Neisseriaceae

Laboratory diagnosis of infections caused by the genus Neisseria

Family Neisseriaceae

• Comensal; exception: Neisseria gonorrhoeae (always pathogenic)

• Colonize mucous membranes

• Common characters:– Gram negative, – shape: reniform cocci – arrangement: pairs (diplo) + tetrades/larger groups– cultivation: optimal growth 35-37°C– Catalase +– Oxydase +


• Genera:– Neisseria

• Neisseria gonorrhoeae• Neisseria meningitidis

– Moraxella• Branhamella catarrhalis

– Kingella

– Acinetobacter

Neiseria gonorrhoeae - Clinical Significance -

• Pathogenic germ: gonorrhea = sexually transmited infection (STI); incubation 2-7 days

• Men: – acute urethritis (abundant urethral secretion + dysuria);

– Complications (due to lack of / delayed treatment): • urethral strictures, • prostatitis, • epididymitis

Neiseria gonorrhoeae - Clinical Significance - continued

• Women: – endocervicitis (purulent vaginal secretion + dysuria) – OR asymptomatic !!

↓– further transmission to sexual partner; – Complications (due to lack of / delayed treatment):

• Salpingitis (inflammation of the falopian tubes) + infertility

• pelvic peritonitis, • PID (pelvic inflammatory disease)• Gonococcal arthritis + skin rash (rare complication by

hematogenous dissemination)

Neiseria gonorrhoeae - Clinical Significance - continued

• Newborns to infected mothers: gonococcal ophtalmia neonatorum (may lead to blindness)

• Prophylactic treatment: erythromycin ointment / silver nitrate

Neisseria gonorrhoeae- Collection of specimens -

• Women – endocervical secretion (during gynecologic exam):– insert valves

– use 2 consecutive swabs (1 for microscopy + 1 for inoculation of culture media);

– insert each swab into cervix and rotate

• Men – urethral secretion (in the morning/at least 1 hour after last miction):– Acute urethritis: direct swab/loop collection (abundant secretion)– Chronic infection: loop / special swab inserted 2 cm into urethra

and rotated

Neisseria gonorrhoeae- Transport of specimens -

• Ideally: direct inoculation on culture media (gonococci are very sensitive to external environment; very low microbial load in collected specimens)

• Alternatively - transport media (semisolid, non-nutritive):– Stuart‘s medium: agar + sodium thioglycolate (delays oxydation),

Ca, Mg salts (osmolar protection of bacterial cells), methylene blue (redox indicator; blue colour = oxydation)

– Amies‘ medium: similar + charcoal (neutralize toxic materials)

• Transportation to lab – a.s.a.p. – tube cap firmly screwed; – keep tube cool but do not freeze!

Neisseria gonorrhoeae- Microscopic examination -

Gram stained smears: - over 95% sensitivity in acute male urethritis; - less sensitive in:

- chronic infections (increased associated microbial flora)- endocervicitis (50-70% sensitivity)

- High no of PMN cells with Gram negative reniform (kidney-like) cocci, arranged in pairs (”coffee beans”; concavities facing each other)

Neisseria gonorrhoeae: Gram stained smear

Gram stained smear: gonococci in PMN cells

N.gonorrhoeae: Gram negative reniform cocci, in pairs + PMNs

Neisseria gonorrhoeae: Cultivation

Fastidious organism = requiring complex cultivation conditions i.e. blood + aminoacids + vitamins

e.g. Thayer Martin medium = Chocolate agar (blood) + antibiotics:- Vancomycin (eliminates sensitive Gram positive cocci)- Colistin (eliminates Gram negative bacilli)- Trimethoprim (eliminates Proteus spp)- Nystatin (eliminates fungi)

- Incubation at 37°C, humidity, CO2 (or candle jar), 24-48 hours

Cultivation in CO2 atmosphere: ”candle jar”

Cultivation in CO2 atmosphere: CO2 incubators; gas pack systems

Neisseria gonorrhoeae: Cultivation- continued -

Colonial characters:

round, 0.5-1 mm,

transparent / grey,

shiny

(best examined

under magnifying

glass)

Neisseria gonorrhoeae: identification

• Oxidase test

• Catalase test

• Gram stained smear from colonies: – pairs of Gram positive reniform cocci

• Biochemical tests:– The CTA test (cystine trypticase agar)

• Agglutination with antisera

The Oxidase test

Principle: the enzyme cytochrome c oxidase oxidizes the reagent TMPD (TetraMethylPhenylenDiamine) to indophenol – purple/dark blue end product; if enzyme is not present TMPD remains colourless.

