Neisseria and Moraxella catarrhalis

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Neisseria and Moraxella catarrhalis

• The Gram Negative Diplococci

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Introduction

• Neisseria and Moraxella (Branhamella) catarrhalis
  are Gram negative cocci with the following
  general characteristics
• Kidney bean shaped
• Arranged in pairs mostly
• Flattened sides of kidney adjacent Oxidase
  positive
• Catalase positive
• Obligately aerobic
• Non-motile some Moraxella species exhibit
  twitching motility (via pilus retraction???)
• N. gonorrhoeae is often called the gonococcus
  or GC and N. meningitidis is often referred to
  as the meningococcus or MC

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Often associated with neutrophils as in this
image N. gonorrhoeae
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continued

• Neisseria utilize sugars oxidatively only (acid
  in O tube)
• M. catarrhalis is asaccharolytic no acid
  produced from sugars
• All Neisseria and M. catarrhalis are commensals
  of humans, except N. gonorrhoeae which is an
  obligate parasite (rare)
• N. gonorrhoeae, N. meningitidis and M.
  catarrhalis are the major human pathogens in the
  group.
• Other Neisseria species (as well as related
  genera Kingella and Eikenella) are rarely
  implicated in human infections
• They are generally nutritionally fastidious
  capnophilic requiring X V factors, and other
  enrichment. The major pathogens are the most
  fastidious in this order N. gonorrhoeae, N.
  meningitidis, M. catarrhalis.

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continued

• The GC and MC grow best on enriched chocolate
  agar. Most commercial CA contains B vitamins and
  amino acids.
• Some strains of GC (known as the AHU strains)
  cannot synthesize the amino acids arginine and
  hypoxanthine, nor the RNA nucleotide uracil.
• Actually, all but GC will usually grow on SBA at
  37oC, although not well.
• If cultured from sites harboring normal
  microbiota, each of these pathogens are easily
  overgrown. Therefore, selective media should be
  used if normal microbiota are expected to be
  present
• All selective and non-selective media should
  contain the AHU enrichments

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Selective Media for GC and meningococci
Medium Selective Agents
1Thayer Martin (TM) 1Modified TM (MTM) 1JEMBEC
1Transgrow New York City Medium
NYC GC-Lect
All contain vancomycin to inhibit Gram positive
bacteria and colistin to inhibit Gram negative
bacteria. TM, MTM, and JEMBEC contain Nystatin
to inhibit yeasts NYC and GC-Lect contain
amphoteracin B for this purpose. All but TM
contain trimethoprim to inhibit Proteus from
swarming
1Chocolate agar plus B vitamins and amino acid
supplements
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Cultural requirements

• GC and MC are very sensitive to temperatures
  below 30oC
• Clinical specimens suspected of harboring them
  should never be refrigerated and culture media
  should be brought to room temperature before
  inoculation
• Plating GC at the bedside yields the highest
  number of positive cultures. Specimens collected
  away from the clinical lab require special
  handling
• The typical transport media such as Stuart and
  Amies maintain viability for only a few hours.
  The inoculated transport media should be
  incubated at body temperature until they can be
  taken to the lab. How practical is this?
• Body temperature incubation is continued in the
  lab for one to two more days at elevated CO2

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continued

• Transgrow and JEMBEC are perhaps the most
  commonly used GC media. They serve as both a
  transport enrichment medium and a culture medium.
  Transgrow is a bottled medium to which CO2 has
  been added from a cylinder of gas.
• JEMBEC is an agar medium that contains sodium
  bicarbonate, and is used in a unique plate with a
  receptacle for CO2 generating tablets. The plate
  is closed and sealed in a zip-lock pouch
  trapped moisture combines with the bicarbonate
  and releases CO2
• Some strains of GC are sensitive to vancomycin
  which is present in all selective media designed
  to recover pathogenic Neisseria species from
  clinical specimens that harbor normal microbiota
• For this reason, it is advisable to use a
  nonselective chocolate medium in addition to one
  of the selective media

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Summary of Cultural Methods

• Never refrigerate specimens for GC or
  meningococci
• Incubate in environment rich in CO2 and high
  humidity
• Use selective media if from non-sterile sites
• Media designed for transport and culture (e.g.
  Transgrow or
• JEMBEC ) are required if a delay is
  anticipated between time of
• collection and incubating the media

