Gram-negative cocci

GRAM STAIN REACTIONS

All COCCI are GRAM POSITIVE Except: NBMV

Neisseria - No
Branhamella - Boyfriend
Moraxella - Muna (para)
Veilonella - Virgin
All BACILLI are GRAM NEGATIVE Except: BL2AC2MEN
Bacillus Actenomyces Mycobacterium
Lactobacillus Clostridium Erysiperothlix
Listeria Corynebacterium Nocardia

NEISSERIA

 Are obligate aerobe, non-motile and non-hemolytic bacteria (gamma hemolytic bacteria)
 Microscopy: Gram-negative diplococcic (pinkish) with coffee or kidney bean shaped, except N. elongata and
N. weaveri
 Culture: Small, gray-white opaque, convex and glistening colonies – fastidious organism requires meadia
containing blood, serum, cholesterol or oleic acid
 Most are non pigmented except for: N. flava, N. flavescens and N. subflava
 Biochemical testing: oxidase (+); catalase (+) except N. elongata
- Differentiate micrococacceae through gram stain
 Are capnophilic (2-8%) and have an optimal growth in a moist temperature
 Are carbohydrate fermenters – primarily glucose and maltose
 Natural Habitat: mucous membrane of the respiratory and urogenital tract
 Sensitive to drying and extremes in temperature – bedside inoculation is needed (place in a plate and use
media immediately or use transport media)
 Major pathogens: N. gonorrheae and N. miningitidis

Neisseria gonorrheae (gonococci)

 Is never considered as a part of the normal flora
 Man is the only known host
 Found in urogenital tract, anorectal area and conjunctiva
 Mode of Transission: Person to person transmission – Sexually Transmitted Disease/Infection
 Microscopy: gram-negative intracellular diplococci
 Culture (CAP): small, tan, transluscent and raised after 24-48 hours of incubation
 Requires iron for growth – the ability to bind to transferin
 Glucose fermenters
 Produces an IgA protease – an enzyme that degrades protein and an antibody in mucous membrane
secreation
 Principal virulence factor: common pili

Clinical infections

Gonorrhea (tulo in tagalog)

- meaning “flow of seed” and “brothel”
 - an acute infection of nanciliated columnar and transitional epithelium
- 2 – 7 days incubation period
- symptoms: purulent discharge(in genitals), lower abdominal pain and dysuria and vaginal bleeding
- other infections caused by gonorrhea:
 Purulent urethritis (male) and cervicitis (female)
 Pharyngitis - inflammation on oropharyngitis
 Anorectal infections – rectal pain and bloody stool (MOT: anal)
 Purulent arthritis
 Conjunctivitis (opthalmia neonatorum) gonoccocal eye infection – common among
newborns babies
- CREDE’s Prophylaxis – given to the baby with this infection and it
contains 1% of AgN or Silver Nitrate
- Erythromycin can also be given to newborn babies to prevent
conjunctivitis

Specimen Collection and Handling

- most important factor in the laboratory diagnosis of N. gonorrhea

- swabs are acceptable if the specimen will be plated within 6 hrs because they are prone to drying
- if cotton swabs are used, the transport medium should contain activated charcoal/calcium– to inhibit the
toxic fatty acids. Calcium Alginate or Rayon Fibers can also be used
 Specimen: pus and secretions from urethra, cervix, prostate, rectal mucosa, throat and joint fluid(purulent
arthritis).
 Use Dacron and Rayon swab for collection
 Rayon Fibers/Swabs – preferred over cotton swabs
- Swabs should be placed in a transport media
- Joint fluid and CSF must be kept at Rt or placed at 37oC
 Laboratory diagnosis
- always perform different biochemical test to conclude that the infection is gonorrhea
 Gram Staining
- gram negative diplococci, kidney or coffee bean shaped is diagnostic
- PMNs or Polymorphonuclear neutrophils
- A gram stain with more than 5 PMN/field but no bacteria seen is suggested of non-gonococcal
uretritis or chlamydia
- If gram negative diplococci is seen inside the PMN, this is a diagnostic already of N. gonorrhea
 Culture for confirmation (for Confirmation)
- BAP & CAP – for sterile specimen
- Direct inoculation
- JEMBEC system best used for culture and transport

Other Culture for confirmation

a. Thayer Martin Agar – contains the ff

- Vancomycin - (kills gram + bacteria)
- Colitis - (kills gram negative except gonorrhea)
- Nystatin - (antifungal)
b. Modified Thayer Martin Agar – all TMA components + trimethoprim lactate (added to prevent proteus
swarming)
c. Martin lewis Medium – all MTM component except nystatin which is substituted by anisomycin
d. New York City medium – used in genital mycoplasma
- Contains vancomycin, colistin, trimethoprim and amphotericin B
 e. INHIBITS

Inhibits TMA MTM MLA NYC

Gram (+) Vancomycin V V V
Gram (-) Colistin C C C
Fungi Nystatin N Anisomycin Ampothericin
Proteus swarming X Trimethoprim TL TL
Lactate

