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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
open this folder and view contentsQuality assurance in microbiology
close this folderPart I. Bacteriological investigations
open this folder and view contentsBlood
open this folder and view contentsCerebrospinal fluid
open this folder and view contentsUrine
open this folder and view contentsStool
open this folder and view contentsLower respiratory tract infections
open this folder and view contentsUpper respiratory tract infections
open this folder and view contentsSexually transmitted diseases
close this folderPurulent exudates, wounds, and abscesses
View the documentIntroduction
View the documentCommonly encountered clinical conditions and the most frequent etiological agents
View the documentCollection and transportation of specimens
View the documentMacroscopic evaluation
View the documentMicroscopic examination
View the documentCulture
View the documentIdentification
View the documentSusceptibility testing
open this folder and view contentsAnaerobic bacteriology
open this folder and view contentsAntimicrobial susceptibility testing
open this folder and view contentsPart II. Essential media and reagents for isolation and identification of clinical pathogens
View the documentSelected further reading
View the documentSelected WHO publications of related interest
View the documentBack Cover
 

Identification

With the exception of contaminants from the environment or from the skin (such as Staphylococcus epidermidis), all organisms isolated from wounds, pus, or exudates should be considered significant and efforts made to identify them. Full identification, however, is not always necessary, particularly in the case of mixed flora.

Bacteria and fungi isolated from pus and exudates may belong to almost any group or species. Identification criteria are given here only for staphylococci commonly associated with pus (“pyogenic”), and for two other pathogens, Pasteurella multocida and Bacillus anthracis, which are rarely isolated from wounds or skin infections, but are very important for the management of the patient. A standard textbook of clinical microbiology should be consulted for a full description of identification methods. In every case, the first step should be to examine well-separated colonies carefully, pick a single colony of each type, prepare a Gram-stained smear, and then characterize the organisms under the microscope.

Staphylococci

Staphylococci are the bacteria most frequently associated with the production of pus. For clinical purposes, staphylococci can be divided into those that produce coagulase and those that do not. The coagulase-producing staphylococci belong to the species S. aureus, which is the species of greatest medical interest. Of the several coagulase-negative species, only two will be considered here - S. epidermidis and S. saprophyticus.

Although S. aureus is part of the commensal microbial flora of the nose (40% of healthy adults are positive), skin, and intestinal tract, this species causes impetigo, boils, abscesses, wound infection, infection of ulcers and bums, osteomyelitis, mastitis (breast abscess), pleural empyema, pyomyositis, toxic shock syndrome, and other types of pyogenic infection.

S. epidermidis is also a common commensal of the skin, nose, and other mucous membranes and possesses a very low pathogenicity. However, its presence in pus should not always be dismissed as skin contamination. Despite its low infectivity, S. epidermidis can cause skin infections at the site of an in-dwelling catheter, cannula, or other device. Infections with S. epidermidis are particularly troublesome in cardiac and orthopaedic surgery involving the insertion of prosthetic devices (artificial heart valves or artificial hips).

S. saprophyticus is recognized to be a common cause of urinary tract infections in young women, being second only to E. coli in some populations.

Staphylococci grow well aerobically on blood agar and form opaque white to cream colonies, 1 - 2 mm in diameter after overnight incubation. They are unique in growing on media with a high salt content, such as MSA. They can be differentiated from streptococci by their morphology and by the production of catalase. Catalase production by staphylococci is shown by picking up a little growth with a capillary tube, placing it in a drop of 3% hydrogen peroxide in the bottom of a small test tube, and closing with a cotton stopper. The appearance of bubbles of oxygen is an indication of catalase production.

Table 11. Differentiation of medically important species of Staphylococcus

 

S. aureus

S. epidermidis

S. saprophyticus

Production of coagulase

+

0

0

Acidification of mannitol on mannitol salt agara

acid (yellow)

neutral (red)

acid (yellow)

Pigment of coloniesa

grey, cream, or yellow

white

white

In vitro susceptibility to novobiocin

susceptible

susceptible

resistantb

 

a Exceptions are possible.
b Inhibition zone of less than 16 mm, using a 5-μg disc in the standardized disc-diffusion method.

The distinctive features of the three main species of Staphylococcus are given in Table 11.

