In this article we will discuss about Staphylococci:- 1. Meaning of Staphylococci 2. Morphology and Staining of Staphylococci 3. Cultural Characteristics 4. Biochemical Reactions 5. Antigenic Structure 6. Toxins and Enzymes 7. Pathogenicity and Clinical Features 8. Laboratory Diagnosis 9. Phage Typing Technique 10. Control Measures.
Contents:
- Meaning of Staphylococci
- Morphology and Staining of Staphylococci
- Cultural Characteristics of Staphylococci
- Biochemical Reactions of Staphylococci
- Antigenic Structure of Staphylococci
- Toxins and Enzymes
- Pathogenicity and Clinical Features of Staphylococci
- Laboratory Diagnosis of Staphylococci
- Phage Typing Technique
- Control Measures of Staphylococci
1. Meaning of Staphylococci:
The Staphylococci are Gram-positive cells arranged in grape like clusters, cause suppuration, abscess formation, pyogenic infection, osteomyelitis, septicaemia, toxic shock syndrome (TSS), food poisoning. There are about 20 species of Staphylococci.
2. Morphology and Staining of Control Measures of Staphylococci:
Staphylococci (Fig. 23.1) are spherical, 1µ in diameter, arranged in clusters. Single cocci, pairs, tetrads and chains are also seen in liquid cultures. Young cocci are stained strongly Gram-positive, old ones may become Gram-negative. They are non-motile and do not form spores.
3. Cultural Characteristics of Staphylococci:
They grow readily on most of the bacteriological media under aerobic or microaerophilic condition at 37°C but form pigment at room temperature (20-25°C). The pigment is believed to be a liporprotein allied to carotene.
Staph, aureus forms deep golden-yellow colonies; Staph, albus colonies are white, and those of Staph, citreus are lemon-yellow. On milk agar the pigment is intense. No pigment is produced anaerobically or in broth. On nutrient agar slant, their growth presents “oil paint” appearance.
On blood agar, most strains are haemolytic and produce B type haemolysis. Similar pink colonies are seen on MacConkey’s medium (Fig. 5.14) due to lactose fermentation. For primary isolation, sheep blood agar is recommended rather than human blood agar as human blood contains antibodies of inhibitors.
4. Biochemical Reactions of Staphylococci:
Staphylococci ferment a number of sugars with acid only. Staph aureus ferments mannitol which is of diagnostic value to differentiate from Staph, albus. It is catalase positive unlike Streptococci, hydrolyses urea, reduces nitrate to nitrite, liquefies gelatin and Methyl Red (MR) and Voges Proskauer (VP) positive but Indole (I) negative.
When grown on media containing egg yolk, Staph, aureus produces dense opaque colonies as they are proteolytic. By culturing on nutrient agar containing phenolphthalein diphosphate, the production of phosphatase can be demonstrated by exposing this culture to ammonia vapour.
The colonies assume a bright pink colour due to presence of free phenolphthalein. This phosphatase test can distinguish Staph, aureus from Staph, albus in mixed culture.
Pathogenic Staph, aureus:
(1) Coagulase positive,
(2) Ferments mannitol,
(3) Produces phosphatase,
(4) Liquefies gelatin,
(5) Produces β-haemolysis on blood agar,
(6) Produces golden-yellow pigment on nutrient agar,
(7) Produces black colonies on medium containing potassium tellurite by reducing potassium tellurite.
5. Antigenic Structure of Staphylococci:
Staphylococci contain peptidoglycan, a polysaccharide polymer, which produces the rigidity to the cell wall and is responsible for the pathogenesis of infection and when this peptidoglycan is linked with teichoic acid, it is antigenic. Cell wall protein A with attached IgG molecule directed against a specific antigen will agglutinate bacteria that have that antigen. This “Coagglutination” test is of diagnostic value.
6. Toxins and Enzymes Produced by Staphylococci:
Staphylococci produce enzymes and toxins.
(a) Catalase:
It is detected by its ability to convert hydrogen peroxide (H2O2) into water and oxygen.
(b) Coagulase:
Pathogenic Staph, aureus produces coagulase, an enzyme-like protein that clots oxalated or citrated human or rabbit plasma in presence of Coagulase Reacting Factor (CRF) contained in plasma. CRF does not occur in other species (e.g. guinea pig). Coagulase is of two types—Bound, or Free Coagulase.
(c) Other enzymes:
Hyaluronidase (Spreading factor), staphylokinase, proteinase, lipase, β-lactamase.
(d) Erotoxin:
Staphylococcal toxins are of three types:
(a) Cytotoxic toxins (haemolysin and leucocidin);
(b) Enterotoxin;
(c) Exfoliative toxin.
a. Haemolysins:
Haemolysin is responsible for pathogenicity; β-haemolysin is haemolytic to sheep cells and exhibits a hot cold phenomenon; Lysis is initiated at 37°C and is evident only on chilling; gamma lysin—the weakest—acts on human, sheep, rabbit erythrocytes; delta lysin is lytic to human cells; alpha and delta lysin are found in human strains of Staphylococci; while beta lysin is from strain of bovine origin.
