MICROBIOLOGY

066-1-MI
FASTIDIOUS GRAM-NEGATIVE
BACTERIA

THE MICHENER INSTITUTE for Applied Health Sciences

194
Acknowledgements

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

Contributors And Reviewers

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

© 2000 by THE MICHENER INSTITUTE for Applied Health Sciences

222 St. Patrick Street
Toronto, Ontario, Canada
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Revised 2002

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 MICROBIOLOGY
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FASTIDIOUS GRAM-NEGATIVE BACTERIA

Table Of Contents

PAGE
OBJECTIVES 1
Haemophilus 1
Bordetella 2
Legionella 3
Brucella 3
Pasteurella 3
Francisella 4

RESOURCE MATERIAL 5

INSTRUCTIONAL MODULES DEVELOPED BY THE MICHENER

INSTITUTE 5

REFERENCES 5

INTRODUCTION 7

A. Haemophilus 9
1. TAXONOMY 9
2. GENERAL CHARACTERISTICS OF THE GENUS 9
3. NATURAL HABITAT 9
4. CLINICAL SIGNIFICANCE 10
5. CULTURE AND ISOLATION 13
6. LABORATORY DIAGNOSIS 14
Direct Antigen Testing 15
X And V Factor Requirements 16
Porphyrin Test 18
Other Tests 18
7. TREATMENT 19
8. SUSCEPTIBIILITY TESTING 19

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 B. Bordetella 21
1. TAXONOMY 21
2. GENERAL CHARACTERISTICS OF THE GENUS 21
3. NATURAL HABITAT 21
4. CLINICAL SIGNIFICANCE 21
5. LABORATORY DIAGNOSIS 23
6. TREATMENT 27
7. SUSCEPTIBILITY TESTING 27

C. Legionella 28
1. TAXONOMY 28
2. GENERAL CHARACTERISTICS OF THE GENUS 28
3. NATURAL HABITAT 28
4. CLINICAL SIGNIFICANCE 29
5. LABORATORY DIAGNOSIS 30
Specimens - Collection and Handling 30
Direct Detection 31
Culture 31
Serological Tests 32
6. TREATMENT 32
7. SUSCEPTIBILITY TESTING 32

D. Brucella 33

E. Pasteurella 36

F. Francisella 37

APPENDIX A - OTHER FASTIDIOUS GRAM-NEGATIVE BACTERIA 40

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OBJECTIVES

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

1. Describe the fastidious growth requirements of the genera in this module by noting whether they are
able to grow on blood or MacConkey agars or whether they require special growth media.

Haemophilus

2. Recognize the hmnan species in the genus - Haemophilus.

3. State the basic characteristics of the genus - Haemophilus.

4. Describe what is meant by typeable and non-typeable strains of H. influenzae.

5. Discuss the natural habitat of members of the genus - Haemophilus.

6. State the primary virulence factor possessed by H. influenzae.

7. Name the two main infections caused by H. influenzae serotype b in children under 5 prior to the
development of H. influenzae serotype b vaccine.

8. Name 5 diseases caused by H. influenzae.

9. Name the growth factors required by various Haemophilus species. Explain the terms X and V
factors. Describe why sheep blood agar does not support good growth of H. influenzae. State why
chocolate agar is adequate for the growth of all Haemophilus species.

10. State the meaning and value of the "Staph streak". Describe what is meant by "satellitism."

11. Describe the atmospheric conditions ideal for the growth of Haemophilus species.

12. Describe the colonial characteristics of H. influenzae.

13. Outline the pros and cons of Direct Antigen Tests (DATs).

14. Explain how X, V, and XV discs are used to identify Haemophilus species. Describe the pattern of
growth for H. influenzae around these discs.

15. Discuss techniques that enhance the reliability of X and V tests.

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16. Explain the theory behind the porphyrin test. Describe the results expected for H. ifluenzae. State
why this test is considered to be more reliable than X factor determination by X and V testnig.

17. Name an antibiotic that is used to treat meningitis caused by H. influenzae.

18. Name the enzyme produced by some strains of H. influenzae that can inactivate ampicillin.

19. Identify various Haemophilus species based upon X and V, porphyrin, beta-hemolysis and other test
results.

Bordetella

20. Recognize the human species in the genus - Bordetella

21. State the basic characteristics of the genus - Bordetella

22. State the natural habitat of B. pertussis

23. Discuss the following details regarding the disease pertussis:

 Name the species most frequently responsible
 Give the common name for this disease
 State how the disease is transmitted
 Describe the 3 stages of infection
 Describe the prevalence in Canada

24. Discuss the following details regarding the diagnosis/management of pertussis:

 Describe the appropriate specimens
 State the pros and cons of DFA
 Name the test that is considered to be the gold standard for pertussis diagnosis
 Give the estimated sensitivity of culture
 Describe the various media types used for isolation of B. pertussis
 Describe the incubation requirements for cultue plates
 Describe the colonial appearance of B. pertussis on BG medium
 Describe the Gram stain characteristics of B. pertussis
 Recognize various tests used to confirm identification of cultue growth
 Name the antibiotic of choice for treatment

Legionella

25. State the basic characteristics of the genus, Legionella.

26. Describe the natural habitat for Legionella species.

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27. Describe the types of infections caused by Legionella species and how they are acquired.

28. Discuss the collection and handling of lower respiratory tract specimens for legionellosis.

29. Name 3 laboratory methods used to diagnose legionellosis. State which method is the gold standard
for diagnosis.

30. Describe diagnosis by culture in terms of media of choice, atmospheric growth requirements, and
colonial morphology of Legionella species.

31. Name the antibiotic of choice for treatment of legionellosis.

Brucella

32. Name 4 species of Brucella and their preferred host.

33. State the basic characteristics of the genus, Brucella.

34. Discuss the following details regarding the disease brucellosis:

 State the various routes of entry into the human host
 Give the risk factors
 State the two strategies that have reduced the incidence
 Name two methods used to diagnose brucellosis

35. Discuss culture of Brucella species from blood specimens.

36. Discuss the risks associated with working with Brucella species. Describe the safety measures
recommended in the laboratory when working with these organisms.

Pasteurella

37. Name the most clinically significant species in the genus, Pasteurella.

38. Describe the natural habitat for Pasteurella species.

39. Describe the 3 general types of disease caused by P. multocida. State the one common risk factor for
these types of infections.

40. Describe the colonial characteristics of P. multocida.

41. State antibiotic of choice for treatment of P. multocida infections.

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Francisella

42. Name the most clinically significant species in the genus, Francisella.

43. Describe the natural habitat for Francisella species.

44. Discuss the primary routes of entry into the human host. State the name of the infection caused by F.
tularensis.

45. Describe the most common presentation of tularemia.

46. Discuss the risks associated with working with Francisella species. Describe the safety measures
recommended in the laboratory when working with these organisms.

47. State the methods used to diagnose tularemia. Describe the difficulties in culturing F. tularensis.

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RESOURCE MATERIAL

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

INSTRUCTIONAL MODULES DEVELOPED BY THE MICHENER INSTITUTE

REFERENCES

1. Askari, S., The Changing Epidemiology of Bacterial Meningitis: Implications for the Clinical
Laboratory, Clinical Microbiology Newsletter, Vol. 20, No.5, March 1, 1998.

2. Balows, A. (Editor), Laboratory Diagnosis of Infectious Diseases, Principles and Practices,

Springer-Verlag, New York, 1988.

