Title: Neisseria and Moraxella catarrhalis
1Neisseria and Moraxella catarrhalis
- The Gram Negative Diplococci
2Introduction
- 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
3Often associated with neutrophils as in this
image N. gonorrhoeae
4continued
- 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.
5continued
- 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
6Selective 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
7Cultural 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
8continued
- 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
9Summary 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
10Colony 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)
11N. gonorrhoeae
12N. meningitidis
13M. catarrhalis
14Microscopic 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
15Gram 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)
16(No Transcript)
17Microscopic 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
18Presumptive 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
19Definitive 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
20Carbohydrate Utilization of Neisseria
Organism CTA Sugar
Tubes Glucose Maltose Lactose
Sucrose N. gonorrhoeae
- - - N.
meningitidis
- - N. lactamica
- Nonpathogenic
- Neisseria Moraxel
la catarrhalis - -
- -
21Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
Acid from glucose only identifying this organism
as N. gonorrhoeae
22Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
Acid from glucose and maltose, characteristic of
N. meningitidis
23Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
Acid from glucose, maltose, and lactose,
characteristic of N. lactamica
24Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
Acid from all four sugars, characteristic of most
nonpathogenic Neisseria
25Carbohydrate Utilization in CTA Medium
Glucose
Lactose
Maltose
Sucrose
No acid from any of the four sugars,
characteristic of M. catarrhalis
26Definitive 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
27Supplemental 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
28Commercial 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
29Virulence 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)
30Pathology 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
31Pathology 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.
32Antimicrobial 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.