and yet more fastidious Gram negative coccobacilli

1
and yet more fastidious Gram negative
coccobacilli

• Pasteurella, Francisella and Brucella
• (but not Yersinia?)

2
Pasteurella introduction

• This genus is the namesake of a bacterial family
  Pasteurellaceae. Over the years members of this
  family have been taxonomically scrutinized and
  reclassified genera and species in and out.
  Two highly significant human pathogens are no
  longer in this genus Yersinia pestis and
  Francisella tularensis. Strangely, the genus
  Haemophilus is currently included in the family
  Pasturellaceae.
• Pasteurella species are non-motile, Gram negative
  (often exhibiting bi-polar staining),
  facultatively anaerobic coccobacilli-rods.
• Pasturella species are common oral resident of
  most cats dogs, and can be found in many other
  animals, in some cases as a pathogen. Some
  species can be transferred zoonotically to
  humans. P. multocida is the primary medically
  significant species.

3
P. multocida Gram stain
4
Pathology

• Although P. multocida has been touted as an
  awesome veterinary pathogen it is not generally
  so serious for humans. The organism causes a
  serious hemorrhagic condition in cattle, a
  cholera-like condition in fowl, severe pulmonary
  abcesses in various animals, and various other
  animal pathologies.
• The organism can be transferred from infected
  animals to humans via bites or through open
  wounds. They usually result in surface tissue
  abcesses at the bite location (bites of cats or
  dogs, or lions or tigers - ya, it said that!)
• Human abcesses caused by P. multocida are due
  largely to endotoxin and are characterized by
  pain, edema, and raised abcess formation.
• Infections can disseminate to joints or
  underlying tissues causing a chronic arthritic
  condition
• Less common human pathologies include mild
  pneumonia, otitis media, and bacteremia.

5
Ascesses of P. multocida introduced zoonotically
by traumatic implantation
6
Cultural characteristics

• Swab-sampled abcesses can be plated on SBA.
  Colonies are smooth, gray, non-hemolytic and
  0.5-2mm (like a lot of things) after 24hrs 35oC.
  Colonies have a characteristic chemical odor on
  protein containing media due to large amounts of
  indole produced from the amino acid
  tryptophan.could be E. coli or Proteus? No
• The organism does not grow on enteric selective
  media, and remember that it is non-motile.

7
P. multocida
8
Identification

• P. multocida is oxidase positive, ONPG positive,
  indole positive, urease negative, and reduces NO3
  ?NO2
• Most produce acid from common sugars except
  lactose.
• Isolates recovered from likely specimens from
  patients that have come in contact with animals,
  and with the following characteristics is strong
  presumptive evidence of P. multocida oxidase
  positive, Gram negative rods that are strongly
  indole positive and fail to grow on Mac or EMB.

9
Other Pasteurella species

• Several other species rarely cause pathololgy in
  humans. Speciation is difficult due to
  similarity between the species in regard to
  symptoms, morphology and biochemical testing.
• Fortunately, speciation is not unecessary as
  treatment regimes are identical.
• Some other potentially human pathogenic
  Pasteurella species include P. pneumotropica
  P. haemolytica P. aerogenes P.
  dagmatis P. canis P. stomatis P.
  caballi

10
Francisella introduction

• The only significant species in this genus is F.
  tularensis.
• F. tularensis is also an animal pathogen,
  primarily of rabbits and squirrels, but other
  mammals are affected as well.
• Rodents are highly susceptible to this organism.
  Tularemia was first described in 1911 by McCoy
  who thought this may be a type of bubonic plaque
  due to its affects on rats.
• Also like Pasteurella, F. tularensis is
  transmitted zoonotically to humans, either by the
  bite of an infected deerfly or tick, directly
  from the blood of an infected animal (through a
  wound farmers, butchers, taxidermists), or by
  eating undercooked meat of the infected animal.
• Recent reports suggest a conjunctival route of
  infection as well. In any case, the infection is
  highly acute and potentially life-threatening.
  Cases are commonly reported in all 50 states but
  most in Arkansas and Missouri
• The cells are small, nonmotile, Gram-negative
  coccobacilli.

11
F. tularensis
12
Pathology

• F. tularensis is a facultative intracellular
  parasite, which induces strong cellular and
  humoral immune responses - results in complete
  life-long B-cell memory.
• Most strains of F. tularensis (especially those
  that infect rabbits) are highly virulent to
  humans making this a significant threat for use
  as a biological weapon. For this reason,
  tularemia is also known as rabbit fever.
• Laboratory workers are often infected while
  working with the organism. Must use P3 or P4
  containment conditions in the lab careful!
  Untreated mortality reaches 30.
• Intradermal infections form a local abscess
  followed by septicemia with rapid spread to the
  liver and spleen.
• GI infections result in a severe typhoid-like
  condition, with local abscess formation in the
  Peyer's patches. It is associated with high
  fever and severe septicemia.

