LISTERIA, FRANCISELLA, BRUCELLA, BACILLUS AND YERSINIA
MBIM 650/720 Medical
Microbiology Lecture: 44 - 45
Links - Zoonoses
know the general morphology and physiology the organisms
know epidemiology and clinical symptoms
understand the mechanisms pathogenesis
know the diagnostic, therapeutic and preventive procedures
READING: Murray, et
al.: Medical Microbiology, 3rd ed., Chapter 25, pp. 209-210;
chapter 29, pp. 240-242 and chapter 34, pp. 271-275.
refers to a disease primarily of animals which can be transmitted to
humans as a result of direct or indirect contact with infected animal
are Gram-negative, nonmotile, coccobacilli. They are strict
aerobes and grow very slowly (fastidious) on blood agar. In the
host, they live as facultative intracellular pathogens.
Brucella abortus - Gram-negative, coccobacillus prokaryote;
causes bovine spontaneous abortion due to its rapid growth in
the presence of erythritol (produced in the plancenta). SEM x
29,650 © Dr
Dennis Kunkel, University of Hawaii. Used with permission
Brucella ovis in epididymis © Bristol
Biomedical Image Archive, University of Bristol. Used with
Epidemiology, transmission and symptoms
primarily a disease of animals and it affects organs rich in the sugar erythritol
(breast, uterus, epididymis, etc.). The organism localizes in these
animal organs and cause infertility, sterility, mastitis,
abortion or resides as carriage. Humans in closed contact with infected
animals (slaughterhouse workers, veterinarians, farmers, dairy workers)
are at risk of developing undulant fever. There are 100-200 cases
of brucellosis seen in the US, although the worldwide incidence is
estimated at 500,000. Four different species of Brucella are
known to infect humans: B. abortus (cattle), B. suis
(swine), B. melitensis (goats/sheep) and B. canis (dogs).
Although brucellosis has been eradicated in most developed countries
through animal vaccination, it persists in many underdeveloped and
and B. canis cause a mild suppurative febrile infection whereas B.
suis causes a more severe suppurative
infection which can lead to destruction of the lymphoreticular organs
and kidney. B. melitensis is the cause of most severe prolonged
recurring disease. The bacteria enter the human host through the mucous
membranes of the oropharynx (ingestion/inhalation routes), through
abraded skin, or through the conjunctiva. Usually infection occurs by
direct contact with infected material, although it may also occur by
ingestion of milk or milk products. The bacteria are engulfed by
neutrophils and monocytes and localize in the regional lymph nodes,
where they proliferate intracellularly. If the Brucella organisms are
not destroyed or contained in the lymph nodes, the bacteria are released
from the lymph nodes resulting in septicemia. The organisms migrate to
other lympho-reticular organs (spleen, bone marrow, liver, testes)
producing granulomas and/or micro abscesses. Symptoms include fever,
chills, sweats, fatigue, myalgia, profound muscle weakness, and
anorexia. Joint involvement occurs often. Brucellosis may be either
acute or chronic. Fatalities (0-3%) generally due to endocarditis.
The symptoms in
brucellosis are due to the presence of the organism and appear 2 - 4
weeks (sometimes up to 2 months) after exposure. While in the
phago-lysosome, B. abortus releases 5'-guanosine and adenine
which are capable of inhibiting the degranulation of peroxidase-containing
granules and thus inhibit the myeloperoxidase-peroxide-halide system of
bacterial killing. The intracellular persistence of bacteria results in
granuloma formation in the reticuloendothelial system organs and tissue
damage due to hypersensitivity reactions, mostly type-IV.
Diagnosis is based
on prolonged (at least a week) presence of undulating fever, myalgia,
and the history of exposure (contact with animals or consumption of
unprocessed material from infected animals). Definitive diagnosis can be
made by culturing blood samples on blood enriched media. The
(fastidious) organisms grow very slowly (4-6 weeks in blood culture). B.
abortus but not other Brucella grow better in 5% CO2
atmosphere. On blood agar, they produce white glistening colonies.
Serology can be used to further confirm the diagnosis.
with rifampin, which penetrates cells with streptomycin or tetracyclin
is used to treat human Brucella infections. The control measures
include animal vaccination and avoidance of infected material.
is a pleomorphic,
Gram-negative, bipolar staining, facultatively aerobic, nonmotile,
bacillus. Optimal temperature for growth is 28EC. It is a facultative
Histopathology of spleen in fatal human plague - Necrosis and
Yersinia pestis. CDC/Dr. Marshall Fox
Histopathology of lymph node in fatal human plague -
Medullary necrosis with fluid and Yersinia pestis. CDC/Dr.
