Nonsporeforming aerobicfacultative Gram positive rods

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Nonspore-forming aerobic-facultative Gram
positive rods

• Corynebacterium, Listeria Nocardia

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Corynebacterium introduction

• Despite being ill-defined in its infancy,
  inclusion in the genus Corynebacterium is now
  restricted (for the most part) to organisms that
  fulfill 5 rules see 3rd paragraph on page 673.
  One of these is having a 32-carbon mycolic acid
  in the envelope called corynomycolic acid.
• Corynebacteria are unique in their morphology.
  As cells divide they snap to form L-shapes
  which come together in groups that resemble
  Chinese letters (palisade).
• Cells are thin rods, often slightly tapered on
  one end (Koryne is Greek for club-shaped) and
  clumped
• Cells may possess metachromatic granules or
  inclusions, most often polyphosphate, usually
  terminal
• They are non-motile and non-acid fast (vs
  Mycobacteria)
• Colonies are dull, irregular, white-yellow and
  chalky look like lumps of damp flower. When
  disturbed they crumble.
• Corynebacteria species are widely distributed in
  natural samples such as soil and water.

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Corynebacterium notice the Chinese letter
(palisade) arrangement of cells. The barred
appearance in polyphosphate granules
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Notice the slight tapering of cells to one end.
They are said to be club-shaped.
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Colony morphology
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Pathology - diphtheria

• Even C. diptheriae which is the most significant
  pathogen in this genus, is a common resident of
  the human microflora. Clearly, it is an
  opportunistic pathogen.
• Non-diptheria Corynebacteria were originally
  thought only to be significant as contaminants,
  but recently have been recognized as pathogens in
  immuno-compromized patients.
• Diptheria cases are constantly decreasing with
  increasing global vaccine usage, but
  non-diptherial infections associated with
  catheters, heart valves, shunts, and urinary
  tract infections, are more prevalent.
• C. diptheriae is primarily a URT pathogen. It is
  commensal in the oropharynx of a high percentage
  of folks. Humans are the only known host
  animal reservoir. The organism also causes
  primary (explain) LRT, GI, epidermal and
  genitourinary pathology, but these are relatively
  rare.

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Pathology virulence - diphtheria

• Diptheria has high virulence it is highly acute
  and contagious (airborne) with a 10-30 mortality
  rate. There are 3 recognized strains in
  decreasing order of virulence gravis,
  intermedius mitis.
• Prior to advent of conjugated vaccine in the
  1940s diptheria MM was high in the U.S. Boston
  epidemic in late 1700s killed 100,000, and 1
  million killed annually worldwide. Now there are
  less than 10 cases annually in the U.S.
• Pathology is primarily associated with the
  diptheria exotoxin which is formed only by cells
  lysogenized by bacteriaphage carrying the tox
  gene. Diptheria toxin is highly potent (3rd)
  why we use a toxoid vaccine. The toxin blocks
  host cell protein synthesis cell death.
• Infections are not systemic but toxemia results
  from dissemination of the toxin all over the body.

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continued

• Diptheria toxin causes necrosis of the pharyngeal
  epidermis and sloughing of a tough, leathery gray
  pseudomembrane which can obstruct the trachea
  and suffocate unattended infants (1 diptheria
  assoc. mortality in infants).
• Major targets of the disseminating toxin are
  heart (CHF 1 mortality in non-infants),
  peripheral nervous system (meningitis) and
  kidneys, but any tissue can be affected broadly
  varying symptoms sometime difficult diagnosis

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Gray pseudomembrane
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Pathology of non-diptheria species

• These species recently linked to human disease.
  Usually IDed to genus level, and to species level
  if isolated from normally sterile site. Often
  difficult to speciate even by reference labs.
  Rapid coryne system now available
• C. jeikeium (formerly CDC group JK) 1
  clinically significant non-diptheria species
  (septicemia), 1 most often isolated from
  normally sterile sites
• C. ulcerans can cause a URT similar to diptheria
  even produces the diptheria toxin. ???
• ? NOT ON TEST ?
• C. pseudotuberculosis (also known as C. ovis)
• C. pyogenes
• C. haemolyticum
• C. aquaticum
• C. pseudodiphtheriticum (also known as C.
  hofmannii)
• Group D2 (also known as C. urealyticum)

