MD-2021 | Manila Central University S.Y.

2018-2019

MICROBIOLOGY • Presence of pili in N. gonorrhoea and meningitides

GRAM-NEGATIVE COCCI: appears to be important for pathogenesis.
Neisseria spp., Veillonella spp., and • Pili expression is associated with virulence, in part
Branhamella (Moraxella) spp. because the pili mediate attachment to non-ciliated
epithelial cells and provide resistance to killing by
Based on the lecture by Dr. Manangan | October 18, 2018
neutrophils.
RULE of THUMB: • N. gonorrhoeae and N meningitides are pathogenic
for humans.
All COCCI are GRAM-POSITIVE except: “NeVer Mind”
• Found associated with or inside PMNs.
Neisseria, Veilonella, Moraxella
• Are closely related, with 70% DNA homology

GONOCOCCI MENINGOCOCCI
NEISSERIAE
Polysaccharide capsule (-) (+)
Plasmids Mostly (+) Rarely (-)
Habitat Genitals URT
Disease Genital infections meningitis

MORPHOLOGY and IDENTIFICATION

• Aerobic, gram-negative bacteria
• Typically coccoid shaped arranged in pairs
(diplococci) with adjacent sides flattened together
• “coffee-bean” morphology on microscopy
• Non-motile, no endospores
• All species are OXIDASE positive
• Most produce catalase properties
• Acid is produced by oxidation of carbohydrates
o Most bacteria produce acid by fermentation
• Carbohydrate utilization is useful in differentiating
pathogenic strain from other Neisseria species
*refer to table below for CHO utilization
• N. gonorrhoeae oxidize glucose while N. meningitidis
oxidize glucose and maltose
A. Typical Organisms
➢ gram negative, non-motile diplococcus
➢ 0.8 um in diameter
➢ Kidney bean shaped (the flat or concave sides are
adjacent)
B. Culture
➢ In 48 hrs on enriched media ( Modified Thayer Martin,
Martin-Lewis, GC-Lect, and New York City
- Both form convex, glistening, elevated,
mucoidcolonies 1-5mm in diameter
- Colonies are transparent or opaque, non-
pigmented, and non-hemolytic

GLUCOSE MALTOSE
N. gonorrhoeae (+) (-)
N. menigitidis (+) (+)

• Non-pathogenic species of Neisseria can grown on

Nutrient Agar incubated at 35 to 37 °C. In contrast, N.
meningitidis has variable growth on Nutrient Agar and
N. gonorrhoeae is a fastidious organism, requiring
complex media for growth
• All strains of N. gonorrhoeae require cysteine and an
Yellow pigmentation (colony N. flavescens
energy source (e.g. glucose, pyruvate, and lactate) for growth) N. cinereal
growth N. subflava
• N. gonorrhoeae does not grow on blood agar but N. lacatmica
does grow on chocolate agar and other enriched Opaque, brittle, wrinkled N. sicca
supplemented media. Optimum temp is 35 to 37 °C colonies
with poor survival of the organism at cooler Non-pigmented, pinkish-gray Moraxella catarrhalis
temperatures opaque colonies
• Pathogenic and non-pathogenic strains have pili that
extend from the cytoplasmic membrane through the *Moraxella catarrhalis was previously known as Neisseria
outer membrane. Pili mediate a number of functions, catarrhalis before it became Branhamella catarrhalis
including attachment to host cells, transfer of genetic
material and motility
•

