By Ms. Kostiuk from Microbiology department

1. Lesson N1: LABORATORY DIAGNOSIS OF STAPHYLOCOCCAL INFECTIONS
1.Scientifically methodical ground of theme
There are several types of symbiosis between cocci and the human body. Saprophytic and conditionally
pathogenic types of staphylococci and streptococci live on the skin, mucous membranes, and in the respiratory
tract. Meningococci may be harboured for long periods in the nasopharynx, and faecal streptococci (enterococci) in
the intestine.
When body resistance is lowered or the skin and mucous membranes are injured, these bacteria penetrate the
body tissues and cause infection. The various cocci possess different organotropic ability. This is distinctly
manifest in meningococci, and gonococci but less so in staphylococci and streptococci. Cocci belong to the
families Micrococcaceae, Streptococcaceae Peptococcaceae, Neisseriaceae.
2.Educational purpose
STUDENTS MUST KNOW:
1. Structure, tinctorial and cultural properties of staphylococci.
2. Antigenic structure of staphylococci, their classification.
3. Fermentative properties and toxin production of staphylococci.
4. Differentiation of staphylococci.
5. Main clinical forms of staphylococcal infections.
6. Methods of laboratory diagnosis of staphylococcal infections and main preparations for specific
prophylaxis and treatment of staphylococcal infections
STUDENTS SHOULD BE ABLE TO:
– prepare the smears from pus;
– stain the smears by Gram’s technique;
– make microscopical examination of the smears;
– inoculate pus on solid medium;
– inoculate a blood specimen in sugar MPB with the purpose of diagnostics of a sepsis;
– inoculate material from a nasopharynx for revealing bacterial carriage.
3.Chart of topic content.
Staphylococci, being resistent to dry conditions and hight salt concentrations, are well suited to their
ecological niche, which is the skin surface of man and animals. Approximately 30% of helthy people are “carriers”
of S. aureus, an even higher number carry coagulase-negative staphilocicci. Staphilococci are also commonly
present on other animals, including farm animals used for milk production, cattle, sheep and gouts; mastitis caused
by S. aureus is common and mostly complication of milking. Contamination food can occur from either human or
animal sources; such contamination can result in staphilococcal food poisoning.
Staphylococci. The staphylococcus, Staphylococcus aureus, was discovered by R. Koch (18,78), and later
isolated from furuncle pus by L. Pasteur (1880). It has been described as the causative agent of numerous
suppurative processes by A. Ogston (1881), and has been studied in detail by F. Rosenbach (1884)
Definition of genus
The chief properties characteristizing the genus Staphylococcus are shown in table N1. Staphylococci resemble
members of genus Micrococcus morohologically, but they differ in DNA base composition. Micrococcus also
deffer in having a strictly aerobic metabolism.
Table N1:Characteristics of the genus of Staphylococcus
Gram-positive cocci
Catalase positive
Divide in more than one plane to form irregular, grape- lake cluster
Capable of aerobic and anaeribic metabolism
Occur widly in the surface of man and other vertebrate animals
Morphology. Staphylococci are spherical in shape, 0.8-1 mem in diameter, and form irregular clusters resembling
bunches of grapes. In smears from cultures and pus the organisms occur in short chains, in pairs, or as single cocci.
Large spherical (L-forms) or very small (G-forms) and even filterable forms may be seen in cultures which have
been subjected to various physical, chemical, and biological (antibiotics) factors.
Staphylococci are Gram-positive organisms which possess no flagella and do not form spores. A
macrocapsule can be seen on ultrathin sections of Staphylococci isolated from infected mice. The nucleoid
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2. occupies most of the cytoplasm and is filled with DNA fibrils. The G+C content in DNA ranges between 30.7 and
39.0 per cent.
Cultivation. Staphylococci are facultative-anaerobes. They grow well on ordinary nutrient media with a pH
of 7.2-7.4 at a temperature of 37 °C but do not grow at temperatures below 10 °C and above 45 °C. At room
temperature with adequate aeration and subdued light – the organisms produce golden, white, lemon-yellow, and
other pigments known as lipochromes. These pigments do not dissolve in water but are soluble in ether, benzene,
acetone, chloroform, and alcohol. They are most readily formed on milk agar and potatoes at a temperature of
20-25° C.
