UNIVERSITY OF COLORADO

1
UNIVERSITY OF COLORADO SCHOOL OF
MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY
TRAINING PROGRAM MICROBIOLOGY VIII
Miscellaneous Gram Negative Bacilli--1 CASE STUDY
1 Recurrent Fever
2
CASE STUDY I Recurrent Fever
The case study presented here occurred in one of
our local hospitals, serving as a teaching
exercise for future reference. A 40-year old man
developed intermittent low-grade fever and a
general feeling of tiredness and weakness that
occurred about 2 weeks following a family
gathering in his home town in rural Mexico. Blood
cultures were drawn during one of the fever
spikes. A positive culture did not alert until
after 5 days of incubation. Although considered
by the clinical staff to be a contaminant, the
specimen was subcultured to blood agar (see next
screen). The one relevant note of clinical
history was the ingestion of home-made cheese
prepared with raw, unpastuerized milk.
3
CASE STUDY I Recurrent Fever
Blood Culture Isolate
The colonies growing on the surface of the blood
agar plate illustrated in the photograph were
observed after 48 hours incubation. They are
relatively small, measuring 0.5 - 1.0mm in
diameter. They are entire, convex, and
glistening, with no evidence of hemolysis.
4
CASE STUDY I Recurrent Fever
Gram Stain Features
The bacterial cells observed in a gram stain
prepared from an isolated colony were tiny,
averaging 0.5 - 0.7 x 0.6 - 1.5um in diameter,
appearing as Gram-negative coccobacilli. (see
photograph). The appearance of the bacterial
cells as observed in direct gram stains of
clinical materials has been described as grains
of sand.
5
CASE STUDY I Recurrent Fever Recommendation
It is the recommendation of Public Health
laboratories that all slowly growing isolates
with the appearance of tiny gram-negative
cocco-bacilli, particularly if poorly staining,
be referred immediately to them for work up,
bypassing attempts to make an in-house definitive
identification. In this case, the clinical
history should have provided an additional
clue. The slow-growing isolate was considered to
be a contaminant. Several attempts were made to
make the laboratory identification of the blood
culture isolate however, it did not key out on
any of the commercial identification systems. It
was ultimately sent out for definitive
identification. Two days later the report of
Brucella species was received. As indicated in
the algorithm on the following page, a rapid
urease reaction is key to making the
identification. This isolate was a very weakly
urease positive on Christensens agar, with the
reaction so weak and delayed that a false
negative reaction occurred in the commercial
system used.
6
ABBREVIATED IDENTIFICATION OF Brucella species
Slow growth of tiny gray-white, shiny
colonies on BA
Tiny pale-staining cocco-bacilli on gram stain
Positive cytochrome oxidase reaction Spot test
Rapid hydrolysis of urea reaction in 1- 4 hours
on Christians agar
History of close contact with animals or
ingestion of raw animal products
History of recurrent fevers
Suspect Brucella species Consult public health
laboratory immediately
7
CASE STUDY I Recurrent Fever Follow Up
Since this isolate had been worked up on the open
bench in the laboratory, and because of the known
ease of inter-human communicability, each of the
laboratory microbiologists were put on a 5-day
course of antibiotics. Again to reiterate, the
recovery of a slowly growing, poorly staining
gram-negative cocco-bacillus is considered to be
suspicious for an agent of bioterrorism and
should be referred to a public health laboratory
shortly after recovery. Brucella species has been
recognized as one of the more common bacterial
agents involved in laboratory-acquired
infections.
8
Brucellosis Review
Brucellosis is a zoonotic disease caused by any
of four Brucella species B. abortus, B.
melitensis, B. suis, and B. canis. Brucellosis is
endemic in many animal species. Animals acquire
disease either sexually or by ingesting
contaminated milk or other animal
products. Humans most commonly develop disease
following ingestion of contaminated milk or milk
products (goat cheese, for example). Cutaneous
infections occur following direct contact with
abraded animal skin. Human infection is less
commonly acquired through the conjunctiva or by
inhalation. Person to person transmission occurs
only rarely, if at all. In the United States,
brucellosis is an occupational disease, most
commonly in abittoir workers, butchers, farmers,
and veterinarians.
9
Brucellosis Clinical Presentations
Incubation period for the onset of until clinical
signs of acute brucellosis is between 7 and 21
days after exposure. The infective dose 10 -
100 organisms. The onset of disease may be
insidious--low grade fever, malaise, weakness,
fatigue, headache, backache and myalgies. There
may be a paucity of physical findings--splenomegal
y (10 - 20 of patients), lymphadenopathy (15 of
patients), and hepatomegaly (lt10). Because of
the ability of the organisms to persist
intra-cellularly, the acute disease usually
extends into chronic illness with periods of
remission interspersed with recurrent episides of
fever, chills, malaise, and other symptoms
mentioned above ("undulant fever").