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C. Growth Characteristics
• Grow best under aerobic condition.
• Have complex growth requirements.
• Most oxidize carbohydrates, producing acid
but not gas
• Produce oxidase
• Oxidase positive –hence oxidase test is a key test for
identifying them
OXIDASE TEST
- Bacteria spotted on a filter paper with
tetramethyparaphenylenediaminehydrochlori
de (oxidase) – turns dark purple (positive result)
• Grows best on media containing complex substances
such as heated blood, hemin, and animal proteins
and in an atmosphere containing 5% CO2
• Growth is inhibited by toxic constituents of media like
fatty acids or salts.
• Rapidly killed by drying, sunlight, moist heat and
disinfectants.
• Produces autolytic enzymes that result in rapid
swelling and lysis in vitro at 25’C and at an alkaline
pH
**the only DNAse positive is M. catarrhalis
**the only LACTOSE positive is N. lactamica
Neisseria gonorrhoea
• Occurs only in humans. Second only to chlamydia as
the most commonly reported sexually transmitted
disease in the U.S.
• Peak incidence is 15-24 years.
• Is transmitted by sexual contact.
• Women have 50% risk of acquiring the infection as
the result of a single exposure to an infected man,
whereas man have a risk of approximately 20% as
the result of a single exposure to an infected woman.
• Risk of infection rises as the person has more sexual
encounters with infected partners.
• Asymptomatic carriage is more common in women
than in men.
ANTIGENIC STRUCTURES
1.Pili(fimbriae)
• Are hair-like appendages that extend up to several
micrometers from the gonococcal surface.
• Enhance attachment to host cells
• Resistance to phagocytosis
2. Por
• Extends through the gonococcal cell membrane.
• Forms pores in the surface → nutrient enters the cell
• Impact intracellular killing – by preventing
phagosome-lysosome fusion.
3. Opa proteins
• Function in adhesion of gonococci within colonies and
in attachment of gonococci to host cell receptors such
as heparin related compounds and CD 66 or
Carcinoembryonic antigen related cell adhesion
molecules.
4. Rmp (Protein III)
• Antigenically conserved in all gonococci.
• Reduction-modifiable protein and changes its
apparent MW when in reduced state.
5. Lipooligosaccharide
• Gonococcal lipopolysaccharide does not have long O
antigen side chains and is called a
lipooligossacharide
• Toxicity in gonococcal infection is largely attributable
to the endotoxic effects of LOS.
• In fallopian tube, it causes ciliary loss and mucosal
cell death.
• Causes molecular mimicry because It resembles
human cell membrane glycosphingolipid and both
react with the same monoclonal antibody →helps
evade immune recognition.
6. Otherproteins:
• Lip(H8)–is a surface exposed protein that is heat-
modifiable like Opa
• Fbp(ferricbindingprotein)–expressed when the
available iron supply is limited
• IgA1protease–splits and inactivates IgA1(a major
mucosal immunoglobulin); Meningococci, H.influenza
and Streptococcus pneumonia elaborate similar
IgA1protease
PATHOGENESIS, PATHOLOGY and CLINICAL FINDINGS
➢ Attacks mucus membranes of the genitourinary tract,
eye, rectum and throat
➢ Men have urethritis with yellow creamy pus and
painful urination
➢ Urethral infection in men can be asymptomatic
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➢ Primary infection in women is in the endocervix and
extends to the urethra and vagina giving rise to
mucopurulent discharge
➢ May progress to uterine tubes causing salphingitis,
fibrosis and obliteration of the tubes
➢ Gonococcal ophtalmia neonatorum – is an infection of
the eye in newborns, acquired during passage thru an
infected birth canal
➢ Prevention of opthalmia neonatorum – instillation of
tetracycline, erythromycin or silver nitrate into the
conjunctival sac
DIAGNOSTIC LABORATORY TEST
A. Specimen
Pus and secretions are taken from the urethra, cervix, and
rectum and conjunctivitis
B. Smears
Gram stained smears of urethral or endocervical exudates
reveal many diplococci within pus cells
C. Culture
➢ Modified Thayer Martin Medium
➢ To avoid overgrowth of bacteria, the selective medium
contains antimicrobial drugs
