Section 4: Chapter 16 Corynebacterium diphtheria

Listeria, Corynebacterium, and Similar Organisms a) Only in humans

b) Transmission: respiratory secretion or
exudates from skin lesions
General Characteristics Corynebacterium jeikeium
 Catalas positive ֍ Common in clinical specimen; proliferates as
 Gram-positive rods skin microbiota of hospitalized individuals
 Non-acid fast ֍ Not considered to be highly virulent
 Non-spore forming ֍ Penetration by IV devices
 Non-branchin rods
Nosocomial pathogens
Oerskovia spp: exhitbit extensive branching and
vegetative hyphae and penetrate into the agar  C. urealyticum
surface  C. amycolatum
 C. striatum
- No aerial hyphae
Rhodococcus (red coccus)
Nocardia spp. and Corynebacterium:
aerobic/facultative anaerobe  Water, soil, and manure of herbivores
 Infects animals and human = inhalation,
Coryneform bacteria: similar morphology with ingestion, or inoculation through skin lesions
Corynebacterium spp
 Infects immunoncompromised people
 Arthrobacter
 Brevibacterium
 Cellulomonas Pathogenesis and Spectrum of Disease
 Cellulosimicrobium Listeria monocytogenes
 Dermabacter
 Exiguobacterium  Ability to survive within phagocytes
 Leifsonia Corynebacterium diphtheria
 Microbacterium
 Rothia  Produces extremely potent cytotoxic exotoxin
 Not all strains are toxigenic
Rhodococcus equiI (Corynebacterium equi):  Toxin gene: present in that have acquired gene
opportunistic pathogen of immunocompromised tox by viral transduction
patients o Incorporation of the toxin gene into the
Listeria monocytogenes: bacteria’s genome
o Blocks protein synthesis in humans =
» Facultative anaerobe cells die
» Catalase-positive o EXTREMELY LETHAL
» Non-branching o Only toxin-producing = cause diphtheria
» Oxisdase negative  Respiratory or cutaneous
» Gram-positive rod
» Misindentified as diptheroids, cooci, roc
» Assoc. with food-borne infections Respiratory diphtheria:
a) Pharyngitis
Epidemiology b) Dysphagia (difficulty in swallowing)
c) Low-grade fever
 Notable pathogens: Corynebacterium diphtheria d) Cervical
and Listeria monocytogenes e) Submandibular lymphadenopathy
Listeria monocytogenes f) Fever
g) General malaise
a. Widely distributed in nature
h) Headadche
b. Occasional pathogen of GI tract
c. Milk, raw vegetable, cheese, and meat
 » Large pseudomebrane composed of cellular
protein by toxigenic cellular killing = appear in
nasopharynx = obstruction in the airway
» Toxin spreads hematogenously
» Induce systemic organ damage = cardia
arrsest
Cutaneous diphtheria
 Endemic in tropical countries
 Toxigenic or nontoxigenic
 Shallow chronic skin lesions
L.monocytogenes
a) Transmission: ingestion of contaminated food
b) Vertical transmission: transplacental or through
an infected birth canal
c) Cross-infection in neonates is assoc. with
contaminated mineral oil – used for bathing
infants
d) Phagocytized by WBCListeriolysin O – major
virulence factor
Listeriolysin O: pore-forming toxin that reduces T-cell
responsiveness
Act A: surface protein; induce host cell actin
polymerization
֍ Forms pseudopod projectios
e) Reaches to the CNS and the placeta = listeriosis
f) Systemic disease manifestations childbirth and
neonatal death, meningitis, bacteremia,
encephalitis, and endocarditis
g) Localized infx: conjunctivitis, skin infection, and
lymphadenitis
h) Febrile gastroenteritis and assoc. with foodborne
outbreaks
i) Corynebacterium urealyticum: assoc. with cystitis
in hospitalized px, and those who had urologic
manipulation and in the elderly
Laboratory Diagnosis
Specimen Collection and Transport
No special consideration required for specimen
collection and transport
Specimen Processing
No special considerations required for specimen
processing
֍ Isolation of L. monocytogenes from placental
