NAME:________________________________________ STUDENT NO.

__________________

026-1-MI
237
ACKNOWLEDGEMENTS

AUTHORS

June Macdonald, B.Sc , A R T

REVIEWER

Glen Carmichael, A.R. T.

© 1988 by THE MICHENER INSTITUTE for Applied Health Sciences

222 St. Patrick Street
Toronto, Ontario, Canada
M5T 1V4

1992 Revised Edition

This material has been prepared and developed by The Michener Institute for Applied Health Sciences.
Reproduction of any part of this material, written, audio, visual or electronic, in any form, without the
written consent of The Michener Institute, is forbidden.

Materials copyrighted to The Michener Institute may be purchased from the Bookstore at the Institute, at the
above address.

2761XPC

238
 MICROBIOLOGY

026-1-MI

COLONIAL APPEARANCE OF BACTERIA

TABLE OF CONTENTS

PAGE

OBJECTIVES 2

RESOURCE MATERIAL 3

TECHNIQUES USED FOR COLONY EXAMINATION 4

VISUAL OBSERVATION 4
COLONY CHARACTERISTICS 5
SIZE 5
FORM 6
ELEVATION 6
MARGIN 7
SURFACE 7
CONSISTENCY OR TEXTURE 7
COLOUR 8
DENSITY OR OPACITY 8
ODOUR 8
HEMOLYSIS 9
LIMITATIONS OF COLONIAL RECOGNITION 10
SPECIES VARIATION AND OTHER FACTORS 10
CONTAMINATION 10

SELF- ASSESSMENT 11

026-1-MI -2-

239
OBJECTIVES

The objectives indicate what you should know, understand and be prepared to explain upon completion of
this module. The self-assessment questions and answers will enable you to judge your understanding of the
material.

Upon completion of this module, the student should be able to:

1. state why skill in recognizing bacteria using colonial appearance is important

2. name the three senses usually used for examining bacterial colonies.

3. describe how to examine colonies using transmitted and reflected light.

4. describe the features that can be observed using reflected and transmitted light.

5. list three pieces of equipment used for examining colonies

6. give the size in mm of: pinpoint, small and large colonies

7. describe the factors affecting the size of bacterial colonies.

8. describe what is meant by a colony's form, elevation, margin and surface and give three examples of
each

9. state how a colony's texture may be determined and give three examples of texture.

10. differentiate between colonies which are opaque, translucent and transparent

11. define hemolysis and differentiate between complete and partial hemolysis

12. differentiate between partial hemolysis and nonspecific greening

13. describe the limitations of colonial descriptions including species variation, patient medication and
contamination

026-1-MI -3-

240
 14. name one bacteria genus or species which may give the following characteristics

 Spreading edge or margin

 Mucoid texture
 Golden yellow colour
 Brick-red colour
 Green colour
 Metallic sheen
 Grape or ammonia - like odour
 Mousey odour.

If you have studied this subject previously, you may test your ability using the self-assessment questions. If
you are able to obtain 90% or greater, you may choose not to do the unit and merely review the sections, or
parts of sections, where weakness may exist. If you obtain less than 90%, it is recommended that the module
be done in its entirety, stressing areas where more review is needed.

RESOURCE MATERIAL

This module includes the information necessary to meet the objectives. For the references listed below

[P] designates sources of prerequisite information

MODULES/SUPPLEMENTS DEVELOPED BY THE MICHENER INSTITUTE

[P] 022-1-MI Introduction to Microbiology

[P] 027-1-MI Bacterial Classification and Taxonomy
[P] 025-1-MI Bacterial Physiology

026-1-MI -4-

MICROBIOLOGY

241
 026-1-MI

COLONIAL APPEARANCE OF BACTERIA

When a bacterial cell multiplies to form a colony, the appearance of the colony is often distinctive for that
bacterium. Skill in recognizing bacteria by their appearance is very useful in preliminary identification for it
saves time so critical for patient treatment and as well, money since unnecessary tests are avoided .

A skilled technologist will examine a colony, mentally note several colonial features and give a possible
identification, all within a few seconds.

TECHNIQUES USED FOR COLONY EXAMINATION

The visual sense is used most often but many technologists find that smell is essential and touch often very
helpful.

VISUAL OBSERVATION

The most useful piece of equipment for colony examination is a fluorescent magnifier lamp. Colonies may
be seen with more detail using some type of magnifying lens and the lamp provides the necessary
illumination. A hand lens may also be used.