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Colony morphology

• GC colonies are 1-2 mm in diameter after 24-48h.
  They are circular, slightly convex, moist,
  translucent and gray-white. After a few
  sub-cultures, GC colonies lose the ability to
  produce pili,causing them to become dryer
• Colonies of meningococci are similar to GC except
  they are a little larger and may be mucoid
• Colony morphology of nonpathogenic Neisseria is
  quite variable
• Most nonpathogenic Neisseria produce some form of
  yellow colonies and tend to be dry and wrinkly
• Colonies of M. catarrhalis are smooth gray-white
  opaque and convex, sometimes with a thin wavy
  periphery. Colonies are often granular, waxy
  and therefore difficult to emulsify
• When lateral pressure is applied to a colony of
  M. catarrhalis using an inoculating loop the
  intact colony slides laterally ( this is known as
  the hockey puck test)

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N. gonorrhoeae
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N. meningitidis
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M. catarrhalis
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Microscopic morphology

• All Neisseria species and M. catarrhalis have
  very similar microscopic morphology as previously
  described
• Infections of GC, meningococcus, and M.
  catarrhalis are usually highly purulent because
  the infection causes profuse PMN activity. The
  cells are often present inside the phagocytes
• Infections of Gram-negative intracellular
  diplococci is suggestive of Neisseria. Any
  Neisseria isolated from a genital specimen, skin
  lesion, or normally sterile sites must be
  identified definitively. GC is never commensal.
• GC infections are found primarily in genital
  specimens, but also in the eyes, sinsuses,
  respiratory tract, and in septic arthritis.
• The meningococcus is found in CSF, blood
  specimens and skin lesions associated with
  dissemination. It is considered commensal in the
  throat and URT.
• M. catarrhalis is most often associated with
  bacterial pneumonia

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Gram Negative Intracellular Diplococci
PMN
Intracellular diplococci
Urethral discharge from men with gonorrhea or CSF
from patients with meningitis (N. meningitidis),
or sputum from patients with pneumonia (M.
catarrhalis)
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(No Transcript)
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Microscopic morphology

• Since each of the 3 pathogens discussed here can,
  on rare occasions, be found in any of these
  sites, the absolute identity can only be
  established by observing cultural and biochemical
  characteristics or performing genetic probes

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Presumptive identification

• The presence of oxidase positive Gram negative
  diplococci in pairs from cultures of selective
  media is very good presumptive evidence of
  Neisseria species or M. catarrhalis
• N. gonorrhoeae is the only species in this group
  that gives a positive superoxol test, basically a
  modified catalase test
• A drop of 30 hydrogen peroxide is added to a
  18-24h culture of the suspected colony a
  non-blood containing medium
• Instantaneous vigorous bubbling is a positive
  test
• Other Neisseria will produce bubbling but it will
  be delayed by a second or two and will be a
  somewhat weaker response
• M. catarrhalis is the only member of this group
  that gives a positive hockey puck test
  remember the waxy colonies

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Definitive identification

• Carbohydrate utilization tests (profiles) are
  conducted
• Cystine-trypticase agar (CTA) basal medium with
  phenol red containing filter sterilized glucose,
  maltose, lactose, and sucrose is used
• A heavy inoculum of a pure culture no more than
  24h old (?) is stab inoculated into the upper
  half of the CTA
• Tubes are incubated without added CO2 at 35oC.
  After 24h the upper portion of the tubes are
  observed for acid
• This procedure will identify the classically
  pathogenic Neisseria, presumptively identify M.
  catarrhalis and group the nonpathogenic Neisseria
  see table on next slide

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Carbohydrate Utilization of Neisseria
Organism CTA Sugar
Tubes Glucose Maltose Lactose
Sucrose N. gonorrhoeae
- - - N.
meningitidis
- - N. lactamica

- Nonpathogenic
- Neisseria Moraxel
la catarrhalis - -
- -
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Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
Acid from glucose only identifying this organism
as N. gonorrhoeae
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Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
Acid from glucose and maltose, characteristic of
N. meningitidis
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Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
Acid from glucose, maltose, and lactose,
characteristic of N. lactamica
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Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
Acid from all four sugars, characteristic of most
nonpathogenic Neisseria
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Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
No acid from any of the four sugars,
characteristic of M. catarrhalis
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Definitive identification