➢ C02 containing transport culture system – if
immediate incubation is not possible
➢ After 48 hours, quick identification on gram-stained
smear, oxidase positivity, and by co-agglutination,
immunofluorescence staining
D. Nucleic Acid Amplification Tests
➢ Direct detection of N. gonorrhea in genitourinary
specimens---preferred test for these sources
➢ Excellent specificity and sensitivity in symptomatic
patients
➢ Advantage: better detection, more rapid tests results
and the ability to sue urine as specimen
➢ Not recommended as test of cure because nucleic
acid persists up to 3 weeks after successful treatment
E. Serology
➢ Antibodies to gonococcal pili and outer membrane
proteins can be detected by immunoblotting, RIA, and
ELISA tests
➢ not useful as diagnostic aids – delay in development
of antibodies in acute infection
IMMUNITY
Repeated gonococcal infections are common
Reinfection–antigenic variett of gonococci.
TREATMENT
➢ Because of antimicrobial resistance, CDC
recommends:
o For uncomplicated genital-oral infections –
Ceftriazone 250 mg IM single dose or
Cefizime 400 mg oral single does
o Additional 1 gram of azithromycin – single
dose or with mg of d oxyxycline p.o. twice a
day for 7 days (for concomitant chalamydial
infections)
➢ Since ceftriaxone is more potent than cefixime, the
CDC no longer recommends cefixime as an effective
treatment
➢ Injectable ceftriaxone 250mg IM once plus
azithromycin or doxycycline is recommended for
uncomplicated urethritis, cervicitis and proctitis
EPIDEMIOLOGY, PREVENTION and CONTROl
• Worldwide in distributiom
• 1955-1970 – 400-500 cases per 100,000
• 74% Declined in 1975-97
• Plateaued for 10 years but since 2009 slightly
increased each year
• Exclusively transmitted by sexual contact
• 20-30% chance of acquiring infection from a single
exposure to infected sexual partner
• Avoiding multiple sexual partners, use of condoms,
early diagnosis and treatment
• Gonococcal opthalmia neonatorum–0.5%
erythromycin ophthalmic ointment or 1% tetracycline
ointment to conjunctiva of newborn
• Silver nitrate–causes conjunctival irritation
Neisseria meningitidis
• Human are the only natural hosts
• Person-to-person spread occurs via aerosolization of
respiratory tract secretions
• Highest incidence of disease is in children younger
than 5 years, institutionalized people, and patients
with late complement deficiencies.
• Meningitis and meningococcemia most commonly
caused by serogroups A,C, and Y pneumonia most
commonly caused by serogroups Y and W135;
serogroups A and W135 associated with disease in
underdeveloped countries.
• Disease occurs worldwide, most commonly in the dry,
cold, months of the year.
• The oral and nasopharyngeal carriage rates are
highest for school age children and young adults, are
higher in lower socioeconomic populations and do not
vary with the seasons, even though disease is not
common during the dry cold months of the year
ANTIGENIC STRUCTURE
- 13 subgroups have been identified by immunologic
specificity of capsular polysaccharides:
- A, B,C,X,Y,and w-135.-cause disease in humans.
- Meningococcal antigens are found in blood and cs for
patients with active disease.
- Outer membrane of n.meningitides consists of
proteins and lps–play major roles in organism
virulence
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- 2 porin proteins (por a and por b)–important in • The immunizing antigens for groups A , B, C, Y and
controlling nutrient diffusion in to the organisms and W125 are capsular polysaccharide
also interact with host cells • A polysaccharide tetravalent vaccine is poorly
- Porins are targets of interest in vaccine development immunogenic in children less than 18 months, does not
confer long lasting immunity, and does not cause a
sustainable reduction in nasopharyngeal carriage
➢ Meningococci are piliated – initiate binding to • A tetravalent conjugate vaccine is approved for 9
nasopharyngeal epithelial cells months to 55 years old → enhances primary response
➢ Lipid A dissachareide of meningococcal LPS – among infants and substantially reduces asymptomatic
responsible in toxic effects found in meningococcal carriage
disease • Routine vaccination of all young adolescents, children