There are two types of light used to observe colonial features

 Reflected
 Transmitted.

Reflected light is best for examination of colony surface features and colour whereas transmitted light
provides information on the density or transparency of individual colonies. i.e. the ability of colonies to pass
or transmit light, and the type of hemolysis to use transmitted light, place the lamp behind the plate so that
the light passes through the plate.

Both transmitted and reflected light may also be obtained using a plate or stereoscopic microscope. This
microscope provides varying magnifications with appropriate illumination to permit detailed colony
examination.

026-1-MI -5-

COLONY CHARACTERISTICS

242
SIZE

Size is generally the first characteristic noted and is frequently described as

 Pinpoint (< 1 mm)

 Small (1-2 mm)
 Large ( > 2 mm)

Factors Affecting Colony Size

Competition between colonies for limited nutrients often produces smaller colonies than when nutrients are
plentiful. For example, colonies of a pure growth of a single bacterial species will be significantly larger in
the third and forth inoculum areas than those found nearer the main inoculum where cell density (and
therefore competition) is greatest.

Most bacteria grow better and therefore appear most typical when grown on enriched rather than basic
media. As well, media containing inhibitory or selective substances tend to produce smaller colonies.

Finally, incubation conditions also play a major role if agar plates are incubated longer than the usual 18-24
hours, colonies will appear larger than normal. Also, temperatures either lower or higher than the species
optimum will diminish colony size. Similarly, nonoptimal atmospheric growth conditions also affect growth
size. Many bacteria have improved growth with CO2, but the typical conditions for reading colonial
morphology of aerobes and facultatives is ambient air. Facultatives generally have smaller colonies when
incubated anaerobically.

Therefore, colonial morphology should always be described on well isolated colonies on noninhibitory
media incubated for 18-24 hours under the colony's ideal growth conditions.

The descriptive terms used for colony features are given for guidance only; it is more important that the
student forms a mental picture of the colony so that a name may be linked or associated with that image.

026-1-MI -6-

FORM

243
Form is the shape or the general outline of the whole colony. The most typical pathogenic bacterial form is
circular.

ELEVATION

Most commonly isolated pathogenic bacteria are convex or raised, however, one important pathogen is often
described as “checker – like”.

026-1-MI -7-

MARGIN

Margin is the edge or periphery of the colony's form. For example, a dog may be big (form) and hairy
(margin).

244
One commonly seen bacterium, Proteus sp, has a spreading margin which literally advances the colony
margin across the plate surface.

SURFACE

The surface appearance of most pathogenic bacteria is usually smooth but colonies may also be described
as: wet, rough, ground glass, dull, glistening, granular and so on .

CONSISTENCY OR TEXTURE

A colony's texture may be determined by touching its surface with a sterile wire or applicator stick.
Common terms used include:

 Butryous (smooth, butter-like)

 Viscid (mucoid)
 Friable (crumbly, brittle)
 Membranous and so on.

Particularly interesting are colonies which produce abundant capsular material; these colonies form strings
or strands when touched with a wire. Some Klebsiella species. often produces these very mucoid colonies
026-1-MI -8-

COLOUR

Most pathogenic bacteria have rather drab colours, either grays or whites, but a few are distinctive and very
useful for presumptive identification
245
  Staphylococcus aureus - golden yellow
 Serratia marcescens - brick-red
 Pseudomonas aeruginosa - green

Not all strains of these colour-producing species will show colour every time but when seen, colour is very
useful. Colour may also be dependent upon medium constituents and incubation conditions.

Some bacteria may have an iridescent or opalescent surface, often called a "metallic sheen". Many
Pseudomonas aeruginosa strains will be detected by their metallic sheen.

DENSITY OR OPACITY

Colonies may also be characterized by their ability (or lack of) to transmit light. This light transmitting
property is determined by holding the agar plate in front of a light source. Opaque colonies pass no light at
all whereas transparent ones are completely clear, passing not only light but detail. Colonies intermediate
between opaque and transparent are translucent; e.g. ground glass is translucent since light passes but no
detail. Most pathogens are either translucent or opaque.