In view of the importance of the coagulase test in the identification of S. aureus, this test is described here in detail. Coagulase is an enzyme that causes plasma to clot. Staphylococcal coagulase exists in two forms: bound coagulase or clumping factor, which is demonstrated in the slide test; and free coagulase, which is demonstrated in the tube test.

 

• Slide test. On a clean slide, emulsify one or a few similar colonies of staphylococci in a drop of saline. The suspension must be fairly thick. Dip a straight wire into plasma and use this to stir the bacterial suspension. Observe for clumping within 10 seconds. False-negative slide tests occur with about 10% of the strains of S. aureus. If the slide test is negative for an isolate that seems to be pathogenic on other grounds (pigment, clinical source), it should be re-examined in the tube test.

• Tube test. Dispense a few drops (0.5 ml) of plasma into a sterile 12 X 75 mm tube, and add two drops of the pure culture in broth. A suspension with an equivalent density may also be prepared directly from growth on blood agar. Incubate the tube at 35 °C for 4 - 18 hours and then examine for clotting.

The plasma used in the coagulase test may be fresh human or rabbit plasma obtained with ethylene diamine tetraacetic acid (EDTA). It should be stored in the refrigerator in small amounts (1 ml), and its performance checked with cultures of S. aureus and S. epidermidis, run in parallel.

Pasteurella multocida

A number of Gram-negative bacilli are transmitted by animal bites and can cause severe infections in human beings, the most prevalent being Pasteurella multocida. P. multocida is a commensal found in the normal mouth flora of many animals. A bite wound infected with P. multocida may give rise to extensive cellulitis, which may extend to a joint, producing arthritis. Osteomyelitis, bacteraemia, and even meningitis have been described.

P. multocida should be looked for specifically in wound discharge if an animal bite is known or suspected of being the cause of the wound. P. multocida is a very small, Gram-negative, nonmotile coccobacillus. It grows well on blood agar at 35 °C but is completely inhibited by the bile salts contained in the enteric selective media, e.g., MacConkey agar. After overnight incubation, the colonies on blood agar are small, nonhaemolytic, translucent, and mucoid (due to presence of a capsule in the virulent form).

Biochemical identification is based on the following characteristics:

 

• fermentation of glucose without gas: P. multocida grows on Kligler iron agar with acidification of the butt;

• oxidase test is usually negative;

• catalase positive;

• nitrate reduced to nitrite (0.1% potassium nitrate in nutrient broth);

• urease negative;

• indole positive - test in broth (TSB) or on MIU after 48 hours incubation;

• highly sensitive to penicillin in the disc-susceptibility test.

Bacillus anthracis

The genus Bacillus is composed of numerous species of aerobic, spore-forming, Gram-positive rods, widely distributed in the soil. The species B. anthracis is of public health importance in skin infections. The other species, when isolated from wounds or pus, are generally contaminants or, at most, opportunists.

B. anthracis is a major pathogen of cattle, sheep, goats, and other domestic animals. It also affects wild animals. Anthrax can affect human beings, particularly people in parts of Africa and Asia working or living in close contact with livestock. Human infection may also derive from animal products containing anthrax spores, such as wool, skins, fur, and bones.

The commonest form of human infection is cutaneous anthrax, which may progress to septicaemia and meningitis. The spore enters through damaged skin and produces a vesicular lesion with a necrotic centre, surrounded by extensive oedema (“malignant pustule”). Large Gram-positive, square-ended, capsulated rods without spores are seen in smears from the vesicular fluid.

B. anthracis grows aerobically. On blood agar it produces large, flat, greyish colonies, up to 5 mm in diameter, with a rough surface texture and irregular edges showing hairy outgrowths (Medusa head). It is important at this stage to differentiate the highly pathogenic B. anthracis from the generally harmless saprophytic species.

The preliminary distinction should be based on the absence of haemolysis, penicillin sensitivity, and the lack of motility in B. anthracis. In contrast, most of the saprophytic Bacillus species are motile and strongly haemolytic. These three features can form the basis of a presumptive identification. For definitive diagnosis, a pure culture of the isolate should be sent immediately to the central veterinary or public health laboratory.

B. anthracis is a highly infectious organism and specimens and cultures should be handled with the greatest care to avoid contamination of the environment and infection of laboratory personnel.

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