Steps of the bacteriological diagnosis in infections caused by bacteria of the genus Neisseria - for the use of medical school students in the second year of study

1. Laboratory diagnosis of infections caused
by the genus Neisseria
http://www.slideshare.net/DanaSinzianaBreharCi/
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
- - - -