On meat peptone agar Staphylococci produce well defined colonies with smooth edges, measuring from 1-2
to 2.5 mm in diameter. Under the microscope the course granular nature of the colonies can be seen, the latter are
opaque and have a dense centre. Their colour epends on the pigment produced by the organisms. Besides the
typical colony types, Staphylococci produce R-, G-, and L-forms. Growth of Staphylococci in meat-peptone broth
produces diffuse opacity throughout the medium and, subsequently, a precipitate. In some cases when there is
sufficient aeration, the organisms form a pellicle on the surface of the broth. Staphylococci grow well on potatoes
and coagulated serum. After 24-48 hours of incubation there is usually abundant growth along the inoculation stab
and liquefaction of gelatin media. On the fourth or fifth day the gelatin medium resembles a funnel filled with
fluid.
On blood agar pathogenic Staphylococci cause haemolysis of the erythrocytes. Rabbit and sheep
erythrocytes are the most sensitive to the staphylococcal haemotoxin.
Fermentative properties. Staphylococci produce enzymes which cause the lysis of proteins and sugars (see
Table 2). There is no indole production in young cultures. The organisms liquefy gelatin, coagulate milk and
occasionally serum, reduce nitrates to nitrites, produce urease, catalase, phosphatase, ammonia, and hydrogen
sulphide. They ferment glucose, levulose, maltose, lactose, saccharose, mannitol, and glycerin, with acid formation.
A connection has been revealed between arginase activity and the level of γ-toxin formation.
Toxin production. Staphylococci produce α-, β-, δ- and γ-haemolysins which are characterized by lethal,
haemolytic, and necrotic activity. Filtrates of staphylococcal broth cultures contain an enterotoxin which causes
food poisoning on entry into the gastro-intestinal tract. Staphylococci excrete exofoliatines which cause
staphylococcal impetigo and pemphigus neonatomm in children.
Leucocidin, a substance which destroys leucocytes, haematoblasts of the bone marrow and nerve cells, is
also produced by the Staphylococci. The organisms also coagulate blood plasma. Their ability to coagulate, plasma
is a stable property and is used for differentiating various strains. Coagulase is thermoresistant. It can be isolated
from staphylococcal broth cultures.
Staphylococci produce fibrinolysin which when added to a blood clot dissolves the latter within 24-48 hours.
The Staphylococci produce hyaluronidase which breaks down hyaluronic acid, a component of connective
tissue.
Coagulase, fibrinolysin, lecithinase, hyaluronidasa and phosphatasa all belong to the group of enzymes
possessing destructive properties. Lecithinase destroys the lecithin protective membranes of the colloidal particles
of a substance found in human, sheep, and rabbit erythrocytes. An anticoagulant which inhibits blood coagulation
has also been derived from the staphylococcal cultures. This staphylococcal anticoagulant is produced in exudates
of inflamed tissues, occurring during staphylococcal infections. Haemagglutmins which cause the agglutination of
rabbit erythrocytes have also been found in staphylococcal culture filtrates. Virulent Staphylococci inhibit the
phagocytic activity of leucocytes.
Many microbiologists believe that the Staphylococci isolated from patients produce alpha-haemolysins,
while the organisms pathogenic for animals (e. g. itesponsible for mastitis in cows) more often produce beta-
haemolysin.
Staphylococcal exotoxin, inactivated by treatment with 0.3-0.5 per cent formalin at 37 °C for 7-28 days, and
injected parenterally to humans and animals, stimulates the production of a specific antitoxin capable of reacting
with the toxin.
Antigenic structure. Polysaccharides A and B have been obtained from a staphylococcal suspension by
treating the latter alternately with acid and alkali and removing the proteins with trichloracetic acid.