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

4. Bisgard, K.. M., Haemophilus influenzae Invasive Disease in the United States, 1994-1995: Near
Disappearance of a Vaccine-Preventable Childhood Disease, Emerging Infectious Diseases, Vol. 4,
No. 2, April-June 1998. [http://www.cdc.gov/ncidod/EID/vol4no2/bisgard.htm

5. Canada Communicable Disease Report, Notifiable Diseases Annual Summary, Supplement, Health
Canada, Vol. 25S6, August 1999

6. Forbes B.A., Bailey and Scott's Diagnostic Microbiology, 10th Edition, Mosby Inc., St. Louis,
Missouri, 1998.

7. Isada, C. M., Infectious Diseases Handbook, 3rd Edition, Lexi-Comp Inc., Hudson, Ohio, 1999

8. Koneman E.W., Color Atlas and Textbook of Diagnostic Microbiology, 5th Edition, Lippincott,
Philadelphia, 1997

9. Lennette, E. H., Editor in Chief, Manual of Clinical Microbiology, 4th Edition, ASM Press,
Washington D.C., 1985.

10. Mohammadkhani, M., A 29-year-old Man with Haemophilus influenzae in the Cerebrospinal Fluid:
Case Report and Review of the Organism, Clinical Microbiology Newsletter, Vol. 20, No.5, March
1, 1998.

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

12. NCCLS M7-A5, Vol. 20, No.2, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
that Grow Aerobically; Approved Standard -Fifth Edition, January 2000.

13. Rothrock, G., et al, Progress Toward Eliminating Haemophilus influenzae Type b Disease Among
Infants and Children - United States, 1987 -1997, Morbidity and Mortality Weekly Report, Vol. 47,
No. 46, November 27,1998.

14. Sciberras, J., Invasive Haemophilus influenzae serotype b Surveillance Documenting the
Progression Towards Elimination, PHERO, Vol. 10, No.1, Ministry of Health, Ontario, January
29,1999

15. The Sanford Guide to Antimicrobial Therapy, 28th Edition, Antimicrobial Therapy Inc., Vienna,
Virginia, 1998.

16. York, K., Abbreviated, Rapid Identification of Bacteria and Yeasts, Clinical Microbiology
Newsletter, Vol. 21, No. 10, May 15, 1999

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MICROBIOLOGY
066-1-MI
FASTIDIOUS GRAM-NEGATIVE BACTERIA

INTRODUCTION

Many gram-negative bacilli, which have the potential to cause serious infections in humans, have unusual
or fastidious growth requirements. Unlike the species/genera found in the family, Enterobacteriaceae, these
organisms are usually unable to grow on media such as MacConkey. Some will not even grow on sheep
blood agar. To recover many of these organisms from clinical specimens, organism-specific media must be
used.

The hallmark of these fastidious bactena is their slow rate of growth. Their detection in blood cultures may
require several days or even weeks of incubation.

Diagnosis of these organisms is often impossible without good communication between the clinician and
the laboratory. For example, a febrile disease (marked by fever) with a history of animal handling is
suggestive of brucellosis (infection caused by Brucella species). When provided with this information, the
laboratory is able to conduct the appropriate tests to rule out Brucella and other infectious agents associated
with the same clinical picture. Without specific clinical information, specimens will be subjected to routine
processing protocols, resulting in the use of inappropriate culture media and incubation conditions. These
fastidious organisms will then slip through the laboratory undetected.

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The genera discussed in this module include Haemophilus, Bordetella, Legionella, Brucella, Pasteurella,
and Francisella. The basic growth requirements of these genera are displayed in Table 1.

\
TABLE 1
SUMMARY OF GROWTH CHARACTERISTICS OF
FASTIDIOUS GRAM-NEGATIVE BACTERIA

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A. Haemophilus

1. TAXONOMY

Eight human species and a number of animal species comprise the genus - Haemophilus.
Species encountered in clinical specimens include H.influenzae, H. parainfluenzae, H.
haemolyticus, H. parahaemolyticus, H. aphrophilus, H. paraphrophilus, H. segnis, and H.
ducreyi. Because H.influenzae is the most clinically significant species, it will be discussed
in greater detail in the following sections.

Strains of H.influenzae which cause serious infections, usually have a capsule. Based upon
serotyping, encapsulated strains can be placed into one of 6 serotypes ("a" through "f”) and
are known as "typeable" strains. Strains, which lack a capsule, are referred to as "non-
typeable" strains.

2. GENERAL CHARACTERISTICS OF THE GENUS

Haemophilus species are small, pleomorphic (variable cellular shape) gram-negative bacilli
that require specific growth factors for growth. Because blood provides a ready supply of
these factors, the name Haemophilus was chosen as the genus name -meaning ''blood-loving''
in Greek.

Some species require one or more compounds found in Iron -containing pigments (eg. hemin
and hematin). These compounds, grouped under the name X factor, are required for the
synthesis of various metabolic enzymes (e.g. catalases and the peroxidases). X factor is
readily available in blood agar. Most Haemophilus species also require nicotinamide adenine
dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) -known in the
laboratory as V factor.

The majority of Haemophilus species need a humid environment with increased CO2 for
optimal growth. Generally, they are able to grow aerobically and anaerobically.

3. NATURAL HABITAT

Most Haemophilus species are part of the normal flora of the upper respiratory tract of
humans and other animals. Some species have the ability to colonize the gastrointestinal and
urogenital tracts.

H. influenzae is found as normal flora in the pharynx of 80% of the human population. Most
strains in the upper respiratory tract of healthy people are non-typeable (i.e. non-
encapsulated). However, capsular strains can be found in healthy people also.

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Approximately 5% of the healthy population harbour strains of H.influenzae type b, the most
pathogenic serotype, with no ill consequences. Colonization with type b stains can be as high
as 60% amongst children attending day care centres.

4. CLINICAL SIGNIFICANCE

Haemophilus species can cause a number of infections in humans (Table 2), ranging from
those that are uncomplicated (e.g. conjunctivitis, sinusitis, and otitis media) to those which
are associated with significant morbidity and mortality (e.g. meningitis, pericarditis, and
endocarditis).

Species associated with human infection include H. influenzae, H. parainfluenzae, H.

aphrophilus, H. paraphrophilus, and H. ducreyi. H. ducreyi causes chancroid, a sexually
transmitted disease characterized by genital ulcers. Haemophilus species other than H.
influenzae are capable of causing serious infections (e.g. endocarditis), but these infections
are relatively rare.

A polysaccharide capsule is the primary virulence factor behind the pathogenicity of H.

influenzae. Like capsules in other species, it enables the organism to resist phagocytosis by
polymorphonuclear leucocytes and neutralization by host antibodies.

Infections due to H. influenzae can be acquired from an external source (exogenous) or from
within the same host (endogenous). Airborne droplets or contaminated hands are the primary
vehicles of spread from external sources. Most children come in contact with serotype b this
way. By the age of 5, exposed children usually have protective antibodies against this
serotype.

Nasopharyngeal carriers of H. influenzae can succumb to disease after a breakdown of

normal host defenses (endogenous origin). Vulnerability, in the case of children, is usually
seen as the lack of protective antibodies against the encapsulated serotypes (notably serotype
b). Adults with an underlying disease (e.g. alcoholism, cancer, AIDS, and chronic obstructive
pulmonary disease) are also prone to serious invasive infections.

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Just over a decade ago, H. influenzae serotype b was the leading cause of bacterial
meningitis and epiglottitis in children under 5 years of age. It was also a major cause of
pneumonia, septic arthritis, osteomyelitis, and pericarditis in the same age group. However,
with the introductoon of widespread vaccination against serotype b in the mid-1980's, there
has been a dramatic decline (>95%) in the incidence of these infections among children.
There were only 6 invasive infections caused by serotype b that were reported to the
Reportable Diseases Information System (RDIS) in 1998 for the province of Ontario.

