The document provides an overview of the laboratory diagnosis of infections caused by the Neisseria genus, highlighting the pathogenic nature of Neisseria gonorrhoeae and Neisseria meningitidis. It details clinical significance, specimen collection methods, and microbiological examination techniques for diagnosing infections like gonorrhea and meningococcal disease. The document also discusses antibiotic susceptibility and the importance of rapid diagnostic methods in clinical settings.

1. Laboratory diagnosis of infections caused
by the genus Neisseria
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neisseriaceae-45658928

2. 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 +

3. Family Neisseriaceae
• Genera:
– Neisseria
• Neisseria gonorrhoeae
• Neisseria meningitidis
– Moraxella
• Branhamella catarrhalis
– Kingella
– Acinetobacter

4. 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

5. 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)

6. Neiseria gonorrhoeae
- Clinical Significance - continued
• Newborns to infected mothers:
gonococcal ophtalmia
neonatorum (may lead to
blindness)
• Prophylactic treatment:
erythromycin ointment / silver
nitrate

7. 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

8. 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!

9. 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)

10. Neisseria gonorrhoeae: Gram stained
smear

11. Gram stained smear:
gonococci in PMN cells

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

13. 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

14. Cultivation in CO2 atmosphere: ”candle jar”

15. Cultivation in CO2 atmosphere:
CO2 incubators; gas pack systems

16. Neisseria gonorrhoeae: Cultivation
- continued -
Colonial characters:
round, 0.5-1 mm,
transparent / grey,
shiny
(best examined
under magnifying
glass)

17. Neisseria gonorrhoeae: identification
• Oxidase test
• Catalase test
• Gram stained smear from colonies:
– pairs of Gram negative reniform cocci
• Biochemical tests:
– The CTA test (cystine trypticase agar)
• Agglutination with antisera

18. 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

19. The Oxidase test:
purple colour = cytochrome c oxidase present

20. 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 (+)

21. 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

22. 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 +

23. Agglutination-based Identification

24. 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

25. Family Neisseriaceae
• Genera:
– Neisseria
• Neisseria gonorrhoeae
• Neisseria meningitidis
– Moraxella
• Branhamella catarrhalis
– Kingella
– Acinetobacter

26. 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”

27. Meningococcal disease
• Nasopharingeal colonization (from infected person;
transmission via respiratory droplets: coughing,
sneezing)
• Bloodstream invasion:
– Meningitis (~55%)
– Meningitis + meningococcemia (~30%)
– Fulminant meningococcemia (~15%)

28. Meningococcemia
Infection causes disseminated
intravascular coagulation
(DIC) →
• ischemic & necrotic
lesions caused by
blood clots +
• hemorrhages
by clotting disorders →
subcutaneous bleeding
(”meningococcal rash”)

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

30. 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)

31. 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

32. Blood collection for hemoculture
Blood injected in 2
sets of sealed bottles
containing liquid culture
medium for aerobic and
anaerobic bacterial
growth

33. 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

34. Collection of blood for hemoculture

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

36. 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

37. Neisseria meningitidis: Gram stained smear
• Smears should be
examined for at least 10
minutes
• Large no of PMNs –
usually indicative of good
prognosis

38. 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)

39. 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)

40. Neisseria meningitidis: nonhemolytic
colonies, OX+, Vancomycin resistant

41. 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)

42. Neisseria meningitidis:
antimicrobial susceptibility
• Not routinely performed in diagnostic laboratories (most
strains are still sensitive to penicillins)

43. Family Neisseriaceae
• Genera:
– Neisseria
• Neisseria gonorrhoeae
• Neisseria meningitidis
– Moraxella
• Branhamella catarrhalis
– Kingella
– Acinetobacter

44. 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)

45. Branhamella catarrhalis:
collection of specimens
Sputum (in respiratory infections):
• Challenging! – must avoid contamination of sputum with
saliva and secretions from upper air ways
Optimal 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

46. Branhamella catarrhalis:
- Microscopic examination -
PMNs,
multiple mucus
filaments,
Gram negative cocci,
in diplo

47. 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

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

49. Neisseria and Moraxella:
Utilization of sugars (acid production)
Germ Glucose Maltose Lactose Sucrose
Neisseria
gonorrhoeae
+
(very weak in
some strains)
- - -
Neisseria
meningitidis
+ + - -
Branhamella
catarrhalis
- - - -