and other tissue
o Cold Enrichment: enhances the
recovery
 Nutrient broth, incuabated at
4°C for several weeks to
months
 Subcultured at frequent
intervals to enhance recovery
Direct Detection Methods
 Grams stain: only procedure used for direct
detection of bacteri
 Most genera (exluding Listeria, Rothia, and
Oerskovia) are classified as coryneform bacteria
 Gram-positive, short/slightly curved rods with
rounded ends with rudimentary branching
 Cells are arranged singly, in palisades” of parallel
cells, in pairs of cells connected after cell division
to form V or L shapes
 “Chinese letter” – group colonies
 L.monocytogenes: short, gram-positive rod that
may occur singly or in short chains, resembles
streptococci
Cultivation
Media of Choice
Corynebacterium spp: grow on 5% S-BA and ChocA
֍ Some do not grow on ChocA but lipophilic
(lipid loving)
o C.jeikeium
o C.urealyticum
o C. afermentans subsp. Lipophilum
o C. accolens
o C. macginleyi
 Produces larger colonies in 5%
S-BA with 1% Tween 80
Selective and differential media for C. diphtheria
֍ Cystine-tellurite blood agar
֍ Modified Tinsdale agar (TIN)
֍ Tellurite agar: with or w/o cysteine
o Cystine: enhance growth of fastidious
organisms including C.diphtheriae
֍ Both have high conc. of potassium tellurite
o Inhibits normal microbiota
 ֍ Growth on Tinsdale agar: differentiated based
on the conversion of tellurite to tellurium
o Results to color variations of gray to
black colonies
֍ C.diphtheriae produce halo on both media
o Presumptively identified by ibserving
brown-black colonies with a gray-
brown halo on Tinsdale agar
֍ Brown halo= use tellurite to produce
hydrogen sulfide
֍ Halo on cysteine-tellurite blood agar: brown =
breakdown of cysteine
֍ Loeffler medium: contains serum and egg
o Stimulates growth of C.diphtheriae
o Production of metachromatic granules
֍ C.diphtheriae grows rapidly on high enriched
agar
o Gray to white, translucent colonies
within 12-18 hours
o Unable to grown in MacConkey agar
 Capable of growth in routine
blood culture broth and
nutrient broths e.g.
thioglycollate or brain-heart
infusion
֍ Lipophilic coryneform= better growth in broth
with rabbit serum
Incubation Conditions and Duration
5% S-BA and ChocA: detectable growth of
Corynebacterium
- Incubated at 35°C; ambient air or in 5%-10%
CO2
- Occurs within 48-72 hours after inoculation
Lipophilic: grows more slowly
- 3 days or more for visible growth on routine
media
Growth of C. diphtheria: cysteine-tellurite agar and
modified Tinsdale agar
- Incubated for at least 48 hours in ambient air
- 5%-10% CO2 inhibits formation of halo on
Tinsdale agar
Colonial Appearance
C.diphtheriae colonies on cysteine-tellurite BA=
black/gray
Modified Tinsdale: black with dark brown halos
Approach to Identification
Except for L.monocytogenes, some Corynebacterium
are problematic and complex
 Multiphasic approach is required for definitive
identification
 Requires biochemical testing,
 Whole-cell fatty acid analysis
 Cell wall diamino acid analysis or 16S rRNA gene
sequencing
o For reference laboratories
 Coryneforms: present in normal microbiota
throughout the body
Indicators of Clinical Relevance
1) Isolation from normally sterile sites or multiple
blood cultures
2) Isolation in pure culture or as the predominant
organism in symptomatic px
3) Isolation from urine as a culture at >10 000
CFU/mL or the predominant organism at >10
000CFU/mL
Coryneforms: mos likely cause of UTI if the pH is
alkaline or with struvite crystals composed of
phosphate, magnesium, and ammonia
API Coryne strip: commercial product for rapid
indentification
Matrix-Assisted Laser Desorption Ionization
Time-Of-Flight Mass Spectrometry
- Identify toxigenic strains of Corynebacterium
spp.