Polysaccharide A was extracted from pathogenic strains isolated from patients with septicaemia,
furunculosis, osteomyelitis, and acute conjunctivitis, etc. Polysaccharide B is found in avirulent, non-pathogenic
strains. Polysaccharides A and B differ not only in their serological reactions but also in their chemical structures.
Antigen C, containing a specific polysaccharide, has been recently isolated. Staphylococcal polysaccharides
demonstrate a marked type specificity. Even in a 1 :1000 000 dilution they give a distinct precipitin reaction. The
protein antigen is common to all species and types of staphylococci.
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3. Three types (I, II, III) of staphylococci have been revealed by the agglutination test and precipitin reaction.
However, a quite number of cultures are unsuitable for serological typing. Recent studies have revealed fifteen
type-specific staphylococcal antigens.
Classification. Staphylococci are included in the class Bacteria, family Micrococcaceae, genus
Staphylococcus.Currently there are some 32 different species of staphylococci. These fall into two main groups on
basis of their ability to clot blood plasma by action enzyme coagulase. S. aureus is far the most important
coagulase-positive species. The coagulase- negative staphylococci are skin commensals, which are now recognazed
as important oportunic pathogens that can cause infections associated with prostheses, catheters and implants (S.
epidermidis, and uritary tract (S. saprophyticus)
Table 2Differentiation of Staphylococci
Species
Main characteristics S. aureus S. S.
epidermidis; saprophyticus
Presence of:
Plasmacoagulase + — —
Phosphatase + + —
Protein A in + — —
superficial antigen
Oxidation Mannitol + — +
Production of alpha- + – –
toxin
Resistance to S S R
novobiocin
Deoxyribonuclease + - -
Note: S, sensitive; R, resistant
Certain strains of the family Micrococcaceae are strict anaerobes. Peptococcus niger, Peptococcus
anaerobius, Peptococcus asaccharolyticus and others are usually conditionally pathogenic for human beings. They
live in the mouth mucosa, in the intestine, urogenital tract, and in other parts of the human body. In weakened
individuals and in people suffering from chronic diseases the anaerobic micrococci may give rise to various
diseases and complications.
Resistance. Staphylococci are characterized by a relatively strong resistance to desiccation, freezing,
sunlight, and chemical substances. After desiccation they can survive for more than 6 months. Repeated freezing
.and thawing do not kill the organisms. They survive for many hours under direct sunlight. Staphylococci maintain
their viability for more than 1 hour at 70 °C. At a temperature of 80 °C they are destroyed within 10-60 minutes
and at boiling point, they instantly perish. A 5 per cent phenol solution kills the organisms in 15-30 minutes.
Staphylococci are very sensitive to certain aniline dyes, particularly to brilliant green which is used for treating
pyogenic skin diseases caused by these organisms. Staphylococci possess high resistance to antibacterial agents, in
70-80 per cent of cases they are resistant simultaneously to 4-5 agents. Cross resistance to antibiotics of the
macrolide group (erythromycin, oleandomycin, etc.) is encountered.
Pathogenicity for animals. Horses, cattle, sheep, goats, pigs, and, among laboratory animals, rabbits, white
mice, and kittens are susceptible to pathogenic staphylococci.
An intracutaneous injection of a culture of pathogenic staphylococci produces inflammation and subsequent
necrosis in the skin of the rabbit. An intravenous injection of a staphylococcal culture filtrate causes a condition
similar to acute poisoning in rabbits, which is characterized by motonc excitation, respiratory disorders,
convulsions, paralysis of the hind extremities, and sometimes, by diarrhoea and urine discharge. After complete
fatigue the animal shortly dies.
Staphylococci or their toxin will cause vomiting, diarrhoea, and weakness in kittens if introduced per os or
intraperitoneally. Functional disorders of the digestive tract arise owing to the effect of tne enterotoxin which is
distinguished from the other fractions of the staphylococcal toxin by its thermoresistance. It withstands a
temperature of 100 °C for 30 minutes. The most reliable test for the presence of enterotoxin is an intravenous
injection to adult cats.
Pathogenesis and diseases in man. Staphylococci enter the body through the skin and mucous membranes.