• Additional tests are required if CTA results are
  not equivocal
• GC and meningococcus will not grow on T-Soy agar
  at 35oC whereas M. catarrhalis and nonpathogenic
  Neisseria will grow
• GC and meningococcus will not grow at room temp
  period but M. catarrhalis and nonpath. Neisseria
  will grow on SBA or CA
• Remember the superoxal test the GC only GC is
  positive
• N. lactamica produces beta galactosidase, are
  therefore is ONPG (o-nitrophenyl beta
  galactopyranoside a lactose analogue) positive
  whereas all the others are negative
• M. catarrhalis is the only member of the group
  that produces DNAase and lipase (tributyrin is a
  lipid used in a lipase test)
• With rare exception, GC and meningococci are the
  only Neisseria types that grow on TM, MTM, or
  other selective Neisseria medium

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Supplemental tests for Neisseria
Growth on TSA at 35oC
DNAse or trybutryin
Superoxol (30 H2O2)
Growth CA at RT
Growth on TM
ONPG
- - -
-
N.gonorrhoeae N.meningitidis M.
catarrhalis Nonpathogenic Neisseria N. lactamica
- - - -
-
- -
-
- - -
-
- - -

Occasional exception
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Commercial products

• Several identification kits are available for
  these bacteria
• Some are miniaturized versions of the
  carbohydrate utilization tests (e.g. Minitek,
  Quadferm Biolog)
• Most of the carbohydrate utilization tests
  function by simple pH indicator reactions
  (Minitek, API, etc). Some are chromogenic tests
  that detect specific enzymes via cleavage of a
  substrate analog yeilding a colored product.
  Others are based upon increased turbidity from
  growth in the presence of the carbs (Biolog).
• Several products are based on detecting bacterial
  antigens in an isolated colony with a specific
  monoclonal antibody (ELISA, coagglutination, or
  immunofluorescence - FlAB)
• DNA probes for GC are also commercially
  available

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Virulence factors

• N. gonorrhoeae - ?infectivity ?severity
  (fortunately)
• Capsule (???) attachment anti-phagocytosis
• Fimbriae/pili specific attachment to urogenital
  columnar epithelial cells
• Lipopolysaccharide (endotoxin) tumor necrosis
  factor
• Cell wall proteins I, II, and III (interfere with
  phagocytosis)
• IgA protease (cleaves IgA on mucosal surfaces)
• ZERO conferred immunity how?
• N. meningitidis
• Capsule
• Lipopolysaccharide (endotoxin)

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Pathology N. gonorrhoeae

• GC is normally acquired by sexual contact, and is
  normally limited to mucosal columnar epithelial
  cells including cervix and urethra (dysuria), but
  also rectum, pharynx, and conjunctiva (corneal
  scaring perforation, usually perinatal).
  Mucosal infections are usually characterized by a
  maked local neutrophilic response copious
  purulent discharge, burning and itching.
• GC LPS stimulates the production of tumor
  necrosis factor, which causes cell damage.
  Endocervical infection can lead to scared
  fallopian tubes leading to tubal (ectopic)
  pregnancy. Progression can lead to sterility.
• GC dissemination and bacteremia manifest most
  commonly as a dermatits-arthritis syndrome, but
  in rare cases can result in endocarditis and
  meningitis

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Pathology continued

• N. meningitidis The meningococcus is the 1
  cause of meningitis in teenagers and young adults
  - a highly acute condition that usually follows a
  mildly symptomatic nasopharyngeal carrier state.
  Acute manifestations result from intracranial
  pressure (-itis), and the infection itself
  mechanism is thought to be similar to GC.
• Early symptoms include fever, malaise, headache
  vomiting, progressing with seizures, altered
  mental status and coma. Fulminant cases have a
  high mortality rate.
• M. Catarrhalis is the 3rd most common cause in
  the US of sinusitis and otitis media in children
  (3-4 million cases annually), and rarely
  progresses to pneumonia. It appears that
  pathology is related to endotoxin similar to the
  Neisseria.

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Antimicrobial Therapy

• GC was generally susceptible to penicillin in the
  US until 1976 when a pen-resistant strain was
  imported from Southeast Asia. This strain
  proliferated and spread within a few years.
  These strains are now detected using the
  cephalosporin (Cefinase) test
• These strains are referred to as CMRNG
  (chromosome mediated resistant N.gonorrhoeae).
  Tetracycline and Spectinomycin chromosomal
  mediated resistance strains also occur.
• Ceftriaxone, a third generation cephalosporin, is
  recommended for therapy by CDC.
• Penicillin is still the drug of choice for
  treatment of infections caused by N. meningitidis
• Most M. catarrhalis strains produce beta
  lactamase, and are therefore pen-resistant.