10
Brucellosis Pathology
Localized disease may involve almost any organ
system--osteomyelitis, splenic abscess,
genitourinary tract, pulmonary disease, and
endocarditis are among the most common
infections. Osteomyelitis usually involves the
vertebrae--disc space abscesses are also
common. Splenic abscesses, with the formation of
circumscribed granulomas that may undergo
secondary calcification, often are
found. Endocarditis is the most common cause of
mortality among patients with chronic disease.
Skin lesions--animal handlers, particularly
veterinarians, are prone to develop cutaneous
lesions.
11
Brucellosis Pictorial Retrospective
The skin lesions evolve as an erythematous
macular, papular, or pustular lesion on the hands
and arms at the sites of direct exposure to
infected animal material. The lesions
illustrated in the photograph are from an
archival slide collection depicting cutaneous
brucellosis on the arm of a farmer who
hand-milked cows.
12
Brucellosis Pictorial Retrospective
Localized disease may involve almost any organ
system, including the spleen, in which multiple
granulomas may be seen. The granulomas in the
photograph range from 1 - 3 cm in
diameter. Initially, multiple splenic abscesses
may be observed, that later develop into
circumscribed granulomas that characteristically
undergo secondary fibrosis and calcification.
13
Brucellosis Pictorial Retrospective
The reaction in lymph nodes, spleen, liver, and
other organs is granulomatous. Although caseous
necrosis per se is rarely observed, focal areas
of stellate necrosis may be observed, with
festooning of macrophages at the periphery , the
so-called Splendore-Hoeple effect (blue arrow).
14
Brucellosis Pictorial Retrospective
The granulomatous reaction often also is
characterized by the presence of numerous
multi-nucleated giant cells (blue arrows). This
reaction occurs because of the ability of
organisms to remain viable intra-cellularly Organi
sms are difficult to demonstrate with tissue gram
stains. Culture is necessary to establish a
diagnosis in most instances. Pathologists should
include chronic brucellosis in the differential
diagnosis of all granulomatous lesions with
histology as seen in the photograph.
15
UNIVERSITY OF COLORADO SCHOOL OF
MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY
TRAINING PROGRAM MICROBIOLOGY VIII
Miscellaneous Gram Negative Bacilli--I CASE STUDY
2 Pancreatic Abscess
Original Presentation CACMLE Teleconference Jan
22, 1992 Elmer W. Koneman, M.D., Paul C.
Schreckenberger, Ph.D. William Janda,
Ph.D. Microbiology Laboratory
University of Illinois/Chicago
16
Pancreatic Abscess
CASE HISTORY The case presented here is from a
report by Bullock and Devitt (J Infection
332-85, 1981) of a 29 year-old alcoholic male
who developed sudden onset of septicemia related
to a necrotic, purulent pancreatic abscess. The
authors indicate in the paper that septic
complications of acute pancreatitis are not
uncommon. Their review of the literature
indicates that 90 of pancreatic abscesses are
culture positive, with E. coli, Klebsiella,
Bacteroides, Pseudomonas, Proteus, Staphylococcus
and Streptococcus species being the bacterial
agents most commonly recovered. Blood cultures
were drawn and became positive in 72 hours with
the organism presented here.
17
Pancreatic Abscess
Illustrated in this photograph is the
appearance of the colonies on the surface of
selective haemophilus recovery culture media,
supplemented with X V factors and bacitracin.
The colonies are off-white yellow,
entire, flat to slightly convex, smooth, and
measuring 0.5 mm to 2.0 mm in diameter after 48
hours incubation at 35o C. Hemolysis is not
observed.
18
Pancreatic Abscess
Illustration of enhanced growth on the surface of
a chocolate agar plate. Classic chocolate agar,
in which erythrocytes are lysed by the heat
treatment during preparation, release Factor V
and Factor X in abundance, suitable for the
recovery of Haemophilus species from clinical
specimens. Commercial chocolate agar, prepared
by synthetically adding derived Factors X and V,
also support the growth of Haemophilus
species. This isolate was identified as
Haemophilus segnis.
The colonies here appear off-yellow, entire,
slightly convex, smooth, and measure between 1.0
and 2.0 mm in diameter after 48 hours incubation
at 35o C in CO2.