When they overcome the lymphatic barrier and penetrate the blood, staphylococcal septicaemia sets in. Both the
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4. exotoxins and the bacterial cells play an important role in pathogenesis of diseases caused by these organisms.
Consequently, staphylococcal diseases should be regarded as toxinfections.
The development of staphylococcal diseases is also influenced by the resulting allergy which in many cases
is the cause of severe clinical forms of staphylococcal infections which do not succumb to treatment.
Staphylococci are responsible for a number of local lesions in humans: hidradenitis, abscess, paronychia,
blepharitis, furuncle, carbuncle, periostitis, osteomyelitis, folliculitis, sycosis, dermatitis, eczema, chronic
pyodermia, peritonitis, meningitis, appendicitis, and cholecystitis.
Diabetes mellitus, avitaminosis, alimentary dystrophy, excess perspiration, minor occupational skin
abrasions, as well as skin irritation caused by chemical substances, are some examples of the conditions conducive
to the formation of pyogenic lesions of the skin and furunculosis.
In some cases staphylococci may give rise to secondary infection in individuals suffering from smallpox,
influenza, and wounds, as well as postoperative suppurations. Staphylococcal sepsis and staphylococcal pneumonia
in children are particularly severe diseases. Ingestion of foodstuffs (cheese, curds, milk, rich cakes and pastry, ice
cream, etc.) contaminated with pathogenic staphylococci may result in food poisoning.
Staphylococci play an essential part in mixed infections, and are found together with streptococci in cases of
wound infections, diphtheria, tuberculosis, actinomycosis, and angina.
The wide use of antibacterial agents, antibiotics in particular, lead to considerable changes in the severity and
degree of the spread of staphylococcal lesions. Growth in the incidence of diseases and intrahospital infections m
obstetrical, surgical and children's in-patient institutions, intensive spread of the causative agent, and increase in the
number of carriers among the medical staff and population have been noted in all countries of the world.
Intrauterme and extrauterine contamination of children with staphylococci has been registered, with the
development of vesiculopustular staphyloderma, pemphigus, infiltrates, abscesses, conjunctivitis, nasopharyngitis,
otitis, pneumonia, and other diseases.
It has been established that staphylococci become adapted rapidly to chemical agents and antibiotics due to
the spread of R-plasmids among these bacteria. The high concentration of drugs in the body of humans and in the
biosphere has resulted in essential disturbance in the microflora and the extensive spread of resistant strains
possessing more manifest virulence. The L-forms of staphylococci are especially marked by increased degree of
resistance to antibiotics.
Table N3 Virulence factors of S. aureus
Virulence factor Activity
Cell wall polymers
Peptidoglican Inhibitor of inflammatory response
Teichoic acid Phage adsorbtion;
Cell surface priteins
Protein A Binds to Fc portion of antibody, inhibiting phagocytosis
(blocks attachment to Fc receptors on white blood cells).
Inhibit phagocytosis of nonopsonized bacteria
Caosules (microcapsules)
Exoproteins
Lysin (alpha,beta, gamma, delta) Impearent of membrane permeability; cytotoxic effects
on phagocytic and tissue cells
Leucocidin Kills white blood cells by producing holes in their
cytoplasmic membrane
Convert fibrinogen to fibrin, may impede progress of
Coagulase leucocytes into infected area by producing clots in the
surrounding capillaries
Enterotoxin Inducing vomiting and diarrhea
Epixdermolic toxin (exfoliation) Separates layers of epidermis, causeng scalded skin
syndrome
Toxic shock syndrom toxin Causes rash, diarrhea, toxic shock (multisystem effect)
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5. Lipase Break down fats by hydrolyzing the bond between
glycerol and fatty acid
Protease Degrade collagen and other tissue proteins
Hyaluronidase Break down hyaluronic acid component of tissue,
thereby promoting extension of infection
Immunity. The tendency to run a chronic flaccid course or relapse is regarded as a characteristic symptom
of staphylococcal infections. This peculiarity gives a basis for concluding that postmfectional immunity following
staphylococcal diseases is of low grade and short duration.