ODOUR

Technologists with highly developed olfactory skills definitely have an advantage in colonial recognition.
These technologists detect subtle scents which may alert them to an organism's presence even if the colony
has not been seen. However most technologists have no problem detecting the grape-like or ammonia-like
odour of Pseudomonas aeruginosa or the mousy smell of Haemophilus influenzae. (When smelling
colonies only partially remove the petri dish lid)

026-1-MI -9-

HEMOLYSIS

Some bacteria produce toxins which diffuse from the colony during incubation and attacking the red blood
cells in media containing whole blood. This phenomenon is called hemolysis .

There are two types of hemolysis

246
  Complete or beta hemolysis in which the red blood cell is completely lysed and

 Partial or alpha hemolysis in which the cell is only partially broken down

True hemolysis is distinguished by a circular zone produced in the blood agar around the colony. Complete
hemolysis is characterized by a clear, yellow-coloured zone lacking intact whole red blood whereas partial
hemolysis by a greenish zone of partially disintegrated cells.

Some organisms produce a greenish discolouration of the medium which is not hemolysis. This nonspecific
greening can be distinguished from true partial hemolysis because the zone is irregular and not a definite
circle. This media discolouration is not a constant feature and is not noted when describing colonies.

026-1-MI - 10 -

LIMITATIONS OF COLONIAL RECOGNITION

SPECIES VARIATION AND OTHER FACTORS

Not all strains of a species will consistently produce typical colonial appearance. Also, patient medication
may affect colonial features such as colour, texture or size. If in doubt a gram's stain should always be done

247
before inoculating any identification media or doing any other tests. It is very easy to go "off track" and
reach a dead end through failure to check the gram stain of an atypical isolate.

CONTAMINATION

The purpose of colonial recognition is the ultimate identification of organisms causing disease.
Unfortunately, all colonies that grow on patient's plates do not necessarily come from the patient.
Contaminants, introduced either during set-up (inoculation) or during medium preparation, must be
differentiated so they are not described as part of the sample or specimen.

A general rule of thumb is that a single colony on a plate is not described unless the colony appears on a
streak line. As well, a single colony type seemingly markedly different from the rest of the colonies, and
sitting as if it has accidentally fallen on the main inoculum area has probably done just that, is also suspect.

Contaminants occurring during media preparation present other problems. Those which enter the molten
medium appear as biconvex disks within the solidified agar. However, contaminants which settle on the
medium surface can grow into small, difficult-to-see colonies which can be picked up during streaking and
be spread across the plate.

In spite of some limitations, skill with colonial recognition (gained through repeated practice observing
colonies) is a major part of a microbiology technologist’s expertise.

026-1-MI - 11 -

MICROBIOLOGY
026-1-MI
COLONIAL APPEARANCE OF BACTERIA

SELF-ASSESSMENT

248
MARKS

[2] 1. Why should you examine only well isolated colonies when describing colonial recognition?

[3] 2. a) Describe how a plate must be held for viewing colonies with transmitted light.
b) Why should colonies be examined with transmitted light as well as reflected light?

[4] 3. Indicate which of the following factors affecting the growth of a facultative bacterium
produces larger or smaller colonies?

a) anaerobic incubation
b) inoculation on blood agar
c) 48 hour incubation
d) growth near the main or primary inoculum

[7] 4. Match the colonial characteristics in Column A with the bacteria in Column B

Column A Column B

I) spreading margin A) Klebsiella sp

II) brick-red colour B) Pseudomonas aeruginosa
III) grape-like odour C) Proteus sp.
IV) golden colour D) Haemophilus influenzae
V) metallic sheen E) Serratia marcescens
VI) green colour F) Staphylococcus aureus
VII) mucoid or stringy

[4] 5. How can you tell if a colony is a possible contaminant?

[4] 6. What is the visual difference between.

a) complete and partial hemolysis?

b) partial hemolysis and nonspecific greening?

____
24 TOTAL

026-1-MI - 12 -

MICROBIOLOGY
026-1-MI
COLONIAL APPEARANCE OF BACTERIA

SELF-ASSESSMENT ANSWERS

249
1. Colonies demonstrate their most typical size and therefore can be more accurately described if well
iso1ated.

2. a) Held so that the light is behind the plate.

b) Transmitted light permits determination of a colony's hemolysis and the light transmitting
properties.

3. a) smaller
b) larger
c) larger
d) smaller

4. I) C
II) E
III) B
IV) D
V) B
VI) B
VII) A

5. - It is not on the streak lines

- It is sitting on top of the main inoculum
- A biconvex colony in the depth of the medium
- It is part of a section of growth which appears markedly unrelated to the initial inoculum and that
appears to begin anywhere on the plate

6. a) Complete hemolysis has a definite zone of clear yellow-coloured hemolysis whereas partial
hemolysis has a green zone around the colony.
b) Nonspecific greening is not true hemolysis and does not produce a definite zone as is seen with
partial hemolysis.