19
Pancreatic Abscess
Staphylococcus aureus and other bacterial
species provide a rich source for Factor V. Note
in the photograph the tiny satelliting colonies
of Factor-V dependent Haemophilus species growing
adjacent to the Factor-V producing staphylococcal
colonies.
20
Pancreatic Abscess
A hemolytic strain of Staphylococcus aureus is
commonly used as a source for Factor V. The
staph streak procedure takes advantage of this
property in the recovery of Factor-V dependent
Haemophilus species on routine sheep blood
agar. Note in the photograph of the surface of a
sheep blood agar plate the growth of tiny
colonies of Haemophilus species adjacent to the
staph streak on the right side. This is known as
the satellite phenomenon.
21
Pancreatic Abscess
Illustrated in this photo-micrograph is a gram
stain prepared from one of the isolated colonies
illustrated in the previous screen. The bacterial
cells are usually described as short,
cocco-bacillary, and poorly staining, often
admixed with pleomorphic and filamentous forms.
These features are well captured in this
photograph.
22
Pancreatic Abscess
The assessment of X and V factor requirements is
performed in many laboratories using reagent
strips selectively impregnated with these
substrates. Note in the photograph that this
Haemophilus species is X-Factor dependent, but
not V factor as it grows only around the strips
containing Factor X, provided either by the X
strip or the XV strip. Currently commercial
tri- and quad-plates are available, supplemented
with X, V, and XV quadrants, providing for
identification based on differential growth
factor requirements.
23
Pancreatic Abscess
Illustrated in this photograph is the porphyrin
test. This test is based on the ability of an
organism to synthesize porphyrin from the base
substrate, delta amino levulinic acid, which in
this case is contained in the culture
medium. Strains that are not X-Factor dependent
can synthesize porphyrin and will produce a red
fluorescence when observed under ultraviolet
light (see photograph). This test is valuable to
exclude extraction of extraneous hemin when
obtaining inocula from the original blood agar
isolation plate.
24
Pancreatic Abscess
Disk test for performing the porphyrin synthesis
test. These filter paper strips are impregnated
with delta amino-levulinic acid. Each has been
inoculated with a bacterial isolate. The
fluorescence observed on the left disk indicates
porphyrin synthesis and non-dependence on Factor
X the absence of fluorescence in the disk to the
right indicates Factor X dependence.
25
Haemophilus Review
IDENTIFICATION OF HAEMOPHILUS SEGNIS No growth on
BA Growth on chocolate agar and Haemophilus
isolation media or as satellite
colonies Gram-negative coccobacilli and
filaments Factor V required ALA and X-V disk
Tests Acid from sucrose and fructose No acid
from ribose and xylose.
Composite photograph illustrating cultural
characteristics for the identification of
Haemophilus species.
26
Haemophilus Laboratory Features
By definition members of the genus Haemophilus
have absolute growth requirements for
nicotinamide adenine dinucleotide (NAD, coenzyme
I, Factor V) and/or iron-containing pigments
(hemin, hematin, X Factor). Sheep erythrocytes,
upon lysis, release NADases that render sheep
blood deficient in V Factor, precluding growth of
any Haemophilus species requiring this factor
(notably H. influenzae and in this case, H.
segnis). This phenomenon does not occur with
rabbit or horse blood. Selective culture media is
set up in most laboratories for the recovery of
Haemophilus factor-dependent species from select
specimens, particularly from sputum and
nasopharyngeal secretions obtained from children
under 2-years old, and from cerebrospinal
fluid. Physicians should let laboratory personnel
know if H. influenzae infections are clinically
suspected so that selective recovery media can be
set up and appropriate follow-up tests be
performed.
27
Haemophilus segnis Recapitulation
Haemophilus segnis is an uncommon isolate in
clinical laboratories, and a rare cause of
infection. The case presented here, however,
serves as a reminder that such isolates on
occasion may be encountered, and the means for
making a definitive identification must be
pursued. Haemophilus segnis is part of the human
upper respiratory flora, and may be recovered
from dental plaques, and on occasion from the
pharynx as a secondary commensal in cases of
acute pharyngitis of other causes. The cause of
a pancreatic abscess infection as reviewed in
this case presentation is an unusual
circumstance. It is a V-dependent but can be
distinguished from Haemophilus influenzae by not
requiring extraneous X-factor, giving a positive
ALA test result, and producing acid from fructose
and sucrose. Haemophilus segnis shares many
features with Haemophilus parainfluenzae. The
majority of the latter biotypes are urease and/or
ornithine positive while H. segnis is negative
for these reactions.