• used to identify bacteria that produce cytochrome c oxidase e.g. Neisseria

Procedure: filter paper soaked with TMPD is moistened with sterile distilled water; pick bacterial colony with loop and smear on filter paper; colour change to dark blue/purple within 10-30 sec = POSITIVE test

The Oxidase test: purple colour = cytochrome c oxidase present

The Catalase test

• Principle: the enzyme catalase decomposes hydrogen peroxide (H2O2) into water and oxygen:

2H2O2 →2H2O + O2 (gas bubbles)

• 2-3 drops of hydrogen peroxide placed directly on a colony

• POSITIVE TEST: rapid effervescence

• Neisseria (+)

The CTA (cystine trypticase agar) test

- Neisseria spp. produce acid from carbohydrates by oxidation → yellow colour

- 4 tubes with CTA base + phenol red indicator- Add 4 carbohydrates (one in each tube):

- glucose (dextrose) – POSITIVE test (N.gonorrhoeae) - Maltose - Negative - Lactose - Negative - Sucrose - Negative

- Inoculate bacterial culture (3-5 colonies) in each tube with different disposable loops

Neisseria gonorrhoeae: oxidation of sugars

CTA test:• Only glucose (dextrose) tube

shows production of acid (yellow turbidity in upper part of tube)

• The maltose, sucrose and lactose tubes show no acidification (red colour persists in the whole tube)

API NH gallery • Only glucose +

Agglutination-based Identification

N.gonorrhoeae: antibiotic susceptibility testing

• recquired due to strains resistant to:– Penicillins (production of penicillinase or other mechanisms)– Spectinomycin (narrow spectrum antibiotic, selectively active

against gonococci)

• therapeutic agents for penicillin-resistant strains: cephalosporins, fluoroquinolones





– Kingella

– Acinetobacter

Neisseria meningitidis- Clinical significance -

• Comensal germ; may colonize human oro- and nasopharinx

• Inter-human transmission via contaminated respiratory droplets

• May cause very severe infections: ”meningococcal disease”

Meningococcal disease

• Nasopharingeal colonization (from infected person; transmission via respiratory droplets: coughing, sneezing)

• Bloodstream invasion:– Meningitis (~55%)– Meningitis + meningococcemia (~30%)– Fulminant meningococcemia (~15%)

Meningococcemia

Infection causes disseminated intravascular coagulation (DIC) →

• ischemic & necroticlesions caused byblood clots +

• hemorrhagesby clotting disorders →subcutaneous bleeding

(”meningococcal rash”)

Meningococcal meningitis

• bacteria invade the CSF: inflammation and irritation of the meninges (membranes surrounding the brain and spinal cord)

Neisseria meningitidis- Collection and transport of specimens -

• Cerebro-spinal fluid (CSF) – collected by spinal tap before antibiotic treatment – turbid aspect suggests bacterial meningitis

+• Blood – for blood culture• Nasopharyngeal exudate

Transport to the laboratory a.s.a.p. (protect from light and temperature variations)

Collection of cerebrospinal fluid (CSF)

Lumbar punction (spinal tap) • patient lies on the side, knees pulled up toward

chest, chin tucked downward • back cleaned and disinfected (iodine) + health

care provider injects local anesthetic into lower spine

• spinal needle inserted into lower back area• needle properly positioned, CSF pressure

measured and sample collected in sterile tube• needle removed, area cleaned, bandage placed

over puncture site

Blood collection for hemoculture

Blood injected in 2

sets of sealed bottles

containing liquid culture

medium for aerobic and

anaerobic bacterial

growth

Collection of blood for hemoculture

• Wear gloves + PPE• Thoroughly wipe skin with antiseptic (chlorhexidine,

iodine, alcohol)• During 3 hours, draw blood by venipuncture from up to 3

different sites at 1 hour interval (3 sets of 2 bottles each) – around 5 ml blood per bottle