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Sample processing, culture ID

• Collect nasopharyngeal throat swab specimen by
  vigorously rubbing lesion.
• Examination of smears stained with the Gram
  method as well as Loefflers methylene blue is
  standard procedure.
• Gram stain of throat sample is useful to
  differentiate Vincents disease (ANUG - fusiform
  Gram (-) bacteria spirocheates) from pharyngeal
  diphtheria pathology
• Loefflers stain is best to see the polyphosphate
  granules characteristic of C. diptheriae

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continued

• Inoculate media see figure 13-2 page 677
• SBA 35oC for 24hrs morphology hemolysis vs Grp
  A Streptoccoci follow up with Gram stain
  catalase
• Loefflers 35oC for 4-8hrs methylene blue for
  morphology and basophilic polyphosphate granules
• Tinsdales 35oC for 24-48hrs dark colonies
  follow up with Loefflers (see above)
• The info above is the most important for
  speciating C. diphtheriae. Additional
  characteristics include production of urease,
  nitrate reduction, gellatin liquefaction and
  catalase are useful.

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Characteristic brown-black colonies formed by C.
diphtheriae on Tinsdale (left) or Tellurite agar
(serum cysteine and/or thiosulfate) due to
sulfate reduction in the presence of selective
antibiotics
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Listeria introduction

• The Genus contains 7 species. Only L.
  monocytogenes (Lm) and L. ivanovii are associated
  with human disease, with the former being
  clinically most important.
• Lm is a short Gram positive rod-coccobacillus.
  It is facultatively anaerobic and exhibits
  tumbling motility.
• Lm can be found in numerous sample types
  including virtually all natural environments,
  animal feces and food products, and feces of
  healthy and symptomatic humans. Soil plant
  detritus are the most likely sources of
  contamination, and transmission is most likely
  foodborne
• Like Brucella, this organism is virtually always
  systemic. Meningitis (meningoencephalitis) is the
  primary pathology but virtually any tissue or
  organ can be involved

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continued

• There are 2000 annual Listeriosis cases in the
  US with mortality lt70. As much as 1/3 of
  morbidity and mortality is in pregnant mothers
  who can also transmit the pathogen
  transplacentally or perinatally to their baby.
• Oddly, 3rd trimester mothers seem most at risk
• Listeriosis is also common in immuno-compromised
  meningitic individuals. These 3 groups make up
  virtually all Listeriosis cases.
• Considering the prevalence of the organism, it is
  likely that infectivity of the organism is low
• L. ivanovii is rarely isolated from human samples
  and its clinical importance remains uncertain.

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L. monocytogenes in macrophage
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L. monocytogenes being phagocytized
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Pathology

• Lm is another intracellular parasite of
  macrophages, or more often of monocytes, hence
  the name. This is responsible for some
  pathology, as well as affording the organism the
  same advantages as it did Brucella.
• Patients have meningitis symptoms (headache )
  and GI symtoms (vomiting pain) as well as back
  pain. Meningitic cases can progress to severe
  and life threatening.
• Prenatal or neonatal listeriosis has multiple
  organ pathology
• Some isolates have recently (and disturbingly)
  demonstrated resistance to tetracycline and
  trimethoprim-sulfamethoxyzole. These have
  classically been used in treatment due to
  effectiveness and ability to cross the
  blood-brain barrier for prenatal cases.
• Little in known about mechanisms of pathology
  beyond being intracellular, hemolytic inducing
  immune response.
• Lm is being researched as the basis for a cancer
  vaccine (Lovaxin) because of its ability induce
  potent immunity and sneak cancer antigen into
  host cells for directed attack.