MICROBIOLOGY
060-1-MI
STAPHYLOCOCCUS

250
 THE MICHENER INSTITUTE for Applied Health Sciences

ACKNOWLEDGEMENTS

Author
John TarBush, B Sc., A.R.T

Contributors And Reviewers

Christine Moore, B Sc., A.R.T

251
© 2000 by THE MICHENER INSTITUTE for Applied Health Sciences
222 St. Patrick Street Toronto, Ontario, Canada
M5T 1V4

Revised 2002

This material has been prepared and developed by The Michener Institute for Applied Health Sciences.
Reproduction of any part of this material, written, audio, visual, or electronic, in any form, without the
written consent of The Michener Institute is forbidden.

Materials copyrighted to The Michener Institute may be purchased from the bookstore at the address above.

DIVISON OF CONTINUING EDUCATION

MICROBIOLOGY

060-1-MI

STAPHYLOCOCCUS

Table of Contents

PAGE

252
OBJECTIVES 1

RESOURCE MATERIAL 2

GLOSSARY 3

TAXONOMY 4

GENERAL CHARACTERISTICS OF THE GENUS 4

NATURAL HABITAT 5

CLINICAL SIGNIFICANCE 5

DISEASES CAUSED BY Staphylococcus aureus 6

Infectious Disease Processes 6
Toxin - Medicated Disease Processes 7

VIRULENCE FACTORS OF Staphylococcus aureus 7

Structural 8
Extracellular Products 8

EPIDEMIOLOGY 9

TREATMENT 10

LABORATORY DIAGNOSIS OF Staphylococcus aureus 11

Specimens 11
Microscopic Appearance 11
Cultural Characteristics 11

IDENTIFICATION 12

SUSCEPTIBILITY TESTING 13
060-1-MI 1 2733

MICROBIOLOGY

OBJECTIVES

The objectives indicate what you should know, understand and be prepared to explain upon completion of
this module.

Upon completion of this module, the student should be able to:

1. Describe the general characteristics of the genus Staphylococcus.

253
 2. State the natural habitat of Staphylococcus aureus in/on the body and the carrier incidence in the
nasopharynx.

3. Briefly describe the skin lesions produced by Staphylococcus aureus.

4. Describe four other infections caused by Staphylococcus aureus.

5. Describe three syndromes produced by Staphylococcus aureus toxins.

6. List three virulence factors associated with Staphylococcus aureus.

7. Briefly state the purpose of epidemolical typing and give examples.

8. Discuss the development of penicillin resistance.

9. Give the clinical significance of Staphylococcus epidermidis and Staphylococcus saprophyticus.

10. Describe the microscopic and cultural appearance ofcStaphylococcus aureus.

11. State the significance of the coagulase test for staphylococcal identification.

060-1-MI 2 2733

RESOURCE MATERIAL

This module includes the information necessary to meet the objectives. For the references listed below:

[P] designates sources of prerequisite informaton.

All others contain additonal information.

REFERENCES

1. Baron, E.J., Medical Microbiology, A Short Course, Wiley-Liss, New York City, N.Y., 1994.

254
 2. Barrow, G.I., Editor, Cowan and Steel's Manual for the Identficaton of Medical Bacteri a, 3rd Edition,
Cambridge University Press, Cambridge, U.K., 1993.

3. Kloss, W.E., Update on Clinical Significance of Coagulase-Negative Staphylococci, Clinical Micro.

Reviews, Vol 7, pages 117-133,1994.

4. Kloos, W.E., Update on Clinical Significance of Coagulase -Negative Staphylococci, Clinicial

Micro. Reviews, Vol. 7, Pages 117 -133, 1994.

5. Lowly, F.D., Staphylococcus aureus Infections, The New England Journal of Medicine, Vol. 339,
No.8, August 1998.

6. Murray, P.R., Editor in Chief, Manual of Clinical Microbiology, 7th Edition, ASM Press, Washington,
D.C., 1999

7. Sherris, John C., editor, Medical Microbiology, An Introduction to Infectious Diseases, Elseiver,
1984.

8. Shulman, Stanford T., Pharr, John, P., Sommers, Herbert M., The Biological and Clinical Basis of
Infectious Diseases, 4th Edition, W.B. Saunders, Toronto, 1992.