 Carbohydrate utilization – standard method of identification

 Medium: CTA = 1% carbohydrate and phenol red indicator
 Detect acid production from glucose, maltose, lactose, fructose and sucrose
 Glucose – N. gonorrhea
 Maltose – N. meningitides
 Lactose – N. lactamica
 Oxidation test
 Superoxol test – 30% hydrogen peroxide (positive test - vigorous bubbing)
 Beta lactamase test
 DNAse test – (negative for N. gonorrhea)
 Immunologic test - employs monoclonal antibody (bacteria – Ag/antigen + monoclonal – Ab/antibody
reaction) (Agglutination indicates a positive result)
 Molecular assay – detects gonococcal antigen or nucleic acid directly from specimen

Neisseria meningitidis (meningococci)

 In our brain, we have our meninges and once it is invaded by meningitides, your meninges will become
infected.
 It has the capability to invade the CENTRAL NERVOUS SYSTEM
 Causative agent of meningococcal meningitis/ meningococcemia(in blood)/ cerebrospinal fever and spotted
fever
 Leading cause of fatal bacterial meningitis (pathogenic)
 Microscopy: intracellular or extracellular gram negative diplococci.
 Culture: mucoid in appearance, bluish gray colonies on BAP, small tan colonies in CAP
 Colonizes the the mucous membranes of nasopharynx and oropharynx

Clinical infection

Meningococcemia
 refers to the presence of N. meningitidis in the blood
 occurs with or without meningitis
 sources of epidemics: MOT: oral secretion and respiratory droplets
 signs and symptoms: frontal headache, stiff neck and fever
 Causative Agent is a bacteria, it is easy to contain unlike the viruses which is prone to mutation
 2004- 1 month epidemic of meningococcemia in Baguio

 Petechial skin lesions (rashes) are due to release of the endotoxin after bacterial cell lysis
 May cause Waterhouse-Friderichsen syndrome
- Deposition of fibrin(blood clot) in small vessels, producing hemorrhage in the adrenals(top of
kidneys) that alters peripheral vascular resistance leading to shock and death
 Deficiency in complement component C5-C8(membrane attack system/MAC)
- Humoral Mediated Immunity/ HMI – is an additional defense of our body
- Is a CASCADE
 - Contains complement C1 – C9 and each has different function but the major
function of these is to LYSE the Bacteria
- Once the bacteria is lysed, it will notify the macrophage and it will engulf and
process it to eliminate it in the body.
 2 types of Immunity
 1. Human Immunity – Proteins that protects the body, complement and antibodies produced
by Bcells
 2. Cell Mediated – WBCs, macrophages, Tcells and NKcells

 Penicillin G/Pfizerpen G – given to patients with meningococcemia

- Physician will treat the meningococcal disease with a high dose in the initial 48 hours of therapy
because meningitis is a lifely complication

Specimen collection and handling

 Specimen: CSF, blood, nasopharyngeal swabs and petechial skin lesions

 Sensitive to SPS, thus the content in blood culture broth should not exceed 0.025%
 Cold sensitive organism
 Ex. CSF -37 degree celsius

Laboratory Diagnosis

 Gram stain – gram (-) cocci

 Culture – BAP, CAP and TMA
 Oxidase test
 CHO utilization - (+ for N. meningitides)
- Glucose, lactose, maltose and etc.
Glucose Lactose Maltose
N. gonorrhea (+) (-) (-)
N. meningitis (+) (-) (+)

Nonpathologic Neiserria Species

 N. cinera – colony morphology resembles N. gonorrheae on CAp

 N. flavescens – yellow pigmented Neisseria spp.
 N. lactamica – found on the nasopharynx of infants and children (+ lactose)
 N. mucosa – seen in airways of dolphins
 N. sicca – have dry adherent and breadcramp like colonies
 N. elongata – rod-shape gram negative
 N. weaver – Rod shape in appearance

Moraxella catarrhalis (Branhamella catarrhalis)

 This specie resembles the Neisseria by exhibiting “gram-negative coccal” morphology

 Most common isolate of the genus Moraxella
 Normal flora of upper respiratory tract (lungs are not involved)
 Microscope: small gram-negative cocci that tend to grow in pair end-to-end with adjacent sides flattened
 Culture: smooth, opaque, gray to white with hockey puck appearance
 Biochemical test: oxidase (+) and catalase (+)
Neisseria Glucose Maltose Lactose Sucrose Nitrate Polysaccharide
spp Reduction from Sucrose
N. animalis - - - + +
N. canis - - - - + -
N. caviae - - - - + -
N. cinerea - - - - - -
N. cuniculi - - - - - -
N. + - - + - +
denitrificans

N. elongate - - - - - -
(Var.
glycolytica)

N. flava + + - - - -
N. flavescens - - - - - +
N. + - - - - -
gonorrheae

N. lactamica + + + + - -
N. + + - - - -
meningitidis

N. mucosa + + - + + +
N. ovis - - + +
N. perflava + + - - - +
N. sicca + + - - - +
N. subflava + + - - - -
N. - - - - + -
catarrhalis