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Culture ID

• Samples include blood, CSF, amniotic or placental
  samples and stool samples.
• If recovery proves difficult, Lm isolate recovery
  is enhanced using a cold-enrichment method. Mix
  sample in TSB, hold at 4oC, and periodically
  plate on SBA or CAN (Columbia Nalidixin) over a
  period of several days to 2 months until recovery
  achieved.
• Colonies on SBA after 24hrs at 37oC appear small,
  translucent and gray with narrow zone of beta
  hemolysis.
• Catalase () can be used to distinguish from S.
  agalactiae in cases of neonatal meningitis.
• Lm exhibits a unique tumbling motility in wet
  mounts.
• Presumptive ID via clinical suspicion,
  morphology, catalase, motility and beta hemolysis
  may be enough
• Lm serology was not successful until recently due
  to problems with cross-reaction was not used
  for regulatory ID of Lm. Improved serological
  methods such as RapidL mono test can be used now
  - chromogenic plate test

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Nocardia introduction

• Nocardia are ubiquitous in the soil worldwide.
  They are transmitted through the soil, either by
  inhalation or traumatic implantation. Soil of
  domesticated animal farms carry large numbers
  growth via feces as nutrient and periodic
  watering, then dust is inhaled.
• Nocardia are in the order Actinomycetales -
  taxomonically related to Mycobacteria. They share
  acid-fastness if the modified Kinyoun stain
  method is used.
• They morphologically resemble fungi having
  branched aerial mycelia and fruiting bodies
  resembling fungi Nocardiaform. For years
  they were classified as fungi prior to molecular
  methods that showed otherwise.
• Most species produce pigmented, waxy or mycelial,
  and biting colonies on an agar surface. Growth
  in broth is grainy (sandy pellet) or waxy (floats
  on top) rather than forming homogeneous
  turbidity.

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Acid-fast stain of Nocardia
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N. asteroides
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Pathology

• The 3 most significant Nocardia species as
  pathogens are N. asteroides (1?), N.
  brasiliensis, and N. madurae.
• Nocardia cause varied pathology. Each can be
  acute or chronic. Most patients are
  immunocompromised but some have no known
  underlying impairment in host defense.
• One is a chronic respiratory infection resembling
  tuberculosis frequently chronic atyptical
  pneumonia symptoms. It often causes pleurisy.
• Another is actinomycetic (vs mycomycetic)
  mycetoma local infections of cutaneous and
  subcutaneous tissue, fascia, and bone involving
  tumefaction, draining sinuses (yogurt-like
  drainage), and sclerotia (hard granules, grains)
  formation - following implantation from soil.

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continued

• A related condition is Madura foot, a chronic
  infection of the bones and soft tissues of the
  foot that can cause gross deformity. It most
  commonly occurs in Sudan, north Africa, and the
  west coast of India among people who walk
  barefoot. A common causative organism is N.
  madurae (sometimes classified as Actinomadura
  madurae).
• Nocardia is a facultative intra-macrophage
  parasite like Brucella (can also inhibit lysosome
  fusion) and others discussed same advantages
  considered Nocardias primary virulence factor).
  Therefore, nocardiosis can disseminate (which
  happens in 50 of cases) via the lymphatics
  causing lymphatic nodule formation, and pathology
  of virtually any organ or tissue. The CNS will
  be affected in about 1/3 of people with
  disseminated nocardiosis.
• Oddly, cells are very difficult to find in CSF of
  patients with CNS pathology path via immune
  hypersensitivity?

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N. asteroides pulmonary infection
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N. asteroides cutaneouslesions and inflamation
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Systemic lymphatic nodules - N. brasiliensis
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Culture ID

• Nocardia are aerobic. They grow on many media
  such as SBA, BHI, Sab-dex, and BCYE (thought to
  be superior for recovery) and have a broad range
  of growth temp from 25-45oC. Makes sense
  considering their home.
• Growth is often slow can take 4 days to 6
  weeks.
• ID via characteristic pathology, growth
  characteristics, colony and cellular morphology,
  and other below.
• Can be distinguished from Mycobacteria via
  presence of branched aerial hyphae with conidia
  (absent in Mycobacteria). Also Mycobacteria are
  strongly acid-fast using the Ziehl Neelsen
  procedure where Nocardia are weakly acid fast
  only with the Kinyoun modification.