When stained using Loeffler’s polychrome (McFadyean) methylene blue the bacilli stain blue and the capsular material stains purple-pink. Giesma stain can also be used when MacFadyean methylene blue is not available.

In smears from aerobic cultures: Bacilli are non-capsulated but contain oval spores (same diameter as the bacilli), giving the organisms a beaded appearance. They occur in chains.

Culture

B. anthracis grows aerobically and anaerobically (facultative anaerobe). The temperature range for growth is 12–45 ^OC with an optimum of 35–37^OC. Spore formation is best between the range 25–30 ^OC.

Blood agar: B. anthracis produces large 2–5 mm in diameter, grey-white, irregular colonies with wavy edges. The colonies are nonhaemolytic or only slightly haemolytic. Saprophytic Bacillus species are markedly haemolytic.

Broth cultures: They are not usually turbid, but they often show a thick skin (pellicle) and a sediment.

Gelatin stab culture: The organism slowly liquefies the gelatin along and out from the line of inoculation. The treelike pattern formed by the liquefaction lines is characteristic of B.anthracis, but the reaction is slow and in practice anthrax bacilli are usually identified microscopically by their morphological appearance.

Antimicrobial susceptibility

Antibiotics with activity against B. anthracis include penicillin, tetracycline, streptomycin, and co-trimoxazole. Workers at risk of infection should be vaccinated.

Bacillus cereus

B. cereus toxin causes food-poisoning, usually in rice or other cereals that have been cooked and then stored in warm temperatures. B. cereus unlike B. anthracis is motile, non-capsulate, and produces haemolytic colonies on blood agar. Non-lactose fermenting, producing pale colonies on MacConkey agar. On egg-yolk agar, B. cereus gives a strong lecithinase reaction. It rapidly liquefies gelatin stabs.

Mannitol egg-yolk phenol-red polymyxin agar (MYPA) is recommended as a selective medium for the isolation of B. cereus from faeces, vomit, or food. After overnight incubation at 35–37 ^OC, large 3–7 mm flat, dry grey-white colonies surrounded by an area of white precipitate are produced.  B. cereus produces beta-lactamase and is resistant to penicillin

and cephalosporins. Antimicrobials with activity against B.cereus include gentamicin, erythromycin, vancomycin and clindamycin.

Corynebacterium diphtheriae

Pathogenicity

C. diphtheriae causes:

_Nasal, nasopharyngeal and tonsillar diphtheria, especially in young children. Often there is marked oedema of the neck. Infection is by inhaling respiratory droplets.

Exotoxin

Virulent strains of C. diphtheriae produce a powerful exotoxin that is absorbed through the damaged mucous membrane into the blood circulation. If not neutralized by antitoxin, the toxin can cause toxaemia with fatal cardiac and neural complications.

Inflammatory membrane                         

At the site of infection there is an acute inflammatory response which leads to the formation of a grey-yellow membrane which becomes necrotic at a later stage. If this membrane extends downwards to the larynx it can blockthe passage of air and cause death from asphyxia.

_ Cutaneous (skin) diphtheria which usually develops when C. diphtheriae infects open wounds.

C. diphtheriae biovars. There are four biovars (biotypes): gravis, intermedius, mitis, and belfanti. These names were used to describe the severity of disease. In the investigation of diphtheria, it is not necessary to differentiate these biovars.

Note: Commensal diphtheroids form part of the normal microbial flora of the upper respiratory tract and skin.

LABORATORY FEATURES

The role of the laboratory is to confirm the clinical diagnosis.

Specimens: Include throat, and, or nasopharyngealswabs to confirm a diagnosis of throat diphtheria, and a skin swab if cutaneous diphtheria is suspected.

Morphology

C. diphtheriae is Gram positive(stains unevenly and weakly). It is markedly pleomorphic. Long, thin, and curved forms can be seen and also short rods and rods enlarged at one end (clubshaped). They often appear in clusters, joined at angles like Chinese letters.

Commensal diphtheroids: These are strongly Gram positive and stain uniformly. They are usually short and show little variation in size and form.

Volutin granules.  In Albert stained smears, particularly from Loeffler serum or Dorset egg cultures, C. diphtheriae often appears beaded due to the presence of darkstaining granules in the rods. These granules, known as volutin or metachromatic granules, in toluidine blue stained smears, the organisms stain pale blue and the granules dark red-purple.

C. diphtheriae is non-capsulate, non-motile, and does not form spores.

Culture

C. diphtheriae is an aerobe and facultative anaerobe. Temperature range for growth is 20–40 ^OC with an optimum of 35–37 ^OC. Loeffler serum medium and Dorset eggmedium: C. diphtheriae grows rapidly on these media, producing significant growth in 4–6 hours.

Note: It is not advisable to use either Dorset egg or Loeffler serum medium as a primary medium for isolating C. diphtheriae because commensal diphtheroids may overgrow the diphtheria bacteria.

Tellurite blood agar: Used as a primary medium for isolating C. diphtheria from throat and nasopharyngeal swabs. C. diphtheriae reduces tellurite and produces grey or grey-black colonies measuring 0.5–2 mm in diameter. Single colonies are generally darker or blacker than those massed together.

Some strains are raised and cone-shaped (especially mitis), others are raised with striated margins and grey centres (especially gravis), and others are small with black centres and clear margins (especially intermedius). Strains can be haemolytic, slightly haemolytic, or non-haemolytic. Mitis strains are beta-haemolytic.

Commensal diphtheroid colonies are grey, non-haemolytic, and measure 0.1–0.8 mm in diameter. Separate colonies are generally paler than those massed together. Some staphylococci and streptococci also produce black colonies on tellurite blood agar.

Tinsdale medium: After 24–28 h incubation, C. diphtheriae colonies are grey-black, raised, and surrounded by a dark brown area as shown in. The brown colour is due to the hydrogen sulphide produced from the cystine interacting with the tellurite. Occasionally commensal diphtheroids and other respiratory tract commensals may grow on Tinsdale’s medium but the colonies are not surrounded by a brown halo like those of C. diphtheriae. Proteus species produce large colonies with a blackening in the medium.

Tinsdale medium in addition to tellurite also contains cystine which makes it more differential for C. diphtheriae (browning is produced in the medium).

Biochemical tests: C. diphtheriae:

● Catalase and nitrate positive.

● Oxidase negative.

● Urease negative.

● Ferments glucose and maltose with acid production. A few strains of gravis and mitis biovars ferment sucrose.

● C. diphtheriae gravis ferments starch with acid production.

Rapid carbohydrate utilization test to identify C. diphtheriae and other Corynebacterium species