However, CDC surveillance data in the United States for 1996 to 1997 indicates that H.
influenzae type b invasive disease still occurs, especially in infants under 6 months of age.
These cases are likely due to reduced levels of protection as a result of incomplete
vaccination (i.e. infant received less than the 3 recommended doses of vaccine). Also,
serotype b invasive disease among older persons (5 years or older) has achieved greater
prominence. Up to 28% of isolates causing invasive disease in this age bracket have been
identified as serotype b.

Non-encapsulated (non-typeable) strains account for the majority of invasive H. influenzae

infections in adults and older children. Lower respiratory tract infections (bronchitis and
pneumonia), with an accompanying bacteremia, are the conditions most often associated
with these strains.

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Major infectious diseases caused by (modified, 5th Edition, Koneman)

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5. CULTURE AND ISOLATION

All Haemophilus species, with exception of occasional strains of Haemophilus aphrophilus,

require either X factor (hemin) or V factor (NAD), or both.

Blood agar, whether it is sheep, horse or rabbit blood, provides ample X factor. However, not
all blood agars are equal when it comes to providing V factor. Metabolically active NAD in
sheep blood agar is found within intact red blood cells and is not available to bacteria for
growth. However, V factor is readily available in rabbit or horse blood agar.

Chocolate agar, the most widely used culture medium for recovery of Haemophilus species,
is rich in both factors. A mixture of basal medium and whole blood (usually sheep) is heated
to 85°C. At this temperature, X factor remains functional and the red blood cells breakdown
releasing V factor. The disadvantage of using chocolate agar for primary isolation of
Haemophilus species is that hemolytic characteristics can not be determined; useful
information when differentiating H. haemolyticus and H. parahaemolyticus (both beta-
hemolytic) from H. influenzae and H. parainfluenzae (both non-hemolytic).

Sheep blood agar may be used for Haemophilus isolation if a "staph steak" is used. A sheep
blood agar plate is first streaked with the clinical specimen. Using an inoculating wire or
loop, a beta-hemolytic strain of Staphylococcus aureus is applied over the areas of streaking.

The beta hemolysin of the S. aureus serves to make hematin (X factor) more readily
available by lysing red blood cells. Also, S. aureus is an organism that liberates NAD into
the surrounding medium during growth. As a result, Haemophilus species requring both X
and V factors (e.g. H. influenzae) will form colonies in proximity to the "staph streak" but
will not grow elsewhere on the plate (Figure 1). Colonies of these species will be largest near
the "staph streak" and become progressively smaller further away. This phenomenon is
called satellitism.

Chocolate and sheep blood agars are non-selective media that can become overgrown with
normal flora. Many commercial media suppliers market antibiotic-containing media
designed to selectively recover Haemophilus species from respiratory tract specimens.
Bacitracin is commonly used as the selective agent in these media.

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Figure 1. H. influenzae showing "Satellitism" along

"staph streak"

As seen from Table 2, H. influenzae can cause many types of infections. The most common
specimens submitted for investigation for H. influenzae include: CSF, blood, middle ear
fluid, sinus aspirates, sputum, urethral swabs, and conjunctival swabs.

Although most Haemophilus species may grow in ambient air, they grow best in a moist
atmosphere containing 5 to 10% CO2. Agar media should be incubated at 35 to 37°C for 48
to 72 hours.

After 24 hours of incubation, colonies of Haemophilus species tend to be small (0.5 to 1 mm

in diameter), translucent, convex and exude a "mousy" or "bleach-like" odour. Encapsulated
strains of H. influenzae have a glistening appearance and are somewhat larger after overnight
incubation than non-encapsulated strains. In mixed cultures, H. influenzae (which requires
both X and V factors) may satellite around organisms like S. aureus and yeasts, which
secrete NAD into their environment during growth.

6. LABORATORY DIAGNOSIS

Tests (and expected results) for identification of Haemophilus species found in humans are
shown in Table 3. For a more comprehensive discussion of identification, refer to the
textbooks listed in the References section of this module.

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Direct Antigen Testing

There are commercial assays that can detect the soluble antigens of H. influenzae serotype b
and other pathogens in body fluids (e.g. CSF, serum and urine). Until recently, many
laboratories performed direct antigen tests (DATs) on CSF samples; to detect the classic
agents of meningitis.
However, the clinical utility of DATs has been seriously questioned.

Many of the classic agents (including H. influenzae serotype b) are not causing as many
cases of meningitis today. The performance characteristics of the various commercially
available DATs have been evaluated and all have shown limitations in sensitivity and
specificity. Positive DATs have been reported for CSF samples that were collected from
patients who had recently received the H. influenzae serotype b vaccine. Also, in the
majority of cases of meningitis, the direct gram stain of CSF has adequate sensitivity.
Consequently, DATs are not performed routinely in most laboratories.

Some laboratories continue to offer DAT when attempting to determine the causative agent
in partially treated cases of meningitis. In this situation, the yield of organism in CSF may be
too low for detection by direct gram stain or culture. A DAT may pinpoint the causative
agent.

Table 3. Key biochemical and physiologic characteristics of Haemophilus species (modified from
Manual of Clinical Microbiology, 7th Edition)

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X and V Factor Requirements

The conventional method for identifying an isolate which exhibits microscopic and colonial
properties consistent with Haemophilus species is to determine the organism's X and V-
factor requirements. One method uses 3 filter paper discs or strips impregnated with X factor
(X), V factor (V) and a combination of X and V factors (XV).

A suspension of the suspect organism is made in saline with a density matching a 0.5
McFarland density. The suspension is swabbed to the surface of a medium free of growth
factors, such as trypticase-soy agar. The growth factor discs or strips are applied to the
surface of the inoculated plate (Figure 2). The plate is incubated in an atmosphere with 5 to
10% CO2 for 18 to 24 hours. Factor requirements are determined based upon the growth or
lack of growth around the 3 discs or strips.

Figure 2. X and V Requirements of H. influenzae according to the X and V disc

method.

Interpretation of growth factors is as follows:

1. Growth around the X and XV discs:

 Indicates that the organism requires only X factor
 Characteristic of H. ducreyi only

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2. Growth around the V and XV discs:

 Indicates that the organism requires only V factor
 Characteristic of H. parainfluenzae, H. parahaemolyticus, H. segnis, and H.
paraphrophilus

3. Growth around ONLY the XV disc:

 Indicates that the organism requires both X and V factors
 Characteristic of H. influenzae and H. haemolyticus (Figure 2)

4. Growth over the entire plate surface:

 Indicates that the organism does not require either X or V factors
 Characteristic of H. aphrophilus and other non-Haemophilus species

Generally, the interpretation of factor requirement tests is clear-cut but misidentifications of H.

influenzae and H. parainfluenzae can occur. To minimize misidentifications, the technologist must
be aware of a few technical points:

 The basal medium used for the test must be deficient in hemin or misleading results will
occur. One study found that trypticase-soy agar was the most reliable medium compared to
Mueller-Hinton and nutrient agars.

 When obtaining the inoculum for the test, avoid sweeping or digging the surface of primary
plates. The inoculum must not contain chocolate (or other blood-contaning medium).
Carryover of hemin or NAD can result in unreliable results. For instance, carryover of X
factor will permit H.influenzae to grow around the V factor and XV discs, resulting in the
incorrect identification of H. parainfluenzae.

 Making the organism suspension in a fluid without growth factors (e.g. saline) will reduce
the impact of carryover through dilution.