Yersinia pestis CDC
Wayson stain of Yersinia pestis. Note the characteristic
safety pin appearance of bacteria CDC
Yersinia pestis - rod prokaryote (dividing); causes bubonic
plague (SEM x20,800) © Dr
Dennis Kunkel, University of Hawaii. Used with permission
transmission and symptoms
Swollen lymph glands (buboes) caused by plague bacteria
in bubonic plague CDC
Male Xenopsylla cheopsis (oriental rat flea) engorged with
The three documented
pandemics of plague (Black Death) have been responsible for the death of
hundreds of millions of people. Today, sporadic infections still occur.
In the U.S., animal (sylvatic)
plague occurs in a number of western states, usually in small rodents
and in carnivores which feed on these rodents.
Humans are infected
by carrier rodent fleas or by contact with infected animals. The flea
acquires the Y. pestis organisms during a blood meal from
infected rodents. These organisms lose their capsule, multiply in the
intestinal tract and partially block the proventriculus. During the feed
on a human host the flea regurgitates some of the organisms into the
wound. The bulk of non-capsular organisms are phagocytized and destroyed
by neutrophils. However, few organisms are taken up by histiocytes which
are unable to kill them and allow them to resynthesize their capsule and
multiply. The encapsulated organisms, when they are released from
histiocytes are resistant to phagocytosis and killing by neutrophils.
The resulting infection spreads to the draining lymph nodes which become
hot, swollen, tender and hemorrhagic giving rise to the characteristic
black buboes whence the name of the disease, bubonic plague
is derived. Within hours the organism spreads into the spleen, liver and
lungs resulting in pneumonia. While in circulation, the organism causes
diffuse intra vascular coagulation resulting in intra vascular thrombi
and purpuric lesion all over the body. If untreated, the infection has a
very high (unto 90%) mortality rate. The organism in exhaled in cough
droplets, infect other humans in close proximity and cause pneumonic
plague, which more difficult to control and has 100% mortality.
factors play direct and indirect roles in Y. pestis pathogenesis.
response (lcr) :
This is a plasmid-coded gene that enables the organism to grow in a
low Ca++ (intracellular) environment. It also coordinates
the production of several other virulence factors, such as V, W and yops
(Yersinia outer proteins).
V and W proteins:
These plasmid-coded proteins are associated rapid proliferation and
A group of 11 proteins, which are coded by plasmids, are essential
for rodent pathogenesis and are responsible for cytotoxicity,
inhibition of phagocyte migration and engulfment and platelet
antigen: It is a
protein-polysaccharide complex which is highly expressed at 37
degrees in the mammalian host but not in the flea and is anti-phagocytic.
Both of these are plasmid-coded proteins. Coagulase
is responsible for micro thrombi formation and plasminogen
activator promotes the dissemination of the organism. It also
destroys C3b on the bacterial surface, thus attenuating phagocytosis.
Diagnosis is based
on appearance of buboes. The diagnosis is confirmed by culture of a
lymph node aspirate. Extreme caution is warranted in handling of the
specimen, as it is highly infectious.
Plague suits. The beak was filled with sweet smelling
oils and vinegar to counteract the smell of plague victims.
Although physicians in the 1300's did not know the cause of
plague, the suits may have been effective to some degree in
that they kept fleas off and the shiny beak may have posed an
obstacle to the entry of fleas. Left From: Bubonic
Plague by Ely Janis Right: © Bristol Biomedical
Image Archive, University of Bristol. Used with permission
strict isolation are the rule. Streptomycin and tetracycline are highly
effective. An effective formalin-killed vaccine is available but is
recommended only for people at a high risk. The disease is
internationally quarantinable and reporting of cases is mandatory.
Control of urban plague is based upon flea and rodent control.
World distribution of plague 1998 CDC
US distribution of plague by county CDC
anthracis is the causative agent of anthrax. It is a Gram-positive,
aerobic, spore-forming large bacillus. Spores are formed in culture, in
the soil, and in the tissues and exudates of dead animals, but not
in the blood or tissues of living animals. Spores remain viable in soil
Robert Koch's original micrographs of anthrax bacillus DOD
Gram stain of anthrax DOD Anthrax Program
Anthrax due to Bacillus anthracis (blood smear) ©
Bristol Biomedical Image Archive, University of Bristol. Used
transmission and symptoms
Anthrax is a major
disease threat to herbivorous animals (cattle, sheep, and to a lesser
extent horses, hogs, and goats). People become infected by the cutaneous
route (direct contact with diseased animals, industrial work with hides,
wool, brushes, or bone meal), by inhalation (Woolsorter's disease),
or by ingestion (meat from diseased animals).
accounts for more than 95% of human cases. Spores enter through small
break in skin, germinate into vegetative cells which rapidly
proliferate at the portal of entry. Within a few days, a small papule
emerges that becomes vesicular. The latter is filled with blue-black
edema fluid. Rupture of this lesion will reveal a black eschar
at the base surrounded by a zone of induration.