9. Volk, W.A., Benjamin, D.C., Kadner, R.J. and Parsons, J. T., Essentials of Medical Microbiology, 4th
Edition, J.P. Lippincott Company, New York, 1991.

MODULES/SUPPLEMENTS DEVELOPED BY THE MICHENER INSTITUTE

[P] 022-1-MI Introduction to Microbiology

[P] 870-071 Factors in the Development of Disease

060-1-MI 3 2733

GLOSSARY

1. Bacteremia: Presence of bacteria in the bloodstream.

2. Commensal: A commensal is an organism that lives in or on another organism (host) without causing
harm to the host.

3. Endocarditis: Inflammation of the valves or the lining of the heart.

4. Enterocolitis:Inflammation of the mucous membranes of the small and large intestines.

5. Enterotoxin: Substance released by an organism that causes damage to intestinal epithelial cells
involved in fluid and electrolyte transport - resulting in diarrhea.

255
 6. Nosocomial: Term describing an infection that develops while a patient is in a health care facility.

7. Opportunistic: When an organism that is normally non-pathogenic has the capacity to produce
disease in a host, the former organism is referred to an "opportunistic"

8. Phagocytosis: Ingestion of one organism by another organism or cell; bacteria is phagocytized by

polymorphonuclear leucocytes, as part of the cellular immune response of a healthy host.

9. Pustule: A raised lesion containing pus.

10. Septicemia: Evidence of acute infection and organ failure related to release of mediators (e.g.
cytokines) into the bloodstream, with or without positive blood culture(s).

11. Symbiosis: The relationship where two organisms living together both benefit from the association.

12. Vesicle: A raised lesion containing serous fluid.

060-1-MI 4 2733

MICROBIOLOGY
060-1-MI
STAPHYLOCOCCUS

TAXONOMY

Recently, there has been considerable upheaval in the classification of catalase-positive, gram-
positive cocci as a result of new nucleic acid identification/typing methods. The most recognized
nomenclature has the genus Staphylococcus belonging to the family Micrococcaceae, along with the
genera, Stomatococcus, Planococcus, and Micrococcus.

There are at least 32 species and 15 subspecies in the genus Staphylococcus.

256
GENERAL CHARACTERISTICS OF THE GENUS

The defining characteristics of Staphylococcus genus are as follows:

 Gram-positive cocci that appear in grape-like clusters, singly, or in pairs or tetrads
 Catalase-positive *
 Usually facultative anaerobes* (energy derived from either aerobic and/or fermentation
pathways)

* Except for catalase-negative S. saccharolyticus and S. aureus subspecies anaerobius which grow
better under anaerobic conditions

Species of the genus Micrococcus are generally considered to be non-pathogenic but can produce
opportunistic infections in immunocompromised patients. Micrococci can be distinguished from
staphylococci based upon Gram stain morphology and glucose fermentation. Micrococcus species have
larger cells than staphylococci and these cells are usually arranged in packets of four (tetrads). Micrococci
are not able to ferment glucose, whereas staphylococci are glucose fermenters. [Refer to module 063-1-MI
for more details concerning the identification of Micrococcaceae.]

060-1-MI 5 2733

NATURAL HABITAT

Staphylococci are found on the skin and mucous membranes of humans. They are a major
component of the normal microflora of humans. They are usually commensals or they exist in a
symbiotic relationship with the host.

Thirty to 50 percent of healthy adults are colonized with S. aureus. Both methicillin-sensitive and
strains of methicillin-resistant S. aureus (MRSA) can be persistent colonizers. Carriage can be much
higher amongst hospital staff; ranging from 50 to 70%.

CLINICAL SIGNIFICANCE

Most staphylococcal infections develop after the breakdown of skin by trauma, inoculation by
needles, or implantation of medical devices (e.g. Hickman catheters).

257
 Certain Staphylococcus species are found frequently as etiologic agents of human infections. These
species include: S. aureus, S. epidermidis, S. haemolyticus. S. lugdunensus, S. warneri, and S.
saprophyticus.

S. aureus is by far the most clinically significant Staphylococcus species. Infections can occur in any
part of the body and are often pyogenic (pus producing). If these infections are left untreated, they
can spread directly to surrounding tissue or infect a number of organs via the blood stream. These
infections will be discussed in more detail in the next section.