 Some Haemophilus species are fastidious and will not grow well under test conditions
making interpretation difficult.

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Porphyrin Test

Some Haemophilus species (e.g. H. parainfluenzae) have the ability to produce their own X factor
using raw materials or substrates found in their environment. These species do not require pre-
formed X factor. Other species (e.g. H. influenzae) lack key metabolic enzymes, which permit the
manufacture of X factor. These species must be supplied with pre-formed X factor. The porphyrin
test determines an isolate's X factor requirement.

In the porphyrin test, isolates are provided with a compound - d-aminolevulinic acid (ALA).
Haemophilus species capable of synthesizing their own X factor are able to convert ALA into
porphobilinogen and porphyrins, two products formed in the hemin biosynthetic pathway. These two
products fluoresce under ultraviolet light. A fluorescent end-point indicates a positive porphyrin test.
The absence of fluorescence, a negative end-point, indicates that the organism was unable to convert
ALA into porphobilinogen and porphyrins.

There are a number of commercial applications of the porphyrin test. A method, using discs
impregnated with ALA, is straightforward and convenient to perform. The impregnated disc is
moistened with water then inoculated with the test organism. After a four-hour incubation period,
the area of the disc with inoculum is observed under an ultraviolet light source (i.e. Wood's lamp)
for fluorescence.

The porphyrin test is probably a more reliable method to determine X factor requirements than the
conventional X and V test. It elimmates the problems associated with carryover and growth factor
content in test media.

Other Tests

Laboratories that have abandoned classical X and V factor testing for the porphyrin test must
determine the V factor requirement by some other means. A medium rich in X factor but devoid of V
factor is required. This can be accomplished by autoclaving the blood/basal medium mixture prior to
plate pouring. This medium is then inoculated as described in the X and V method. A source of
NAD, either a V factor disc or a "staph streak", is applied to the plate. Growth near the V factor
source and absence of growth away from the source is proof of a V factor requirement.

In addition to determining growth factor requirements, determination of hemolysis on horse or

rabbit blood agar and biochemical tests can be performed to identify isolates to species level (Table
3).

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The organism's ability to hemolyze horse or rabbit erythrocytes is useful for distinguishing
H. influenzae from H. haemolyticus and H. parainfluenzae from H. parahaemolyticus. Fermentation
of 1% glucose, sucrose, lactose and mannose in supplemented phenol red broth is used in the
identification of all Haemophilus species.

Multi-test kits that incorporate biochemical and enzyme detection tests are available commercially
also. These kits appear to provide reliable identification of most Haemophilus species. Isolates
resembling H.influenzae can be confirmed as being H. influenzae using a commercially available
DNA probe (Accuprobe: Gen-Probe, San Diego, California).

7. TREATMENT

The recommended treatment for meningitis and other serious infections caused by H. influenzae is
the use of extended-spectrum (3rd generation) cephalosporins such as cefitriaxone and cefotaxime.
The same regimen, extended 4 to 6 weeks, is used to treat osteomyelitis and endocarditis.
Cefuroxime (a 2nd generation cephalosporin) is effective for pneumonia. Local, non-typeable, β-
lactamase-negative H. influenzae infections (e.g. otitis media) can be treated with ampicillin.
However, in geographical areas where a high incidence of ampicillin-resistant H. influenzae is
reported, other antibiotics such as amoxicillin-clavulanate, trimethoprimsulfamethoxazole, or an oral
2nd generation cephalosporin should be considered.

8. SUSCEPTIBILITY TESTING

In the 1960s, virtually all Haemophilus infections were treatable with ampicillin. Since then,
ampicillin resistance has become common. A large American study in 1997 established that 36% of
isolates of H. influenzae were ampicillin-resistant due to the production of the enzyme-β-lactamase.

Because β-lactamase production is the primary mechanism of ampicillin resistance, the β-lactamase
test is routinely performed on clinically significant isolates of H.influenzae. Rapid β-lactamase tests
are available commercially. Nitrocefin-based tests are ideal, but acidimetric assays are also
acceptable. Isolates that are positive for β-lactamase can be reported as resistant to ampicillin.

Negative β-lactamase results do not predict ampicillin susceptibility with 100% accuracy. Rare
strains of H. influenzae (approximately 0.1 %) are ampicillin-resistant due a mechanism other than
the production of β-lactamase.

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However, since this resistance mechanism is so uncommon, most laboratories report ampicillin as
susceptible based upon a negative β-lactamase test. To detect this rare form of resistance, the
susceptibility of ampicillin is determined by disc diffusion or a MIC method.

Resistance among H. influenzae isolates to other antibiotics has also emerged over the past 30 years.

NCCLS has developed standard disc diffusion and MIC methods for the susceptibility testing of
Haemophilus species. NCCLS (M100-S10, January 2000) recommends that susceptibility to
ampicillin, one of the 3rd generation cephalosporins, and chloramphenicol be determined and
reported for all isolates of H. influenzae recovered from patients with lifethreatening infections (e.g.,
meningitis, bacteremia, epiglottitis, and facial cellulitis).

Because of increasing levels of resistance to trimethoprimsulfamethoxazole and other antibiotics

some laboratories have elected to perform susceptibility testing on all clinically significant strains of
H. influenzae for epidemiologic or surveillance purposes.

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B. Bordetella

1. TAXONOMY

The genus Bordetella comprises 6 species: Bordetella pertussis, B.parapertussis, B.

bronchoiseptica, B. avium and two recently described species, B. hinzii and B. holmesii.

2. GENERAL CHARACTERISTICS OF THE GENUS

Bordetella species are small gram-negative coccobacilli that grow only under aerobic
conditions. They grow optimally at 35 -37°C, generally do not use carbohydrates for energy,
and are inactive in most biochemical tests. All species are catalase-positive, and have only
simple nutritional needs. B. pertussis is the most fastidious species.

3. NATURAL HABITAT

Humans are the only host for B. pertussis. This species has an affinity for the ciliated
epithelium of the respiratory tract, which is found below the larynx and in the nasopharynx.
B. parapertussis is found in both humans and lambs. B. bronchiseptica, although
predominantly found in a wide variety of animals, may occasionally cause infections in
humans. B. avium, B. hinzii, and B. holmesii are primarily found in the animal world.

4. CLINICAL SIGNIFICANCE

B. pertussis causes the syndrome known as pertussis or "whooping cough." The word
"pertussis" means violent cough. The organism is transported from the infected person to a
susceptible host by aerosolized droplets (small packets of moisture). An estimated 90% of
nonimmunized individuals become infected after infectious droplets gain entry into their
respiratory tract.

Classical pertussis has 3 stages. The prodromal or catarrhal stage appears 5 to 10 days
after the organism gains entry in the host and is characterized by non-specific "cold" or "flu"
symptoms. The disease is highly contagious at this stage and cultures collected at this time
are positive more often than in later stages. After 7 to 14 days, a rapid-fire cough develops,
which marks the beginning of the paroxysmal stage. A "whoop" at the end of each coughing
attack and vomiting are also seen at this stage. The "whoop" sound is created as air is
forcefully drawn back into the lungs through a narrowed airway after a coughing attack.
Fever and signs and symptoms of systemic illness are usually absent. This stage, which may
last 2 to 6 weeks, leads to the convalescent stage.

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During this stage (duration, several weeks) the cough gradually decreases in frequency and severity.

