Culture - Pathology

Haemophilus sp.
H. influenzae
Normal flora of human
upper resp. tract
Person-to-person, droplets;
sometimes endogenous
H. ducreyi
Not normal flora; only
present during infection
Person-to-person; sexual
Other Haemophilus spp.
Normal flora of human
upper resp. tract
Spread of endogenous strain
to non-resp. tract sites; less
common than H. influenzae
Clinical characteristics
H. influenzae
Major virulence factor is polyribitol phosphate capsule
- enhance resistance to phagocytosis
- serologic typing based on antigenic characteristics
- six capsule types: a, b, c, d, e, or f
- type b is the most commonly associated with
serious human infection
- infections are often systemic and life-threatening:
meningitis, epiglottitis, cellulitis with bacteremia,
septic arthritis, and pneumonia
Also produce factors that promote attachment to human
epithelial cells
Clinical characteristics, cont.
H. influenzae
Non-typeable strains do not produce a capsule
- virulence mediated through attachment (pili, etc.)
- infections are typically less serious and more
localized: otitis media, sinusitis, conjuctivitis, and
- pneumonia and bacteremia in adults with underlying
medical conditions
- isolated from patients with cystic fibrosis
Clinical characteristics, cont.
H. ducreyi
Virulence factors also uncertain but probably include
capsule, pili and toxins involved in attachment and
penetration human epithelial cells
Etiologic agent of chanchroid
- genital lesions beginning as tender papules
that progress to painful ulcers with several
satellite lesions
- regional lymphadenitis
- primarily occurs in lower socioeconomic groups
in tropical areas
Clinical characteristics, cont.
H. ducreyi
Chanchroid, cont.
- can be distinguished from syphilitic lesions
that are painless
- presence of genital ulcers increases risk of HIV
Clinical characteristics, cont.
Other Haemophilus spp.
Mainly low virulence, opportunistic pathogens
Cause infections similar to H. influenzae but much
less common
H. aphrophilus is an uncommon cause of brain abscesses
and endocarditis
- H of HACEK; subacute bacterial endocarditis
Laboratory Diagnosis
Specimen collection
• Can be isolated from most clinical specimens
- relatively high bacterial load in blood
of children with bacteremia
• Susceptible to drying and temperature extremes
• For H. ducreyi, specimen should be plated within
10 min. of collection
Laboratory Diagnosis, cont.
Direct detection
• Gram stain: most are small, faintly staining,
gram-negative coccobacilli
H. ducreyi often described as “school of fish”
- mostly seen in lymph node specimens
Laboratory Diagnosis, cont.
Direct detection
• Latex agglutination can be performed on CSF or urine
- can be falsely-positive for recent vaccinees
- sensitivity is equivalent to Gram stain
Laboratory Diagnosis, cont.
• Haemophilus require hemin (X factor) and NAD
(V factor)
Chocolate agar contains both
5% Sheep blood agar only contains hemin
Laboratory Diagnosis, cont.
• S. aureus produces NAD as a metabolic product
- Haemophilus will satellite around colonies
of S. aureus when growing on BAP
Laboratory Diagnosis, cont.
Culture, cont.
• Growth conditions:
Haemophilus spp.: 35 – 37°C, 5-10% CO2, 3 days
H. ducreyi: 33 – 35°C, 5-10% CO2, 7 days
- also require supplemented media
• Colony morphology:
Small and translucent
Exude a “mouse nest” odor
Laboratory Diagnosis, cont.
• Growth characterics on solid media
• Gram stain morphology
• X and/or V factor requirement
• Satelliting
• Porphyrin test
Antimicrobial Susceptibility Testing and Therapy
• Routine testing can be performed using disk diffusion
or broth dilution
• Special supplemented media required
• Beta-lactamase testing routinely performed
• Test panel limited because of lack of resistance to
later generation cephalosporins
• Cefotaxime or Ceftriaxone are drugs of choice
• Routine vaccination with protein-polysaccharide
conjugated vaccine (Hib)
• Significant reduction of serious, life-threatening
infections in children
• Recommended starting at 2 months of age
Bordetella sp.
B. pertussis
Not normal flora; only
present during infection
Person-to-person; airborne
transmission via cough
B. parapertussis
Not normal flora; only
present during infection
Person-to-person; airborne
transmission via cough
B. bronchiseptica
Normal flora of animal
upper resp. tract
(dogs, cats, rabbits)
Exposure to contaminated
droplets following close
contact with animals
Clinical characteristics
B. pertussis and B. parapertussis
- cause URT infections in humans with almost identical
symptoms, epidemiology and therapeutic management
- Pertussis (whooping cough)
- optimal recovery requires special culture media
B. bronchiseptica
- opportunistic infection in compromised patients with
history of close animal contact (pneumonia, bacteremia,
UTI, meningitis, endocarditis)
Clinical characteristics, cont.
• Pertussis primarily caused by B. pertussis, rarely by
B. parapertussis; former cause more severe disease
- higher infection rates and increased duration
of symptoms
• Prior to vaccine, epidemic disease occurred in 2 – 5
cycles; still occurs in unvaccinated populations
• Adults and adolescents can serve as reservoirs and
transmit to unvaccinated or vaccinated with waning
Clinical characteristics, cont.
Multiple virulence factors with various functions
Filamentous hemagglutinin
Pertussis toxin
Adenylate cyclase toxin
Tracheal cytotoxin
Outer membrane inhibits host lysozyme
Siderophore production to circumvent host iron sequestering
Clinical characteristics, cont.