This lesion is called a malignant pustule, however, no pus or pains
are manifested. The lesion is classically found on the hands, forearms
or head. The invasion of the bloodstream will lead to systemic
dissemination of bacteria.
Cutaneous Anthrax DOD Anthrax Program
results form inhalation of B. anthracis spores which are
phagocytized by the alveolar macrophages where they germinate and
replicate. The injured host cell and organisms infect the hilar lymph
node where marked hemorrhagic necrosis may occur. The patient may
manifest fever, malaise, myalgia, and a nonproductive cough. Once in
the hilar lymph node, infection may spread into the blood stream.
Respiratory distress and cyanosis are manifestations of toxemia. Death
results within 24 hours. This form of anthrax is of significance in
the biological warfare.
: Ingestion of
meat-derived from an infected animal leads to organism proliferation
within the gastrointestinal tract, invasion of the epithelium, and
ulceration of the mucosa. The invasion spreads to the mesenteric lymph
nodes and then to the blood. Initially there is vomiting and diarrhea
followed by blood in the feces. The invasion of the blood is
associated with profound prostration, shock, and death. Because of
strict control measures, this form of anthrax is not seen in the U.S.
Anthrax, blood clot passed from anus © Bristol
Biomedical Image Archive, University of Bristol. Used with permission
factors of B. anthracis include a number of exotoxins and the
A plasmid-encoded, heat-labile, heterogeneous protein complex made up
of 3 components: (1) Edema Factor (EF); (2) Lethal Factor
(LF); and (3) Protective Antigen (PA). In vivo, these
three factors act synergistically (for toxic effects). The protective
antigen binds to surface receptors on eucaryotic cells and is
subsequently cleaved by a cellular protease. The larger C-terminal
piece of PA remains bound to the receptor and then binds either EF or
LF, which enters the cell by endocytosis. Edema Factor, when inside
the cells binds calmodulin-dependent and acts as adenylate cyclase.
Lethal factor's mechanism of action involves activation of macrophages
and production of cytokines which cause necrosis, fever, shock and
death. Individually, the three proteins have no known toxic activity.
Antibodies to protective antigens prevent PA binding to cells stop EF
and LF entry.
The capsule consists of a polypeptide of D-glutamic acid which is
encoded by a plasmid and is antiphagocytic. It is not a good immunogen
and even if any antibodies produced, they are not protective against
of anthrax can be confirmed by direct examination or culture. Fresh
smears of vesicular fluid, fluid from under the eschar, blood, or spleen
or lymph node aspirates.are stained with polychrome methylene blue and
examined for the characteristic blunt ended, blue-black rods with a pink
capsule. In case of a negative finding, the specimen can be cultured on
blood agar plates. Cultured organism stains as Gram-positive long thin
Penicillin is the
antibiotic of choice. Antibody to the toxin complex is neutralizing and
protective. There are two vaccines available. One is for use for
immunizing cattle and other herbivorous animals and the other for
Listeria monocytogenes - rod prokaryote that causes
listeriosis, meningitis and food poisoning ©
Dr Dennis Kunkel,
University of Hawaii. Used with permission
sheep: listeriosis © Bristol Biomedical Image
Archive, University of Bristol. Used with permission
is a facultative intracellular, Gram-positive coccobacillus which often
grows in short chains. It is different from other Gram-positive
organisms in that it contains a molecule chemically and biologically
similar to the classical lipopolysaccharide, the listerial LPS. The
organism forms beta hemolytic colonies on blood agar plates and
blue-green translucent colonies on colorless solid media. Upon infecting
a cell (macrophages and parenchymal cells), the organism escapes from
the host vacuole (or phagosome) and undergoes rapid division in the
cytoplasm of the host cell before becoming encapsulated by short actin
filaments. These filaments reorganize into a long tail extending from
only one end of the bacterium. The tail mediates movement of the
organism through the cytoplasm to the surface of the host cell. At the
cell periphery, protrusions are formed that can then penetrate
neighboring cells and allow the bacterium to enter. Due to this mode of
cell-cell transmission, the organisms are never extracellular and
exposed to humoral antibacterial agents (e.g., complement,
antibody). L. monocytogenes is readily killed by activated
Listeria monocytogenes organisms in neutrophil (blood smear) ©
Bristol Biomedical Image Archive, University of Bristol. Used with
monocytogenes is a ubiquitous organism found in the soil,
vegetation, water, and in the gastrointestinal tract of animals.
Exposure to the organism can lead to asymptomatic miscarriage or disease
in humans. At greatest risk for the disease are the fetus, neonates,
cancer patients and immuno-compromised persons. In the U.S., a number of
recent outbreaks have been traced to cheese, cole slaw (cabbage), milk,
and meat. The organisms can grow at 4 degrees C which means that
organism replication continues in refrigerated foods. Laboratory
isolation can employ a cold enrichment technique.