Today, classical pertussis occurs mainly in unimmunized or under-immunized children. In the United States,
40 to 50% of reported cases occur in young infants. Approximately 30% of cases occur in adults.
Pertussis is endemic (persistent background level of disease) in vaccinated countries like Canada but
superimposed "spikes" of disease occur at 3 to 4 year intervals. In 1997, there were 4,439 cases of pertussis
reported across Canada (approximately 16 cases per 100,000 residents).

An estimated 25 to 30% of infected children exhibit mild or non-classical forms of pertussis. These
undiagnosed cases serve as a reservoir for further transmission. Another major reservoir in vaccinated
countries are infected or colonized adults. Infected adults usually present with a milder form of pertussis
(persistent cough without whooping), probably due to residual protection from childhood vaccination.
These adults are often misdiagnosed with other lower respiratory illnesses. Other vaccinated adults (or older
children) can become transiently colonized or come down with an asymptomatic infection. It is recognized
that vaccine immunity wanes over time and is estimated to last about 12 years.

Complications of pertussis include secondary bacterial infections (e.g. pneumonia and otitis media),
cyanosis (oxygen deprivation of tissues), and encephalopathy.

B. parapertussis can cause a non-specific ''bronchitis'', a pertussis-like illness in children, or classical

pertussis. Pertussis caused by this organism is less severe than the pertussis of B. pertussis. B.
bronchiseptica may be isolated as a commensal in the upper respiratory tract or it may cause a variety of
infections (e.g., meningitis, peritonitis, sepsis, pneumonia, and endocarditis) primarily in
immunocompromised hosts. Both B. hinzii and B. holmesii have been recovered from human clinical
specimens and are capable of causing rare infections in individuals with a weakened immune system.

Pertussis is often difficult to diagnose, especially if the characteristic whooping cough is absent. Pertussis
should be considered in the differential diagnosis of any individual with a prolonged cough illness,
regardless of the vaccination status.

Many of the laboratory techniques used to pinpoint the diagnosis of pertussis lack sensitivity, specificity or
both. As a result a clinical case definition is often used instead. The Centers for Disease Control (CDC)
have defined pertussis based on any cough illness lasting 14 or more days (without apparent cause) with

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any of the following: coughing attacks, inspiratory ''whoop'', or vomiting subsequent to a coughing
attack.

5. LABORATORY DIAGNOSIS

Laboratory confirmation of B. pertussis should be attempted in all suspected cases. The most
commonly used diagnostic techniques include direct fluorescent antibody (DFA) and
nasopharyngeal culture. Serological tests are also useful but will not be discussed here. Nucleic acid
detection techniques (e.g. PCR) show tremendous promise but they remain expensive and are not
yet standardized.

Respiratory secretions from the upper respiratory tract are targeted for the direct bacteriological
diagnosis of pertussis. Nasopharyngeal collections are preferred over throat swabs because ciliated
epithelial cells are found in the nasopharynx but not in the throat.

According to studies, nasopharyngeal (NP) aspirates yield more positive cultures than
nasopharyngeal swabs and are ideal specimens for performing one or more types of tests. To obtain
a NP aspirate, a flexible catheter is passed into the posterior region of the nasopharynx and
suctioning is applied through the catheter with a syringe. If NP swabs are collected, they must be
provided with an appropriate transport medium; to prevent drying of the swab during transit to the
laboratory.

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The DFA test uses commercially available species-specific antibodies, labeled with a fluorescent
compound (fluorescein), that detect the antigens of B. pertussis and B. parapertussis. Smears of
clinical specimens are prepared on microscope slides, heat fixed, then stained with the fluorescent
antibody reagent. The organisms appear under a fluorescent microscope as small coccobacillary rods
with strong peripheral fluorescence surrounding a dark centre.

Advantages of the DFA test include: 1) non-viable organisms can be detected, and 2) a rapid result is
attainable. The disadvantages of DFA testing include: 1) cross-reactions with other non-Bordetella
species results in false-positives, 2) studies have shown that specimens with small numbers of
organisms are often reported negative for pertussis organisms by a DFA test but positive by culture,
and 3) the interpretation of stained smears is difficult and somewhat subjective.

Due to the lack of sensitivity and specificity, the DFA test is not recommended as a stand-alone
confirmatory test. It should only be performed in parallel with culture. In the long term, PCR testing
will likely replace DFA since the PCR test combines rapidity with a much higher sensitivity.

Culture is the accepted gold standard for diagnosis of pertussis in the routine laboratory. However,
it should be noted that sensitivity of culture only approaches 60 - 80% in the best of hands. To be an
effective diagnostic tool, the laboratory must pay attention to the smallest of details. Adequate
quality control of both isolation media and identification procedures is essential.

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B. pertussis is not able to grow on basic blood or chocolate agars. Media constituents such as
unsaturated fatty acids, metal ions and peroxides inhibit this species. Starch, charcoal, blood, or
other protective substances must be added to isolation media for optimal recovery B. parapertussis,
which is less fastidious than B. pertussis, is able to grow on both blood and chocolate agars.

The original medium used to isolate Bordetella pertussis was Bordet-Gengou (BG) agar. However,
BG plates have a short shelf-life and must be used within 5 days of manufacture. Newer medium
formulations have been introduced over the past 30 years. Regan and Lowe developed a medium
containing charcoal and horse blood that has demonstrated superiority to BG in several studies.
Regan-Lowe (RL) agar can be used in a semi-solid form as a transport/enrichment medium and in a
solid form for organism isolation. RL media is stable for up to 8 weeks after manufacture.
Cephalexin, the selective agent of choice, can be added to RL to reduce the growth of background
flora. However, some strains of B. pertussis may be inhibited by cephalexin. Consequently, some
experts advocate that specimens be setup onto both selective and non-selective media.

Optimally, media should be inoculated directly at the time of specimen collection onto both
selective (i.e. cephalexin-containing) and non-selective media.

A liquid enrichment medium for NP swabs may be used so more bacteria will be eluted off the
swab.

A reliable approach for handling swab specimens is as follows: 1) inoculate selective and non-
selective plates at time of specimen collection and incubate immediately, 2) immerse the swab(s) in
a slant of semi-solid RL (enrichment step), 3) incubate the slant at 35°C for 48 hours, then 4)
remove the swab from the RL transport and inoculate a second set of selective and non-selective
plates.

Once inoculated, culture plates should be incubated at 35°C for 7 days in ambient air without added
carbon dioxide. Incubation for up to 12 days may improve the sensitivity of culture significantly.
Adequate moisture is essential. Pouring thick culture plates and wrapping plates with gas-permeable
tape are effective measures for maintaining moisture.

Three to four days are required before colonies of B. pertussis appear. Colonies of B. parapertussis
usually become visible after 2 to 3 days of incubation. Fresh clinical isolates of B. pertussis and B.
parapertussis appear as smooth, shiny, convex colonies on BG agar, winch have been likened to a
droplet of mercury. On RL agar, colonies are small, domed, and shiny, with a mother-of-pearl
opalescence (varied colours like that of an opal).

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The colonial morphology of B. bronchiseptica may resemble that of B. pertussis. One difference,
however, is that mature colonies of B.bronchiseptica develop in only 24 hours of incubation.

When performing gram stains of colonial growth, counterstaining for 1 to 2 minutes is

recommended to increase the intensity of staining. B. pertussis appears as small, pale-staining,
coccobacilli, while cells of B. parapertussis and B. bronchiseptica are more rod-shaped.

Suspicious colonies are subjected to oxidase testing. B. pertussis and B. bronchiseptica are oxidase-
positive; B. parapertussis is oxidase-negative. Species-specific antisera can be used to make the
final identification. Both fluorescent and agglutinating antisera are available commercially. Testing
with an antiserum versus B. pertussis is performed on oxidase-positive colonies and B.
parapertussis antiserum is used for oxidase negative growth.