Immunity acquired after staphylococcal diseases is due to phagocytosis and the presence of antibodies
(antitoxins, precipitins, opsonins, and agglutinins).
The inflammation restricts the staphylococci to the site of penetration and obstructs their spreading
throughout the body. At the centre of inflammation the organisms undergo phagocytosis. Neutralization of the
staphylococcal toxin by the antitoxin is an important stage of the immunity complex. As a result of capillary
permeability, the antitoxin penetrates from the blood into the inflammation zone and renders harmless the toxin
produced by staphylococci. Thus, the phagocytic and humoral factors act together and supplement each other.
The presence in the human organs and tissues of antigens which are also common in the staphylococci
(mimicry antigens) is among the causes of low immunity. This causes a state of immunological tolerance to
staphylococci and their toxins, which provides favourable conditions for uninhibited reproduction of the causative
agent in the patient's body. The wide use of antibacterial agents promotes intensive selection of staphylococcal
strains resistant to the natural inhibitors of the microorganism.
Laboratory diagnosis. Test material may be obtained from pus, mucous membrane discharge, sputum,
urine, blood, foodstuffs (cheese, curds, milk, pastry, cakes, cream, etc.), vomit, lavage fluids, and faeces.
The material is examined for the presence of pathogenic staphylococci. Special rules are observed when
collecting the material since non-pathogenic strains are widespread in nature.
Laboratory studies comprise the determination of the main properties of the isolated staphylococci (i. e their
morphologic, cultural and biochemical characteristics), as well as their virulence. For this purpose the following
procedures are carried out. Smears are made from pus and stained by the Gram method. Pus is inoculated onto
blood agar and meat-peptone agar containing crystal violet. In cases of septicaemia blood is inoculated into glucose
broth.The isolated pure culture is tested for its haemolytic (by inoculation onto blood agar plates),
plasmacoagulative (by inoculation into citrated rabbit plasma), and hyaluronidase activities. Virulemce is
determined in rabbits by intracutaneous injection of 400 million microbial cells. Necrosis develops at the site of
injection within 24-48 hours. Pigment production of the isolated culture is also taken into account. For revealing
sources of infection, particularly food poisoning and outbreaks of sepsis in maternity hospitals, serological typing
and phage typing are carried out. To ensure effective therapy the isolated cultures are examined for sensitivity to
antibiotics.
In cases of food poisoning presence of the enterotoxin in th isolated staphylococcal culture is tested for by
intravenous injection of the culture filtrate to adult cats. In cases when intoxication is due to ingestion of the milk
of a cow suffering from mastitis, the culture grown on starch medium is tested directly for toxin production as a
means of detecting staphylococci of animal origin. When the causative agent cannot be detected (osteomyelitis and
other diseases), the patients' serum is tested for agglutinins.
Treatment. Staphylococcal diseases are treated with antibiotics (penicillin, phenoxymethyl penicillin,
tetracycline, gramicidin, etc.), sulphonamides (norsulphazol, sulphazol, etc.), and antistaphylococcal gamma-
globulin.
When treating patients suffering from staphylococcal infections one should bear in mind that it is necessary
to relieve intoxication and improve the immunological defence forces of the body (infusion of glucose, plasma,
blood transfusion, injection of cardiac stimulants).
In cases of chronic staphylococcal lesions specific therapy is recommended: autovaccines, staphylococcal
anatoxin, antitoxic serum, and diphage containing staphylococcal and streptococcal phages.
Staphylococcal produce strains resistant to sulphonamides, antibiotics, and bacteriophage, which advances
the wide distribution of staphylococcal infection. This variability is of particular importance in the therapy of
staphylococcal pyogenic diseases. The medical services produce semisynthetic preparations of penicillin and
tetracycline which are effectively used for the treatment of these diseases.
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6. Prophylaxis. The general precautionary measures include: hygiene in working and everyday-life conditions,
treatment of vitamin deficiency, prevention of traumatism and excess perspiration, observance of rules of hygiene
in maternity hospitals, surgical departments, children's institutions, industrial plants and enterprises, particularly
canneries, observance of personal hygiene and frequent washing of hands in warm water with soap.