S. epidermidis, S. haemolyticus, S. lugdunensis, and S. warneri are coagulase-negative staphylococci

(refer to the IDENTIFICATION section) which can cause a number of nosocomial infections.
Patients infected with these species are often immunocompromised and have had surgery or
intravascular manipulations (eg., implantation of catheters).

S. epidermidis, the most frequently encountered coagulase-negative species, has been linked to
bacteremia, natural (native)/prosthetic valve endocarditis, surgical would infections, and to urinary
tract, cerebrospinal fluid, prosthetic joint, peritoneal dialysis, opthalmologic and intravascular
catheter-related infections.

S. haemolyticus has been implicated in native valve endocarditis, septicemia, peritonitis, urinary tract
infections, and wound, bone and joint infections. S. lugdunensis is capable of causing endocarditis,
urinary tract infections, arthritis, endophthalmitis, and catheter infections. S. warneri has been
implicated in cases of endocarditis, septicemia and osteomyelitis, and catheter infections.
S. saprophyticus can cause urinary tract infections, especially in young sexually active females.

060-1-MI 6 2733

DISEASES CAUSED BY STAPHYLOCOCCUS AUREUS

Diseases caused by S. aureus are either a result of an infectious process or the action of toxins liberated by
the organism upon the host (toxin-mediated).

Infectious Disease Processes

A very common S. aureus infection is the boil or furuncle. A boil often develops after blockage of a hair
follicle or sebaceous gland. The end result is a painful abscess, with a central area of necrosis, which
remains walled-off from surrounding tissue.

Boils often heal spontaneously. However, in some hosts the boil infection may spread to nearby deeper
tissues (subcutaneous) producing a lesion known as a carbuncle. Multiple draining sinuses, leading from
deeper tissue to the skin surface, characterize this type of infection. They are found mainly on the back and
neck. Surgical drainage and antibiotic therapy may be required to eliminate this type of infection.
258
S. aureus may produce a vesicular and/or pustular infection of the face, involving the eyes, nose, lips and
limbs, known as impetigo. This highly contagious skin disease is most commonly associated with children
during warm weather. It is usually caused by S. aureus or Streptococcus pyogenes, alone or in combination.

S. aureus may produce bronchopneumonia with multiple abscesses in immunocompromised patients, the
elderly, and infants under one year of age. Frequently, this type of infection arises in children with measles,
influenza and cystic fibrosis.

Other localized infections include paronychia (infection of the nail bed) and surgical incision, cut/wound
and burn infections.

S. aureus is often found in the blood stream (bacteremia) of immunosuppressed patients, in patients with
abscesses or in patients following surgery or implantation of foreign devices. Bacteremia often leads to
septicemia and a host of other systemic infections.

S. aureus is a leading cause of bone infection or osteomyelitis. Organisms from cutaneous lesions can gain
access to bone via the blood stream. Up to 35% of cases of endocarditis (infection of the valves or lining of
the heart) are due to S. aureus.

060-1-MI 7 2733

Toxin-Mediated Disease

Certain strains of S. aureus release an exfoliative exotoxin into the blood stream, which can attack the
epithelium. Skin is sloughed off in large sheets resulting in a "scalded skin" appearance. This disease,
termed scalded skin syndrome, occurs in children and immunocompromised adults.

Many cases of toxic shock syndrome (TSS) are caused by S. aureus. An abrupt onset, fever, rash, diarrhea,
vomiting and often an irreversible shock leading to death are characteristics of this disease. The disease is
prevalent in young, menstruating females who use certain brands of absorbent tampons. The condition,
however, can occur in men as well as women following localized infections, surgery or insect bites. TSS is
caused by S. aureus strains that produce an exotoxin called toxic shock syndrome toxin 1(TSST-1).

S. aureus is the most common cause of food poisoning in North America. Some Strains are able to grow in
food, producing significant levels of an enterotoxin. Foods most frequently contaminated include custards,
cream desserts, or potato salads containing mayonnaise. If not refrigerated properly, these foods provide a
good medium for bacterial growth.

259
Symptoms of severe diarrhea and vomiting occur two to six hours after ingestion of toxin-containing food.
Staphylococcus aureus food poisoning is regarded as a food intoxication rather than a food infection since
illness is caused by the enterotoxin and not the organism itself. The toxin is heat stable: Consequently,
heating contaminated food does not inactivate the toxin.