• After drawing the blood, dispose of the syring needle and attach new, sterile needle

• Disinfect cap of each culture medium bottle and inject 5 ml blood/bottle

Collection of blood for hemoculture

Automated systems for detection of bacteria in blood and other normally sterile body fluids

Neisseria meningitidis- Microscopic examination -

CSF – after centrifugation:• Supernatant – testing for meningococcal antigens

(antisera for group identication – rapid tests)

• sediment smeared on slides - Gram staining:

↓

• High no of PMNs + pairs of Gram negative, reniform cocci, concavities facing each other, both intra- and extracellular

Neisseria meningitidis: Gram stained smear

• Smears should be examined for at least 10 minutes

• Large no of PMNs – usually indicative of good prognosis

Neisseria meningitidis: cultivation

• CSF sediment inoculated on:– Blood agar/chocolate agar – nonhemolytic, grey, transparent,

smooth colonies, 1 mm

• Blood inoculated on liquid media (see above): examination and reinoculation during 5-7 days

• Nasopharyngeal exudate

• Muller-Hinton agar; Selective media (antibiotic content: e.g. vancomycin)

Neisseria meningitidis: identification

• Colonial characters (see above)

• Gram stained smear from suspected colonies

• Tests:– Oxidase– Utilisation of sugars (acid production): Glucose (Dextrose),

Maltose, Lactose, Sucrose (CTA sugar test; API id systems)– Serogroup identification (agglutination with antisera)

Neisseria meningitidis: nonhemolytic colonies, OX+, Vancomycin resistant

N. meningitidis: CTA sugar test

• POSITIVE tests for maltose and dextrose (turbidity + yellow colour in upper part of 2 tubes on the left)

• Negative tests for lactose and succrose (bacterial growth, no colour change in 2 tubes on the right)

Neisseria meningitidis: antimicrobial susceptibility

• Not routinely performed in diagnostic laboratories (most strains are still sensitive to penicillins)





– Kingella

– Acinetobacter

Branhamella catarrhalis- Clinical significance -

• Comensal of the upper respiratory tract• May cause angina, otitis, synusitis, pulmonary infections,

endocarditis, meningitis• Often involved in co-infections with Streptococcus

pyogenes, Streptococcus pneumoniae bronchopneumonia complicating viral infections (influenza, measles, whooping cough)

Branhamella catarrhalis: collection of specimens

Sputum (in respiratory infections):• Challenging! – must avoid contamination of sputum with

saliva and secretions from upper air waysOptimal moment: in the morning (higher amount of sputum

secreted during the night and stagnant in lower respiratory ways)

Indirect method:• Patient energically rinses mouth with saline solution • Coughs and expectorates in sterile container (Petri dish)Direct method:• Bronchoscopy / tracheal punctioning

Branhamella catarrhalis:- Microscopic examination -

PMNs,

multiple mucus

filaments,

Gram negative cocci, in diplo

Branhamella catarrhalis: cultivation and identification

• Blood agar, 35°C, CO2 atmosphere: grey, nonhemolytic, 3-5 mm, colonies

• Identification:– Gram smear from suspected colonies

– Tests:• Oxidase +• Catalase +• Utilization of sugars (acid production): CTA sugar test

– Negative for all 4 sugars

Branhamella catarrhalis: nonhemolytic colonies, OX+, no acid from sugars

Neisseria and Moraxella:Utilization of sugars (acid production)

Germ Glucose Maltose Lactose Sucrose

Neisseria gonorrhoeae

+ (very weak in some strains)

- - -

Neisseria meningitidis

+ + - -

Branhamella catarrhalis

- - - -

Neisseriaceae

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