If testing with antisera provides clear-cut results, no additional testing is required. If not, other
identification methods must be used, as shown in Table 4.

6. TREATMENT

Erythromycin is the agent of choice for the treatment and prophylaxis of pertussis. Best results are
achievable if the agent is used during the catarrhal and early paroxysmal stages of disease.
Trimethoprimsulfamethoxazole is used if erythromycin is not an option.

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7. SUSCEPTIBILITY TESTING

There are no standardized susceptibility testing methods available for the fastidious, slow growing
bordetellae. Because B. pertussis and B. parapertussis remain predictably erythromycin-susceptible,
there is currently no need for routine susceptibility testing.

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C. Legionella

1. TAXONOMY

At least 40 species have been recognized in the genus Legionella. Some species have been
isolated only from the environment and others have been isolated from both human and
environmental sources. The most clinically significant species is L. pneumophila. It has been
broken down into 3 subspecies - subspecies pneumophila, fraseri, and pascullei - and 15
serogroups.

These organisms remained virtually unknown to man until 1976 when a devastating outbreak
of pneumonia struck American Legion members attending a convention in Philadelphia. The
outbreak claimed the lives of 15% of the conventioneers. The newly discovered infectious
agent, Legionella pneumophila, was named after the legionnaires. The pneumonia caused by
the organism was called "legionnaires' disease."

2. GENERAL CHARACTERISTICS OF THE GENUS

Legionella species have the following characteristics:

 Faintly staining, thin, gram-negative, non-spore-forming bacilli
 Giemsa or Gram-Weigert stains yield better results than the Gram stain
 Require cysteine and iron salts for growth
 Utilize amino acids for growth
 Unable to ferment or oxidize carbohydrates
 Most species are motile

3. NATURAL HABITAT

Legionella species are widespread in the environment but exist primarily in aquatic habitats.
They have been isolated from the majority of natural water sources investigated, including
lakes, rivers and marine waters, as well as moist soil. More importantly, they have been
recovered from man-made water systems, including air-conditioning ducts and cooling
towers; drinking water supplies; large, warm-water plumbing systems; humidifiers;
whirlpools; and medical equipment in hospitals.

Legionella infections are acquired exclusively from environmental sources. The majority of
infections begin with the inhalation of infectious aerosols. Supermarket misting systems for
produce, nebulizers filled with tap water, and showers have been implicated as vehicles

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of transmission. No person-to-person spread has been reported.

Legionella species are not part of the normal flora of humans. Recovery of these organisms
from clinical specimens is usually associated with overt disease.

4. CLINICAL SIGNIFICANCE

The term legionellosis is used to describe the wide range of infections caused by Legionella
species. Health Canada reported 81 cases of legionellosis per 100,000 residents for the
nation in 1997. However, the prevalence rate in Canada is likely significantly higher when
considering the difficulties associated with making a diagnosis. Approximately 85% of
documented cases of legionellosis have been caused by L. pneumophila.

Infections can be placed into four categories: i) sub-clinical infection, ii) non-pneumonic
disease (Pontiac fever), iii) pneumonia, and iv) extrapulmonary inflammatory disease.

A large proportion (from 5 to 10%) of the general population worldwide has antibodies to
Legionella without an overt infection. These infections fall under the sub-clinical category.
The second type of infection is the epidemic, non-pneumonic illness known as Pontiac fever.
This selflimiting infection is marked by fever, cough, flu-like symptoms and the absence of
pulmonary infiltrates.

Pneumonia, most often referred to as legionnaires' disease, is the most common

manifestation of Legionella infection. At risk are people who are immunocompromised,
older than 60, or heavy smokers. The clinical presentation is variable. Often chest
radiographs show infiltrates, and the patient has a high temperature, chills and a non-
productive cough. Today, pneumonia due to Legionella is recognized as one of the three
most common causes of community-acquired pneumonia (i.e., contracted outside health care
facilities). It is also a well-recognized agent of hospital -acquired (nosocomial) pnuemonias.
The case mortality rate is estimated to range from 15 to 30%.

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In severe cases of Legionella pneumonia, organisms frequently spread from the lungs to
other parts of the body via the bloodstream causing extrapulmonary infections such as
pericarditis, hepatic abscess, and endocarditis.

5. LABORATORY DIAGNOSIS

The diagnostic approaches available for the diagnosis of Legionella infections include direct
detection of bacterial antigens or nucleic acids in specimens, isolation of the bacterium in
culture, and detection of a serological response.

Specimens - Collection and handling

It is important that the laboratory is notified whenever legionellosis is being considered in a

diagnosis to ensure that specimens are properly transported and processed for maximal
recovery or detection of these organisms.

Lower respiratory tract secretions should be collected by the usual methods and submitted to
the laboratory at ambient temperature as soon as possible. The appropriate number or timing
of sputum specimens has not been extablished. If Legionella is suspected, then multiple
sputums should be sent for testing because samples may contain only a few bacteria. If a
delay in processing of ≤ 48 hours is anticipated, samples are stable under refrigeration. For
longer delays (several weeks), samples should be frozen at -70°C. Transport media, buffers,
or saline should be avoided as they may be inhibitory. Legionella species are hardy
organisms even though they have fastidious growth requirements. Delayed specimens should
not be rejected because Legionella species have been recovered from specimens after several
days of storage at room temperature.

Blood cultures should be collected from seriously ill patients with documented pneumonia.
Wound material and tissue are also indicated when localized disease outside of the
respiratory is suspected. Urine and other body fluids may be submitted for Legionella
antigen detection. Serum samples are collected for serological studies. An acute serum (taken
close to onset of symptoms) and convalescent serum (taken 6 weeks after the acute sample)
are the specimens of choice.

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Direct Detection

Direct Gram stains of clinical specimens are not usually informative due to the poor uptake
of the counterstain by Legionella bacilli.

Legionella species can be detected directly in preparations of clinical specimens using the
direct fluorescent antibody (DFA) test (see discussion in Bordetella section). Due to its low
sensitivity (25 -70%) and the possibility of false-positives, DFA is not useful in most clinical
situations.

Legionella antigen can be detected in urine and other body fluids by a number of methods.
These methods are limited in that only certain serotypes are targeted and diagnosis of acute
disease using a urine sample is not possible due to the prolonged excretion of antigen - well
after the acute phase.

A DNA probe test, which targets a genus-specific region of Legionella RNA, is

commercially available from Gen-Probe (San Diego, California). The sensitivity of the probe
is similar to that of the DFA test but the specificity is reported to be better. It is not sufficintly
sensitive to replace culture.

Culture

Culture is the "gold standard" for diagnosis of legionellosis. The sensitivity is comparable to
other methods but the specificity is 100%, a level not yet achieved by other methods.

Legionella species do not grow well if at all on standard culture media. The medium of
choice is buffered charcoal yeast extract agar supplemented with α-ketoglutarate or BCYEα.
Non-selective and selective media (BCYEα with and without antibiotics) are recommended
for specimens from non-sterile sources. Plates are incubated in a humid atmosphere for up to
14 days.

Colonies of Legionella usually appear after 2 to 3 days of incubation. Under a dissecting

microscope, they have a "mottled" surface, an iridescent red-blue-green sheen, or a faceted
"cut-glass" appearance. If a gram stain of the suspect colony shows thin, pale-stainig gram-
negative bacilli, the colonies should be subcultured onto media with and without cysteine -
to determine cysteine dependence. For a full discussion of the various methods used for
genus and species-level identification, consult one of the textbooks listed in the reference
section of this module.