13
Intradermal tularemia
14
continued

• Secondary pathologies following ID or GI
• Glandular / lymphatic infections develop in
  virtually all systemic cases
• Pneumonic infections develop in 10-20 of cases
• All infection are highly acute and potentially
  shock inducing. The organism causes
  cell-mediated immune hypersensitivity causes
  most of the pathology.
• Factors contributing to the impressive virulence
  of the organism are not understood.
• Studies suggest that infection is limited to
  reticuloendothelial tissues and peripheral blood,
  although pathology is frequently systemic. This
  is odd considering that no exotoxins have been
  identified
• Possession of an odd LPS suggests that endotoxin
  may play a major role in pathology.

15
Lymphatic tularemia
16
Cultural characteristics

• Franciscella is nutritionally fastidious.
  Cultivation is difficult and slow. Primary
  isolation media include cysteine-glucose-blood
  agar, cysteine heart agar, chocolate agar,
  Thayer-Martin and horse blood based media. It
  grows poorly if at all on SBA
• Growth is enhanced by addition of cysteine and
  growth in an elevated CO2 environment
• Colony morphology 1-3 mm, gray-white to
  bluish-gray with a smooth flat surface at 48-72
  hrs. May take up to 7 days to see colonies for
  morphology.
• Plates must be incubated at 37C for at least 3
  weeks before being discarded as negative.
• The organism grows readily in developing chick
  embryos.

17
F. tularensis on cysteine heart agar
18
Identification

• Difficulty in culturing this organism makes
  classical ID impractical, although evedence of
  characteristic morphology is certainly beneficial
  
• Nutritionally and biochemically F. tularensis
  bears a close resemblance to Brucella, but it can
  be differentiated from members of this genus on
  the basis of serology and DNA homology tests.
• Smears of pathologic material or blood cultures
  may be stained using the direct fluorescent
  antibody technique. Hemagglutinins appear in
  serum samples some 10 to 12 days after infection
  and slowly increase in titer for up to 8 weeks. A
  rising titer is always diagnostic of active
  disease.

19
Brucella introduction

• Brucella is a worldwide zoonotic pathogen that
  causes a variety of conditions with varying
  pathology, both chronic and acute. For this
  reason it is often misdiagnosed.
• Discovered in spleens of British soldiers dying
  of Malta fever from consumption of goats milk
  cheese.
• Found in many wild animal species ranging from
  antelope to fish, and domesticated livestock such
  as sheep, goats and cattle - 1livestock
  pathogen in some cases places.
• Primary means of transmission is from infected
  animals through the skin (like Francisella), by
  consumption of contaminated dairy or meat from
  infected animals.
• Less commonly transmitted via the respiratory
  tract or through the conjunctiva.
• Like tularemia, this pathogen is easily
  transmitted to lab workers

20
continued

• Prior to modern times human Brucellosis was
  prevalent around livestock animal products
  industry although vaccination and culling of
  geriatric or otherwise suspicious members of the
  herd has improved things.
• Cases now rare in the US, but still common in
  Latin America, Asia, and Africa.
• Brucella are Gram (-), aerobic, non-motile
  coccobacilli
• Brucella taxonomy is unresolved but 6 species are
  recognized named by the animal species they
  infect - B. suis (hogs), B. melitensis (goat,
  sheep), B. canis (dogs), B. abortus (cattle), B.
  ovis (rams) B. neotomae (rats)
• Only B. melitensis, B. abortus and B. suis are
  important as human pathogens, probably in that
  order worst to least

21
Brucella
22
Pathology

• Brucellae are primarily reticuloendothelial
  pathogens.
• Common human symptoms include joint pain
  (especially back), splenomegaly, anemia, and
  recurrences with undulant fever weeks after
  apparent successful treatment.
• Recurrences may be due to latent cells existing
  as intra-macrophage parasites. These can result
  in formation of macrophage giant cells in
  lymphatic tissues causing lymphatic granulomas
  and possibly other tissue or organ involvement
• Most cases recover entirely within 3-12 months
  but some enter an ill-defined chronic stage with
  various symptoms of any organ system. Often
  misdiagnosed (leukemia, ?)
• Some of the pathology is caused by the
  significant host defense hypersensitivity induced
  by the organism
• Abortion is NOT a feature of pathology in humans.

23
Virulence

• Being an intracellular parasite gives it
  protection from host immunity. They are uniquely
  adapted to live multiply inside
  monocyte-macrophages - dominates the pathology.
  This also allows the organism to be systemic
  (never local) by disseminating through lymphatic
  tissues
• A prevalent LPS layer containing a unique
  cyclic-glucan is an important part of virulence
  although little is known
• The organism is not encapsulated

24
Brucella lesion on leg of caribou
25
Brucella spontaneous abortion
26
Culture, ID, diagnosis

• Blood is the sample of choice. Brucellae are
  difficult to culture 20-50 isolation rate by
  experienced labs.
• Visible colonies on SBA, CBA or BCYE incubated at
  35oC can sometimes be present in 5-7 days but may
  take as long as 4-6 weeks! Colonies are
  unexceptional but can present in either a
  smooth or rough form depending on the nature
  of their LPS.
• Serology is the mainstay of laboratory diagnosis,
  but result interpretation is particularly
  difficult for this organism for several reasons
  including cross-reaction of Brucella and
  tularemia antigens. A confident diagnosis is
  often impossible.
• Accurate diagnosis must consider clinical
  suspicion (undulant fever, etc) and patient
  history with animals.

27
Brucella melitensis colonies