2 months of age or older with functional or surgical
PATHOGENESIS, PATHOLOGY and CLINICAL FINDINGS asplenia, persons with complement deficiencies
➢ Humans are the only natural hosts • Those who are travelers to or residents of highly
➢ Nasopharynx – portal of entry endemic areas, those with community outbreak, lab
➢ Nasopharynx → attach to epithelial cells (pili) → blood worker, and other at risk groups
stream (bacteremia) → URT infection
TREATMENT
➢ Fulminant meningococcemia is more severe, with a • Penicillin G is the drug of choice
high fever and a hemorrhagic rash, the patient may • Chloramphenicol or cefotaxime or ceftriaxone for those
have DIC and circulatory collapse (Waterhouse allergic with penicillins
Friderichsen syndrome)
EPIDEMIOLOGY, PREVENTION and CONTROL
➢ Meningitis – most common complication of
• Occur in epidemic waves
meningococcemia; begins with intense headache,
• Serogroup A for majority of outbreaks in sub-Saharan
vomiting and stiff neck and progress to coma within a
Africa, where serogroup B often cause sporadic
few hours
infection
• Interepidemic periods – 5-30% of normal population
➢ During meningococcemia – there is thrombosis of
are carriers
small blood vessels with perivascular infiltration and
petechial hemorrhages • 70-80% during epidemics
• Treatment with oral penicillin does not eradicate the
carrier state
DIAGNOSTIC LABORATORY TESTS • Chemoprophylaxis: Rifampicin 600 mg orally 2x a day
A. Specimens: blood, CSF, puncture material from petechiae for 2 days
Nasopharyngeal swab for carrier surveys • Alternative: ciprofloxacin in adults 500 mg as single
dose. And Ceftriaxone 125 mg IM as single dose
B. Smears: typical Neisseria within PMN or extracellularly • Reduction of personal contacts. Avoidance of crowding
or administration of vaccines
C. Culture: CSF – chocolate agar incubated at 37’ C in an
atmosphere of 5% CO2; MTM with antibiotics (vancomycin,
colistin, amphotericin) favors growth of Neisseria, inhibits many
other bacteria, and used for nasopharyngeal cultures OTHER NEISSERIAE
**MTM – modified Thayer martin Medium enriched with
antibiotics
N. lactamica Rarely cause disease but is
PRESUMPTIVE TEST: important bec it grows in the
Identified by gram stain and oxidase test selective media
Spinal fluid and blood – yield pure culture – Can be cultured in the
can be identified by carbohydrate oxidative nasopharynx and most often
reactions and agglutination with type specific found in children; oxidizes
or polyvalent serum lactose
N. sicca, N. subflava, N. Normal microbiota of the
D. Serology: antibodies to meningococcal polysaccharides --- cinirea, N. mucosa and N. respiratory tract, particularly
measured by latex agglutination or hemagglutination tests or flavescens nasopharynx, and very rarely
by bactericidal activity produce disease
N. cinirea Resembles N. gonorrhoea

IMMUNITY
• Associated with the presence of specific, complement
dependent, bactericidal antibodies in the serum
• Antibodies develop after subclinical infections or
injections of vaccines

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Moraxella catarrhalis

- Was previously named Branhamella catarrhalis and

before that N. catarrhalis
o Member of the normal microbiota in 40-50%
of healthy school children
o Causes bronchitis, pneumonia, sinusitis,
otitis media, and conjunctivitis
o Cause of infection in immunocompromised
patients
o Most strains produce beta-lactamse
o Can be differentiation form Neisseria by its
lack of carbohydrate fermentation and by its
production of DNAse; it produces butyrate
esterase, which forms the basis of rapid
fluorometric tests for identification

VEILLONELLA

- Are non-motile, gram-negative diplococci that are the

anaerobic counterpart of Neisseria
- Well-know for its lactate fermenting abilities
- Normal bacterium in the intestine and oral mucosa of
mammals, maybe found in the vagina as well
- Often regarded as contaminants
- They are often associated with oral infections, bite
wounds; head, neck, and various soft tissues
infections
- Implication as pathogens in infections of the sinuses,
lungs, heart, bone and CNS

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