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Serological Tests

Serological tests are useful for diagnosis of legionellosis in individuals who do not have
pneumonia or are unable to produce lower respiratory tract specimens. These tests are an
adjunct to microbiologic diagnosis and are not definitive on their own.

A fourfold rise in titer, moving from the acute to the convalescent sample, provides evidence
of a recent infection. A single titer ≥ 1:256 is evidence of an infection at an undetermined
time. However, due to the relatively high prevalence of antibodies to Legionella
pneumophila in the general population, paired sera are preferred to a single sample.

The disadvantages of serological tests are: 1) the diagnosis is usually retrospective, available
only after seroconversion, 2) false-negative results occur in at least 15% of patients who
seroconvert later than 6 weeks, 3) false-positive results occur when antibodies directed
against other pathogens cross-react with Legionella antigens.

6. TREATMENT

The standard therapy for legionellosis is erythromycin. Rifampin is combined with

erythromycin for treatment of serious infection. The newer macrolides, azithromycin and
clarithromycin, are also used in this situation.

7. SUSCEPTIBILITY TESTING

Studies have found that in vitro susceptibility testing results are a poor predictor of clinical
outcome.

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D. Brucella

The taxonomy of Brucella species is open to debate. Based on physiologic and biochemical
characteristics, the following species have been defined: Brucella abortus, B. suis, B. melitensis; B.
canis, B. neotomae, B. ovis, and a probable new species B. maris. However, at the genetic level there
is not much that separates these species. It has been proposed that all of the above species could be
brought together in a single species - B. melitensis. Until these taxonomic issues are resolved, most
microbiologists will continue to use the 7 species names above in non-taxonomic settings (i.e. in
patient reports).

The following characteristics describe members of the genus Brucella:

 Intracellular parasites in humans and other animals
 Gram-negative cocci or short rods; appearing like "fine sand" in Gram smears
 Non-motile
 Strict aerobes; increased CO2 is usually required for growth
 Most strains require specific growth factors such as amino acids
 Catalase positive, nitrate positive, and oxidase positive

Brucellosis (infection with a Brucella species) strikes the genitourinary tract of sheep, goats, pigs,
cattle, dogs and other animals causing abortion. Each species has a preferred host(s) as shown in
Table 5. Domestic animals serve as the main reservoir for brucellosis in humans. Brucellosis is
considered to be a zoonosis because it is a disease in humans that is acquired from animals or animal
products.

Table 5. Preferred host(s) for Brucella species.

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Brucella gain entry into the human host via ingestion, through breaks in the skin, through mucous
membranes (including the conjunctiva) and inhalation (i.e., in slaughterhouses or laboratories). They are
carried by the lymphatic and circulatory systems to the liver, spleen and bone marrow. Focal infections at
these locations can persist because the organism is able to survive inside phagocytic macrophages and
reticuloendothelial cells found in these tissues.

The clinician should suspect brucellosis in any individual with fever and anyone of the following risk
factors: exposure to livestock; a history of consuming unpasteurized milk, cheese or other dairy products; a
history of travel to endemic areas; or occupations involving the handling of animals (veterinarians), meat or
carcasses (abattoir workers), and live cultures of Brucella species in laboratories.

Brucellosis may present as an acute, chronic, or localized infection. The incubation period varies from 5 to
35 days or more, with an average of 10 to 14 days in acute cases. In acute disease bacteremia is common.
Signs of infection include fever, sweats, chills, weakness, headache and anorexia. An enlarged spleen and
lymphadenopathy occurs in 20 to 30% and 10 to 20% of cases, respectively. Complications can emerge in
cases that remain untreated, which include osteomyelitis, endocarditis, splenic abscess, nodular lung
lesions, prostatitis, and meningoencephalitis. Brucellosis may become chronic, lasting for more than a year.
Irregular cycles of fever that repeat over several months are not uncommon in chronic cases.

Brucellosis is worldwide and known by a variety of names, such as: undulant fever, Bang's disease,
Gibraltar fever, Mediterranean fever and Malta fever. The incidence of disease has declined in developed
countries following the institution of mandatory pasteurization of dairy products and immunization of cattle
with a B. abortus vaccine. In the past few years, only 100 cases have been reported annually in the United
States. However, many infections go undetected or undiagnosed due to the insidious onset of symptoms and
the similarity of brucellosis to other infections.

It is important that the laboratory is notified whenever brucellosis is being considered in a diagnosis for the
following reasons:
 To ensure that specimens are processed properly for optimum recovery
(i.e. the use of proper culture media and prolonged incubation)
 To avoid accidental exposure of laboratory workers handling specimens, because Brucella species
are highly infectious (i.e., considered class III pathogens)

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Blood and bone marrow aspirates are the clinical specimens of choice for the diagnosis of brucellosis by
culture. Blood cultures may be positive after the second day of fever. Multiple blood cultures should be
collected during the febrile period. In latter stages of brucellosis, blood cultures are often negative. Other
specimens important in the diagnosis of brucellosis include lymph node aspirates, urine, joint fluid, spinal
fluid, abscess aspirates, and liver or spleen biopsies.

For serological diagnosis, two serum samples should be collected; one early in the disease (acute-phase
specimen) and a second serum sample 14 to 21 days later (convalescent-phase specimen). A single titer of ≥
160 or a fourfold increase in titer is considered significant.

Bi-phasic Casteneda bottles have been used for many years to recover Brucella species from blood. A
Casteneda bottle contains a slant of enriched agar (solid phase) that is partially submerged in a broth
medium (liquid phase). However, a number of automated blood culture systems such as BACTEC 9000
(Becton Dickinson Microbiology Systems), BacT/Alert (Organon Teknika Corp.), and ESP (Accumed
International Inc.) can be used - with varying success. As a general rule, the incubation period in these
systems is extended to 21 -28 days. Blind subcultures are recommended for maximal yield; one early (i.e. at
48 hours) and one upon completion of the incubation period.

Brucella species grow slowly on most standard culture media (e.g., brucella, blood, and chocolate agars)
when incubated at 35°C in 5 to 10% CO2 under humid conditions. Colonies are small, convex, smooth,
translucent, non-hemolytic and slightly yellow and opalescent after at least 48 hours of incubation. Brucella
species do not grow on MacConkey or other "enteric" media.

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A presumptive identification (i.e., "a possible Brucella species") can be made when a slow-growing,
faintly staining, small coccobacillus is isolated from blood or bone marrow cultures from a patient
with risk factors. Identification to species-level should be performed by a reference laboratory with
the appropriate Class III facilities and experienced personnel.

Treatment of brucellosis requires combination antibiotic therapy (e.g. doxycycline plus either
gentamicin or streptomycin) for an extended period. Tetracycline and trimethoprim-
sulfamethoxazole are effective agents as well.

E. Pasteurella

The genus Pasteurella belongs in the family Pasteurellaceae along with two other genera
-Haemophilus and Actinobacillus. Pasteurella multocida is the Pasteurella species most frequently
encountered in human specimens. The other species are rarely associated with human disease.

Both healthy and diseased wild and domestic animals are the reservoirs for most Pasteurella
infections in humans. The natural reservoir of P. multocida is the upper respiratory tract of healthy
dogs, cats, rabbits, swine and birds. P. multocida is found in the oral cavities of 70 to 90% of healthy
domesticated cats and 40-66% of domesticated dogs.