- Rapid identification and source tracking of
L.monocytogenes: identification in dairy
products
Molecular Methods
☼ Ribotyping
☼ Pulsed-field gel electrophoresis
☼ Multilocus sequence typing
o Exhibited improved sensitivity and
effective for identification during
outbreaks
☼ PCR for quantitative detection of
L.monocytogenes in food producs
☼ L.monocytogene DNA: hly gene – encodes
listeriolysin o in CSF and tissue
(fresh/paraffin? Can be detected by molecular
assays
Comments on Specific Organisms
Halo on Tinsdale agar and Urea hydrolysis: used to
separate C.diphtheriae
Definitive identification of C.diphtheriae:
demonstration of toxin production by isolate
Toxin detection Methods:
a) Guinea pig lethality test
a. Determines whether diphtheria
antitoxin neutralizes the lethal effect of
a cell-free suspended of the organism
b) Immunodiffusion test
c) Tissue culture test
a. Demonstrate the toxicity of a cell-
culture free suspension in tissue
culture cells
b. Neutralization of the cytopathic effect
by diphtheria antitoxin
d) Enzyme immunoaasay (EIA)
e) PCR to detect antitoxin gene
Toxin testing: performed in reference laboratories
Catalase-positive, nonmotile, nonpigmented, and
esculin and gelatin negative: clinically relevant
strains
Irregular, gram-positive rod – strictly aerobe, is
nonlipophilic, oxidizes/does not utilize glucose =
Leifsonia aquatic or Arthrobacter, Brevibacteriu, or
Microbacterium spp.
Tween 80/serum: enhance growth by lipids, useful
for preliminary identification
 C. jeikeium
 C. urealyticum
o Resistant to several antibiotics
L.monocytogenes – identified by observing the
motility by direct wet mount
 End-over-end tumbling motility when
incubated in nutrient broth at RT for 1-2 hours
 Motility: umbrella-shaped patterm
 Ferments glucose and VP + and esculin +
 Differentiated by CAMP test
o Identification of S.pyogenes
Listeriosis: small, gram-positive, catalase positive rod
with narrow zone of beta hemoloysis in blood or CSF
- Presumptive evidence
Serodiagnosis
a. Anti-listeriolysin O Ab: detected in listerioris
b. IgM Ab: undetetable
Antimicrobial Susceptibility Testing and Therapy
 L.monocytogenes: no resistance of
therapeutic drugs
 AST not routinely necessary
CLSI document M45: information guidelines for
testing of Corynebacterium spp
֍ Some strains of Corynebacterium spp: required
48 hours of incubation
o Insufficient growth/ isolate appears
susceptible to beta-lactams at 24 hours,
o Incubate medium for 48 hours before
results reporting
Prevention
 Immunization with multidose diphtheria toxoid
o Inactivation of the toxin with
formaldehyde
 Combination vaccines
o Diphtheria
o Tetanus
o Pertussis
 2 of these are given to children <7 year olds
(DTap, DT)
 For older children and adults: Tdap and Td
 Td boosters: every 10 years to maintain
protection
Treatment
Diphtheria anti-toxin (DAT): hyperimmune antiserum
produced in horses
 Preparation of antibodies capable of toxin
neutralization before its entry into the
patient’s cell
 Administered as soon as complete
presumptive test is done
 Test for skin scratch test in forearm
IM penicillin/course of oral erythromycin
  For 14 days for px with symptoms for
diphtheria
 Prophylaxis for person exposed to diphtheria
Nonimmunized: should begin primary series of
immunizations
Properky wash raw vegetables and throuughly cook
vegetables and meat to prevent listeriosis
Immmnocompromised & pregnant women: avoid
eating soft cheese to prevent food-borne listeriosis
RTF e.g. hot dogs or cold cuts – heated thoroughly
before consumption and stored only for a short
period of time
L. monocytogenes: can replicate during refrigeration t
4°C.