VIRULENCE FACTORS OF STAPHYLOCOCCUS AUREUS

S. aureus possesses several properties which contribute to it's disease-producing capability (pathogenicity).
These properties, called virulence factors, are not exhibited by all strains. The following discussion
highlights some of the primary virulence factors.

060-1-MI 8 2733

Structural

Protein A: This protein remains part of the cell wall of S. aureus or is liberated into the environment. It may
interfer with the neutralization action of host antibodies and phagocytosis by polymorphonuclear leucocytes.
Also, this protein can activate complement (eliciting immediate and delayed-type hypersensitivity reactions)
and facilitate adherence of the bacteria to host surfaces.

Capsule: Most strains produce a polysaccharide coating or capsule which may hinder the ingestion of the
organism by polymorphonuclear leucocytes (phagocytosis) and promote the adherence of the organism to
prosthetic devices.

Extracellular Products

Hemolysins: Alpha, beta, delta, and gamma-type hemolysins have been characterized. These proteins are
toxic to a wide variety of host cell types, including polymorphonuclear leucocytes, macrophages and
platelets. Alpha-hemolysin can cause tissue necrosis and the lysis of erythrocytes. This lytic action versus
erythrocytes is seen in the laboratory as the zone of clearing around colonies of S. aureus grown on sheep
260
blood agar. The delta-hemolysin can activate enzyme(s) in the intestinal mucosa that result in the outpouring
of fluids into the intestinal lumen (diarrhea).

Enzymes: Both free and bound coagulase is thought to interfere with the neutralizing action of host
antibodies and leucocyte phagocytosis. Lipase, hyaluronidase, and protease are enzymes which break
down various tissue components; thereby facilitating the spread of the organism. At least 3 different types of
beta-lactamases are produced by S. aureus, which can render the organism resistant to penicillin and
ampicillin.

Toxins: Leucocidin is toxic to polymorphonuclear leucocytes. Enterotoxins A though E are heat-stable

proteins that are responsible for the clinical features of staphylococcal food poisoning or enterocolitis. They
inhibit water absorption in the bowel (causing diarrhea), induce vomiting and are toxic to cells of intestinal
mucosa. Exfoliatins have the ability to dissolve components in the epidermal layer, resulting in the "scalded
skin syndrome" Toxic shock syndrome toxin 1 (TSST-1) is responsible for the clinical features of toxic
shock syndrome.

Despite having this arsenal of virulance factors, S. aureus is not usually a threat to individuals with a normal
immune system. This is evident from the fact that there is a high carriage rate in the general healthy
population.

060-1-MI 9 2733

EPIDEMIOLOGY

Antibiotic-resistant strains (i.e. resistant to one or more classes of antibiotics) are an infection control issue
in acute and long-term care facilities. Methicillin-resistant S. aureus (MRSA) can cause nosocomial
infections that are difficult to treat with current antibiotics. Many health care facilities adopt infection
control measures (e.g. patient isolation) to prevent the acquisition or spread of MRSA in the patient
population.

Health care workers have instigated many MRSA outbreaks by shedding organisms from colonized body
sites (i.e. nose or throat) into the patient environment or by direct contamination of patients due to poor
handwashing technique. It is imperative that health care workers wash hands between patient visits and
cover open sores that may shed organisms.

In the course of investigating an outbreak of S. aureus or MRSA (methicillin-resistant S. aureus) in a health

care facility, infection control practitioners may request that multiple isolates from multiple patients be
tested to determine if they are epidemiologically related or are unrelated (different strains). It may be
necessary to go beyond colonial morphology, biochemical tests results (biotype) or the antibiotic

261
susceptibility profile (antibiogram) to determine if isolates are descendants of the same strain. There are a
wide range of typing methods to choose from.

One method, called phage typing, uses viruses that are capable of infecting and lysing bacterial cells. These
viruses are called bacteriophages. Certain species of bacteria (e.g. S. aureus) can be characterized by their
patterns of resistance or susceptibility to a standard set of bacteriophages.

Until recently, phage typing was the authoritative system for typing S. aureus. However, due to the technical
demands of this technique and its relatively poor reproducibility, most laboratorians have switched to the
newer DNA-based methods such as plasmid analysis, pulsed-field gel electrophoresis analysis, and
restriction fragment length polymorphisms analysis.