Leucocidin is lethal to white blood cells.
b. Enterotoxin:
There are six soluble enterotoxins (A-F)—produced by Staph, aureus. Ingestion of 25 µg of preformed enterotoxin B causes food poisoning-nausea, vomiting, diarrhoea within six hours. Toxic shock syndrome toxin 1 (TSST-1)—which is same as enterotoxin F and pyrogenic exotoxin C—is associated with fever, shock, and multisystem involvement including a desquamative skin rash.
c. Exfoliative Toxin:
Exfoliative toxin produces generalized desquamation of the staphylococcal scaled skin syndrome.
7. Pathogenicity and Clinical Features of Staphylococci:
Staphylococci cause pyogenic lesions in man; furuncles, stye, boils, abscess, carbuncle, impetigo, pemphigus, neonatorum, sepsis in wound, burn, breast abscess, nosocomial infection, deep infection (osteomyelitis, tonsillitis) pharyngitis, sinusitis, pneumonia).
Staphylococcal food poisoning results due to consumption of preformed toxin elaborated by Staph, aureus on contaminated food-nausea, vomiting, diarrhoea. In bullous impetigo, pemphigus neonatorum, Ritter’s disease, toxic epidermal necrolysis, there is stripping of superficial layers of skin.
In TSS, there is multisystem/illness characterised by acute onset of high fever, hypotension, vomiting, diarrhoea and rash with desquamation. Severe cases may lead to acute renal failure. It is associated with the use of tampon of menstruating women.
8. Laboratory Diagnosis of Staphylococci:
(a) Specimens:
Surface swab, blood, tracheal aspirate or spinal fluid for culture depending upon the localisation of the process.
(b) Smear:
Typical staphylococci are seen in stained smears of pus or sputum. It is not possible to distinguish morphologically Staph, aureus from other Staphylococci.
(c) Culture:
Specimens planted on blood agar plates give rise to typical colonies in 18 hours at 37°C, but haemolysis and pigment production may not occur until several days later and are optimal at room temperature. Specimens contaminated with bacterial flora can be cultured on media containing 10% NaCl; the salt inhibits most other normal flora but not Staph, aureus.
(d) Catalase Test:
It is carried out by placing one drop of H2O2 solution on a slide and mixing it with a small amount of bacterial growth. The formation of bubbles (the release of O2) indicates positive test.
(e) Coagulase Test may be of Two Types:
(i) Slide test;
(ii) Tube test.
(i) Slide Test:
Detects “bound” coagulase. From an agar plate, the organism is emulsified in two drops of saline placed on a slide. If no spontaneous agglutination, a drop of undiluted human or rabbit plasma is added to one of the emulsions and mixed gently. Prompt clumping indicates the presence of bound coagulase. False positive results are caused by citrate utilizing bacteria, enterococci, pseudomonas.
(ii) Tube Test:
Citrated rabbit (or human) plasma diluted 1: 5 is mixed with an equal amount of broth culture and incubated at 37°C. A tube of plasma mixed with sterile broth is included as a control. If clots form in 1-4 hours, the test is positive and detects free coagulase liberated by pathogenic Staph, aureus.
(f) Serological and Phage Typing Tests:
The serology has little practical value; the phage typing is used for epidemiological study.
Bacteriophage Typing:
Staph, aureus strains are distinguished by their susceptibility pattern to lysis by different phages. A set of over 28 bacteriophages are employed, most strains are lysed by more than one phage.
Thus, several hundred phages of Staph, aureus can be identified. The phages are divided into five groups:
Group I 29, 52, 52 A, 79, 80
Group II 3A, 3B, 3C, 55, 71
Group III 6, 7, 42E, 47, 53, 54, 75, 77, 83A, 84, 85
Group IV 42D
Group V 94, 96
9. Phage Typing Technique:
The strain of staph, aureus to be tested is inoculated on a nutrient agar plate to produce a “Lawn” culture by a four hour growth and the plate is dried. Then a set of over 28 phages are applied over marked areas in a routine dilution (RTD) in a quantity of 0.2 ml each. The plate is incubated overnight.
The culture will be seen to be lysed by a phage or a number of phages denoting the phage type of the particular strain of staph, aureus. Phage types are designated according to the phages capable of lysing the bacterial strain. The phage types prevalent in most parts of India are 52/52 A/80/81.
10. Control Measures of Staphylococci:
1. Source isolation.
2. Detection of staphylococcal lesion and carriage among doctor, nurses and other staff and their isolation and treatment.
3. Sterilisation of instruments and disinfection of the environment.
4. Regular use of antiseptics of skin and wounds of patients and the hands of nursing staff.
5. Judicious use of antibiotics.
Staph, Aureus:
It is usually sensitive to flucloxacillin, erythromycin, lincomycin, vancomycin and cephalosporin’s and many develop rapidly drug resistance. Eighty per cent of the hospital strains are at present penicillin resistant. It also shows multiple drug resistance and chronic infection should be treated by more than one drug.
Penicillin Resistance:
It is due to beta lactamase production which is plasmid-coded and transmitted by transduction.
Multiple Drug Resistance:
Several drug resistance genes present in a single plasmid are responsible.
Treatment:
As drug resistance is so common among Staphylococci, the appropriate antibiotic should be chosen by antibiotic sensitivity test.