A history of exposure to animals, especially cats or dogs, appears to be at the root of most P.
multocida infections in humans. This species is associated with three general types of disease:

1. Local wound infections:

 Usually acquired after a cat bite, cat scratch or dog bite
 Clinical features include swelling, cellulitis, purulent drainage at the wound site and
local lymph node swelling

2. Chronic pulmonary disease:

 Usually occur in elderly patients with underlying pulmonary disease (e.g. lung
carcinoma)

3. Systemic infections:
 Usually in immunocompromised patients
 Bacteremia seeded from local wound infections
 infections include endocarditis, meningitis, and brain abscesses

Pasteurella species occur as gram-negative coccobacilli or rods. They are all non-motile, facultative
anaerobes that can use carbohydrates as an energy source.

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Most species recovered from clinical specimens are catalase, oxidase, indole and sucrose positive.

P. multocida is usually not difficult to isolate and identify. It grows well on blood and chocolate
agars but usually fails to grow on MacConkey agar. After 24 hours incubation in increased CO2,
colonies on blood agar appear gray, smooth, convex, and non-hemolytic; measuring 0.5 to 2.0 mm
in diameter. Isolates from respiratory tract specimens may be mucoid due to capsule production.
Many commercial identification systems are capable of identifying P. multocida with reliability.

Although penicillin is generally considered to be the antibiotic of choice for the treatment of P.
multocida infections; alternative drugs that have been recommended include ampicillin, amoxicillin-
clavulanic acid, extended-spectrum cephalosporins (e.g., ceftriaxone), trimethoprim-
sulfamethoxazole, tetracycline, and ciprofloxacin. There is no standardized susceptibility testing
method for Pasteurella species.

F Francisella

Currently, the taxonomy of the genus Francisella is in a state of flux. Members of the genus show
no relationship with the brucellae and pasteurellae according to genetic and physiologic analyses.
Two species have been proposed, as follows:

F. tularensis
biovar tularensis (type A)
biovar palaearctica (type B)
biovar novicida (once a separate
species)

F. philomiragia

F. tularensis is the most clinically significant species. It is the agent of human and animal tularemia.
Generally, human tularemia is a zoonosis - a disease contracted from animals. Since 1965, the
number of cases of tularemia in the United States has been relatively low at 0.05 to 0.15 per 100,000
residents.

F. tularensis is widely distributed in nature and has been isolated from hundreds of animals,
including numerous mammals, birds, arthropods, and domesticated animals, as well as water, mud
and animal feces.

Biovar tularensis predominates in North America. Its reservoir is the cottontail rabbit and it is
frequently transmitted by ticks. This biovar is highly pathogenic in humans. Biovar palaearctica is
found in Europe, Asia, Japan and North America.

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It causes infections in beavers, voles and muskrats. This biovar is not as virulent as biovar tularensis.

Humans can contract tularemia in a number of ways:

 by handling the carcasses or skin of infected animals (entry through breaks in skin)
 after being bitten by a tick or deerfly (inoculation through skin)
 after being bitten by carnivores that have eaten infected animals (inoculation through skin)
 conjunctival exposure (entry through mucous membrane)
 inhalation of aerosolized organisms (entry through respiratory tract)
 ingestion of infected, undercooked meat or contaminated water (entry through gastrointestina1 tract)

Tularemia is characterized by sudden onset with non-specific symptoms including fever, chills, headache
and generalized aches. Cough and abdominal symptoms may also occur. Tularemia can be divided into 7
major syndromes, which reflect the mode of organism entry. The ulceroglandular syndrome, characterized
by a skin ulcer and local lymphadenopathy, originates at the point of entry of the organism following a tick
or deerfly bite, animal bite or the handling of infected animals. This syndrome accounts for 70 -85% of all
cases of tularemia.

It is important that the laboratory is notified whenever tularemia is being considered in a diagnosis for the
following reasons:
 To ensure that specimens are processed properly for optimum recovery (i.e. the use of proper culture
media and prolonged incubation)
 To avoid accidental exposure of laboratory workers handling specimens, because F. tularensis are
highly infections for they are class III pathogens

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Diagnosis of tularemia by culture is difficult and unreliable. One study discovered that a positive
culture was only achievable for approximately 10% of patients with tularemia.

F. tularensis requires specific growth factors, is slow-growing, and can be overgrown by competing
flora. The organism will grow on commercially prepared chocolate agar that is supplemented with a
source of cysteine and other nutrients (e.g. IsoVitelex). Colonies on supplemented chocolate agar are
blue-gray, smooth, and slightly mucoid (due to capsule production); reaching 2 mm in diameter in 2
to 4 days of incubation.

Most cases of tularemia are diagnosed by serological methods (e.g., standard tube agglutination,
haemagglutination, and enzyme immunoassay). A single titer ≥160 and a four-fold rise in titer
between the acute serum (collected at the onset of symptoms) and convalescent serum (taken 2 to 3
weeks after the acute sample) are presumptive and diagnostic, respectively.

The antibiotic of choice for the treatment of tularemia is streptomycin. There is no standardized
susceptibility testing method for Francisella species.

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APPENDIX A: OTHER FASTIDIOUS GRAM-NEGATIVE BACTERIA

1. "HACEK" Organisms:

''HACEK'' is a mnemonic formed from the first letter of the following organisms. These organisms
are all part of the normal oropharyngeal or urogenital flora of humans. They are found mainly in
association with endocarditis, bacteremia, and polymicrobic wound infections.

Haemophilus aphrophilis
Actinobacillus
Cardiobacterium
Eikenella
Kingella

CHARACTERISTICS:
 Slow growers
 Detection in blood cultures may take up to 2 weeks or longer
 Increased CO2 is required or enhances growth
 Optimal growth achieved on enriched media
 No growth on MacConkey
 Hemin enhances recovery in culture

Haemophilus aphrophilis/paraphrophilus (H)

 Has a "grade school paste" odour

 Endocarditis following oral or dental complications

Actinobacillus actinomycetemcomitans (A)

 Closely related to Haemophilus

 Colonies adherent to agar; star appearance in older colonies; adherent to sides of culture tubes
 Oxidase negative; catalase negative; nitrate positive; urea negative
 Endocarditis; peridontal disease in adolescents; spread to other organs; bacteremia

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Cardiobacterium hominis (C)

 Unusual Gram stain; Gram variable, retains crystal violet at poles of cell
 Cells pleomorphic: "tear-drop", "dumbell" and "lollipop" shapes; forms rosette clusters or picket
fence formations
 Oxidase positive; catalase negative; nitrate negative; urea negative
 Infects diseased heart valves, endocarditis
 History of dental work preceeding endocarditis
 Isolated from blood cultures

Eikenella corrodens (E)

 Pits agar; ''bleach smell"

 Slender gram-negative rods or coccobacilli
 No carbohydrate utilization
 Oxidase positive; catalase negative
 Associated with dental and peridontal infection
 Root canal infections, head and neck infections, meningitis, endocarditis, mixed wound infections,
osteomyletitis

Kingella species (K)

 Plump gram-negative rods/coccobacilli in pairs and short chains

 B-hemolytic; pits agar
 Causes endocarditis, bone and joint infections
 Related to poor oral hygiene

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2. Miscellaneous Organisms

Capnocytophaga species

 Gram-negative fusiform rods

 Grows on blood and chocolate agar - not MacConkey
 Colonies yellowish-tan; film around colony
 Normal oral flora
 Cause localized juvenile periodontitis
 Sepsis in immunosuppressed

Streptobacillus moniliformis

 Gram-negative; long filaments that form loops and coils

 Microaerophilic
 Found in oropharynx of rodents
 Also carried by dogs and cats
 Transferred to humans via bites - called "rat bite fever"
 High fever, chills, headache, muscle aches

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