060-1-MI 10 2733

TREATMENT

When penicillin was first used in the early 1940's almost all strains of S. aureus were penicillin-susceptible.
By the early 1960's, many strains were becoming penicillin-resistant via the enzyme -penicillinase (one type
of beta-lactamase). Today, more than 90% of isolates in Canada are resistant to penicillin.

Emergence of penicillin resistance prompted scintists to develop semi-synthetic penicillins, such as oxacillin
and methicillin, which are resistant to the action of penicillinases. Within a few years of their inception,
strains began to emerge worldwide which were methicillin-resistant. Today, methicillin-resistant S. aureus
(MRSA) occurs in acute and long-term care facilities. It should be noted that many coagulase-negative
staphylococci (CNS) are also methicillin-resistant.

Resistance to methicillin confers resistance to all beta-lactam antibiotics, including: all penicillins,
cephalosporins, beta-lactam / beta-lactamase inhibitor combinations (e.g. amoxicillin-clavulanic acid), and
imipenem. Also, many MRSAs and methicillin-resistant CNS are resistant to one or more classes of
antibiotics, such as the quinolones, aminoglycosides, and sulfonamides. To successfully treat MRSA and
methicillin-resistant CNS infections, vancomycin is usually the drug of choice; an antibiotic which is
relatively expensive and is associated with significant side effects. To eradicate MRSA from a colonized
patient, a combination of antibiotics may be used.
262
Lesion drainage, debridement (surgical removal of tissue), and removal of implanted devices are also part
specific treatment strategies.

060-1-MI 11 2733

LABORATORY DIAGNOSIS OF Staphylococcus aureus

Specimens

Specimens from almost any site may yield Staphylococcus aureus in culture. Clinical significance is
highly correlated with the recovery of this species from suppurative lesions (eg. abscesses, boils, and
infected surgical incisions).

Microscopic Appearance

Staphylococcus aureus is seen in specimens as gram-positive cocci arranged in clusters in association

with pus cells (Figure 1).

Figure 1 Direct Gram Stain Of Specimen containing S. aureus

263
Cultural Characteristics

Staphylococcus aureus grows rapidly at 37°C producing a colony 2-3mm in diameter on blood agar
after 24 hours of incubation. Colonies may be golden coloured and βeta-hemolytic on blood agar. Often they
show neither a golden colour nor hemolysis. Many of the MRSA strains are grey and non-hemolytic

Figure 2 Colonies or S. aureus

060-1-MI 12 2733

IDENTIFICATION

There are a number of key conventional tests used to distinguish S. aureus from other staphylococcal species
seen in human infections:

264
The detection of coagulase production continues to be the most widely used and accepted test for the
preliminary identification of staphylococci. S. aureus is the coagulase-positive species associated with
human infection. Other coagulase-positive staphylococcal species exist in nature (S. intermedius, S. hytcus
and S. scheiferi) but they are rarely isolated from clinical specimens.

Two different versions of the coagulase test can be performed: the tube test and the slide test. Plasma is
required for both tests. Rabbit plasma containing EDTA (an anticoagulant) is widely used.

To perform the tube test, the test organism is suspended in a small volume of plasma contained in a test tube,
then incubated. If coagulase is present a fibrin clot will be formed. Any degree of clotting constitutes a
positive test. The top and bottom tubes in Figure 3 show a positive and negative endpoint, respectively. This
procedure is capable of detecting free coagulase or enzyme released into the environment.

The slide test is performed by making a heavy uniform suspension of the test organism in distilled water,
adding 1 drop of plasma, and watching for clumping or agglutination. This test detects only "clumping
factor". Approximately 10 to 15% of S. aureus strains may yield a negative result because they lack bound
coagulase. Consequently, all slide "negatives" must be confirmed by the tube test. Strains deficient in
clumping factor will usually produce free coagulase resulting in a positive tube test.

060-1-MI 13 2733

Figure 3: Coagulase Test

265
SUSCEPTIBILITY TESTING

The National Committee for Clinical Laboratory Standards (NCCLS) publishes up-to-date
guidelines, which outline which antibiotics to test and report for staphylococcal isolates.

Susceptibility testing of Staphylococci can be accomplished using manual methods: agar dilution,
broth dilution and disk diffusion. In addition, a number of automated or semi-automated systems
exist (e.g. Vitek, Microscan).

Special procedures are used to detect methicillin resistance and beta-lactamase production in S.
aureus and coagulase-negative staphylococci.

266