Routine disinfection of hospital premises (surgical departments, maternity wards) and bacteriological
examination of the personnel for carriers of pathogenic staphylococci resistant to antibiotics are also necessary.
In some cases specific prophylaxis by means of immunization with the staphylococcal anatoxin may be
recommended for individuals subject to injury or infection with antibiotic-resistant staphyloc
4. Student’s independent study program
1. General characteristics of gram-positive cocci. Structure, staining properties of staphylococci. Cultivation.
2. Antigenic structure of staphylococci. Classification of staphylococci.
3. Fermentative properties and toxin production of staphylococci. Virulence factors of staphylococcus
(toxins, plasmocoagulase, fibrinolysin, hyaluronidase, lecithinase, DNase, proteinase etc).
5. Differentiation of staphylococci.
6. Main clinical forms of staphylococcal infections.
7. Peculiarities of obtaining of tested material and bacteriological examination stages of sepsis, enteritis,
furunculosis and other pyo-inflammatory processes, which are caused by staphylococci.
8. Staphylococcal carriage and hospital infections.
9. Value of staphylococcal phagotyping, procedure’s technique.
10. Active and passive prophylaxis of staphylococcal infection
5. Students’ practical activities:
1. To prepare the smears from pus, to stain them by Gram’s technique.
2. To familiarize with cultivation methods of staphylococci and streptococci (blood MPA, yolk-salt agar,
MPB etc.).
3. To familiarize with the tests for determination of virulence factors of bacteria (plasmocoagulase, DNase,
hyaluronidase etc.).
Coagulase test plasma is clotted by Staphilococcus aureus
Staphilococcus epidermidis Staphilococcus aureus
Plasma
Incubate
370 C
Clot
4. To inoculate a specimen of pus onto yolk-salt agar.
Using your loop, streak your unknown bacteria (pus) for isolation on a plate of yolk-salt agar.
5. To inoculate patient’s blood with suspicion on a sepsis in sugar MPB.
Take a tube with patients‘ blood, open its and flame the edges of a tube. Take a flask with sugar MPB and do
the same actions. Pour a blood into a flask. Sterile the cotton wool plugs and edges of a tube and a flask in the
flame and close them. Sign a flask (type of material, date, no of group).
6. Control questions and tests
Choose the correct statements:
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7. 1. To staphylococci have next properties: a – in smears they occur in pairs; b – Gram-positive; c– form
spores; d – form irregular clusters resembling bunches of grapes; e – Gram-negative.
2. Growth onto nutrient media: a – might grow onto ordinary media; b – there are haemolysis zones on
blood agar around colonies; c – they produce pellicle on the surface of sugar broth; d – grow on media
with bile; e – on blood agar don’t cause hemolysis.
3. Toxin production of staphylococci: a – produce an alpha-, beta- delta-hemolysins; b – don’t produce
enterotoxins; c – produce Hyaluronidase, plasmocoagulase; d – don’t produce an exfoliative toxin; e –
have plasmids.
4. Choose disease, which are caused by staphylococci: a – Rheumatic Fever; b – sepsis; c – phlegmon; d –
Scarlet Fever; e – pyodermia.
5. The bacteriological preparations, which are used for creation active and passive immunity: a –
staphylococcal anatoxin; b – antistaphylococcal human immunoglobulin; c – staphylococcal toxin; d –
antistaphylococcal antitoxic serum; e – staphylococcal autovaccine.
6. Main method of laboratory diagnosis of staphylococcal infections: a – serological method
(precipitation test); b – bacteriological method; c – express method (ELISA); d – allergic method; e –
serological method ( CFT).
7. For revealing a staphylococcal carriage it is necessary: a – to take material from a nasopharynx; b–to
isolate of a pure culture; c-to carry out CFT; d- to carry out phagotyping of Staphylococcus aureus.
7. List of literature:
1. I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, Lugansk, 2004,
chapter15, p.3-11.
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