Spectrum of disease
Mild cold
Several weeks
Severe coughing
1 to 4 weeks
 Symptoms
Symptoms in adults tend to be milder and are misdiagnosed
as bronchitis; also tend to be mixed with other pathogens
Laboratory Diagnosis
Specimen collection
• Nasopharyngeal wash or swab (Calcium alginate or
dacron on a flexible wire shaft)
• Swabs should be immediately inoculated onto media
and direct smears made at the bedside
• Swabs not directly inoculated should be placed in
transport if time to lab is extended
Laboratory Diagnosis, cont.
Direct detection
• DFA of smear using polyclonal Abs against B. pertussis
and B. parapertussis
• Sensitivity is limited (50 – 70% at best), so should always
be used in conjunction with culture
• PCR methods (home-brew and commercial assays) are
increasing in use and are replacing culture as gold standard
- specificity has been an issue
DFA for Bordetella
ASM Color Atlas of Bacteriology
Laboratory Diagnosis, cont.
• Historical gold standard
• Selective media required
- Potato infusion agar with glycerol and sheep blood
Methicillin or cephalexin
- Charcoal agar with 10% horse blood
Laboratory Diagnosis, cont.
Culture, cont.
• 35 – 37°C, 5 – 10% CO2, hold for 10 – 12 days
- most isolates are detected in 3 – 5 days
• Colonies are small, shiny; resemble mercury drops
• Gram stain shows small, faintly staining gram negative
- confirm identity with DFA reagents
- can distinguish between B. pertussis and
B. parapertussis
B. pertussis on Regan-Lowe agar
ASM Color Atlas of Bacteriology
Gram stain of B. pertussis
w/ antibiotics
w/o antibiotics
ASM Color Atlas of Bacteriology
Antimicrobial Susceptibility Testing and Therapy
• Not routinely performed because Erythromycin and
Azithromycin are active and remain drugs of choice
• Whole-cell vaccines have been used historically
- adverse reactions and waning immunity
• Acellular vaccines have been developed and include
booster doses for older children and adults
Neisseria and Moraxella
– General characteristics
• Gram-negative diplococci, oxidase-positive
– Epidemiology
• Table 45-1
– Pathogenesis
• Table 45-2
– Other Neisseria are saprophytes
Gram stain of Neisseria
Gram stain of Moraxella
– Laboratory Diagnosis
• Specimen collection and transport
– No special considerations for Moraxella
– Pathogenic Neisseria are sensitive to drying and temp
– Swabs are acceptable for GC culture if plated in 6 hrs.
» best method for GC culture is direct inoculation
» Describe JEMBEC plates
– Blood cultures as per routine, although Neisseria inhibited
by high conc of SPS
• Specimen processing
– JEMBEC should be incubated as soon as received in lab
– Body fluids should be kept at RT or 37C before culture (not
– Vol >1 ml should be concentrated and plate the sediment
(e.g. joint fluid or CSF)
– Laboratory detection
• Direct detection
– Gram stain
» shows GN diplococci for both genera; Moraxella tend
to be bigger and fatter
» GNDCs in PMNs from the urethral discharge of
symptomatic male is diagnostic for GC
» Normal vaginal and rectal flora has GNDCs so
diagnosis requires confirmation
– Antigen detection
» not recommended; poor sensitivity
– Molecular detection
» Amplified methods are more sensitive than nonamplified methods
» Increased detection of GC overall
» Can test for CT at the same time
» Cannot be used as evidence in medico-legal cases
» We use B-D Viper automated instrument
– Laboratory detection
• Culture
– Media of choice
» N. meningitidis, Moraxella and saprophytic Neisseria
grow well on BAP, CAP
» GC requires enriched CAP on primary culture
» Selective media have been developed to inhibit normal
flora and allow N. meningitidis and GC to grow
» Modified Thayer-Martin
» IsoVitaleX, colistin, nystatin, vanco, trimethoprim
» Martin Lewis is similar
– Incubation conditions and duration
» 35-37C, 3 - 7% CO2, humid, 72 hrs
» this CO2 conc can be achieved in incubator or candle
– Colony appearance
Culture of Neisseria
Culture of Moraxella
– Laboratory detection
• Approach to identification
– Biochemicals
» Moraxella: glucose -, maltose -, lactose -, butyrate
disk +, ox +
» GC: g +, m -, l –, ox +
» NM: g +, m +, l –, ox +
» Saprophytes: + + + or any other combo
» Culture confirm and ID must be unequivocal in abuse
» Saprophytes are not routinely identified (i.e. from
respiratory cultures
– Serotyping
» Mening: A, B, C, Y, W135
– Susceptibility testing and therapy
• Moraxella
– testing not routinely performed because many options
» beta-lactams; b-l/b-lactamase inhib; cephs;
macrolides; quinolones; bactrim
• GC
– routinely not performed because most labs use molecular
so no isolate
– resistance is a Public Health issue so surveillance
mechanisms exist
» penicillin resistance is widespread
» ceftriaxone resistance not documented
» quinolone resistance is emerging problem
• N. meningitidis
– not routinely performed; resistance rare
– pen, cephs
– Prevention
• Vaccine available for A, C, Y, W135
– military recruits, college students, asplenics > 2 y.o.
• Chemoprophylaxis with rifampin, cipro, or
ceftriaxone for close contacts of patients with
meningococcal disease
– no chemo prophylaxis for pneumococcal mening
• Eye antibiotics for neonates to prevent gonococcal
eye infections
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