Listeriosis has been
categorized in two forms: 1) neonatal disease and 2) adult disease.
Neonatal disease can occur in two forms: the early onset disease,
acquired transplacetally in utero and late onset disease
acquired at birth or soon after birth. In utero acquired
infection (granulomatosus infantiseptica) causes abscesses
and granulomas in multiple organs and very frequently results in
abortion. Exposure on vaginal delivery results in the late onset
disease resulting in meningitis or meningo-encephalitis with sepsis
within 2 to 3 weeks.
Infection in normal adults results in self-resolving flu-like
symptoms and/or mild gastrointestinal disturbance. Chills and fever
are due to bacteremia. In immunosuppressed individuals it can
produce serious illness, leading to meningitis. It is one of the
leading causes of bacterial meningitis in patients with cancer and
in renal transplant recipients. In the elderly, the early symptoms
may go unnoticed and the infection may lead to acute manifestations
of sepsis (high fever, hypo-tension). A complication of the
bacteremia is endocarditis.
Listeriolysin O, a
ß-hemolysin, is related to streptolysin, and pneumolysin and is
produced by virulent strains. It disrupts the phagocytic vacuole and is
instrumental in cell-cell transmission of the organism. The toxin is
oxygen labile and immunogenic.
indicated when blood and CSF monocytosis is observed. The organism can
be isolated on most laboratory media.
alone or in combination with gentamycin have been effective. Immunity is
tularensis is a small, Gram-negative, non-motile, encapsulated,
pleomorphic coccobacillus (short rod). It is a facultative intracellular
parasite which grows poorly or not at all on most laboratory media and
requires a special glucose cysteine blood agar for isolation. Care must
be taken in handling the sample because of the low infectious dose.
tularensis is the causative agent of tularemia (a reportable disease
in the U.S.). Unlike plague, tularemia occurs routinely in all 50 of the
United States. Its primary reservoirs are rabbits, hares, and ticks. Man
most commonly acquires tularemia via insect bites (ticks primarily, but
also deer flies, mites, blackflies, or mosquitoes) or by handling
infected animal tissues. Human disease (rabbit or deer fly fever) is
characterized by a focal ulcer at the site of entry of the organisms and
enlargement of the regional lymph nodes.
Thumb with skin ulcer of tularemia. CDC/Emory U./Dr. Sellers
As few as10 - 50
bacilli will cause disease in humans if inhaled or introduced
intradermally, whereas a very large inoculum (~108 organisms)
is required for the oral route of infection. Incubation period is 3 - 10
days. A small skin papule usually develops at the site of entry.
Ulceration occurs together with fever, chills, malaise, fatigue, and
usually lymphadenopathy. Bacteremia usually occurs and the bacilli then
grow intracellularly in the reticuloendothelial system. Dissemination of
the organisms through the bloodstream permits focal lesions to develop
in numerous organs. The patient will normally exhibit one of several
clinical syndromes. Ulcerogalndular form is most common (70 -
85%) in which a painful ulcerating papule which has a necrotic center
and raised periphery develops at the site of infection. Other forms are
glandular (lymphadenopathy without ulcer), typhoidal, pneumonic,
oculoglandular and rarely oropharyngeal (pharyngotoncillitis with
The capsule of the
organism renders it resistant to phagocytosis. Intracellularly, the
organisms resist killing by phagocytes and multiply. Most of the
symptoms are due to cell-mediate hypersensitivity.
is difficult to visualize in direct smears. The organism can be isolated
from specimens of sputum, or lymph node aspirates inoculated on
chocolate blood agar. Blood cultures are often negative. The organism
grows very slowly and hence must be incubated for several days.
The identity of the organism is confirmed with specific antisera.
Streptomycin is the
drug of choice for all forms of tularemia. Untreated, cases have a
fatality rate of 5 - 15%. A live attenuated organism vaccine is
available but its use is restricted to those persons who are at risk.
Immunity appears to be cell mediated. One must avoid handling infected
animals, watch out for ticks and utilize clean water supplies.
This is an
occupational disease of butchers, meat processors, farmers, poultry
workers, fish handlers: swine and fish handlers are particularly at
risk. The causative agent, Erysipelothrix rhusiopathiae, is a
Gram-positive anaerobic rod which infects through skin abrasion while
handling contaminated animal products or soil. Generally, the organism
produces an inflammatory skin lesion, on fingers or hand, which is violaceus
and has a raised edge. It spreads peripherally, as the discoloration in
the central area fades. The painful lesion is pruritic
and causes a burning or throbbing sensation. It lacks suppuration
and thus is distinguishable from staphylococcal erysipelas. Diffuse
cutaneous infection and septicemia are rare. The organism can be
cultured easily on most laboratory media. It is easily treatable with