Laboratory diagnosis in infections produced by the genera:

Vibrio, Campylobacter, Helicobacter, Pseudomonas

http://www.slideshare.net/DanaSinzianaBreharCi/vibrio-campylobacter-helicobacterpseudomonas

Genus Vibrio

• Family Vibrionaceae:

– Genera: • Vibrio – species clinically significant for human pathology:

– Vibrio cholerae– Vibrio parahaemolyticus– Vibrio vulnificus

• Aeromonas

• Plesiomonas

Vibrio cholerae

Classification (~structure of ”O” antigen):

2 serogroups: • O1 (epidemic cholera):

– 3 subtypes:• Ogawa• Inaba• Hikoshima

– 2 biotypes:• Classic• El Tor

• Non-O1

(non-epidemic cholera-like illness)

Clinical forms: - Diarrhoeic disease (favourable

evolution)- Severe forms with acute

dehydration (choleric enterotoxin)

Vibrio cholerae- Collection and transport of specimens -

• Faeces – as early as possible (within the first 24 hours after onset of symptoms)

• Vibrios - High sensitivity to dryness – inoculation onto transportation media (Cary Blair and alkaline peptone water – pH 8.6)

Vibrio cholerae- Direct examination -

• Macroscopic exam of stool: watery (~60/day), (”rice water diarrhoea”)

• Microscopy: wet mount under dark fileld microscopy: motile comma-shaped vibrios actively moving on the microscopic field

Vibrio cholerae- Gram stained smear -

• Gram negative, comma-shaped bacilli

Vibrio cholerae- Cultivation and Isolation -

• Enrichment liquid medium: alkaline peptone water – incubation at 37°C, 6 hours

• Reinoculation on solid selective media:– TCBS agar (Thiosulphate Citrate Bile salts Sucrose agar):

sucrose fermentation (yellow colonies)– Blood agar: complete hemolysis– MacConkey: lactose-negative colonies

Vibrio cholerae

• TCBS agar (thiosulphate citrate bile salts sucrose agar): sucrose fermentation (large, yellow colonies)

Vibrio cholerae on blood agar: medium size colonies (2-3 mm) with complete

hemolysis

Vibrio cholerae on MacConkey agar

• moderate size (1-3 mm), lactose-negative / slightly pink colonies (resembling ”late”/”slow” lactose fermentation)

Vibrio cholerae: biochemical tests identification

Vibrio choleraeAntigenic structure based identification

• Serogroups– (O1/nonO1)

• Serotypes– (Ogawa/Inaba)

• Biotypes– (Classic/El Tor)

Vibrio cholerae- Antibiotic sensitivity -

• High sensitivity to tetracyclines, nalidixic acid, norfloxacin• Antimicrobial sensitivity testing – required to monitor

acquired resistance of certain strains

Vibrio parahemolyticus

• Clinical significance: – acute gastroenteritis after

consumption of contaminated seafood

– skin infections, conjunctivitis (occupational related – workers manipulating contaminated seafood)

Vibrio parahemolyticusCultivation and Isolation

TCBS agar:

sucrose is NOT

fermented

(green colonies)

Vibrio vulnificus

• Clinical significance: – primary sepsis following

severe, purulent infection of skin lesions (occupationally related: workers manipulating contaminated seafood, fish)

• Bacteriological diagnosis: – similar steps as above– Important differential

character: lactose fermentation! (”the lactose-positive vibrio”): RED colonies on MacConkey

Genus Campylobacter

• Clinical significance: acute diarrhoeal disease; risk factors: long term hospitalization, long term antibiotic treatments (disbalance of intestinal microbiota)

• 11 species: – C.jejuni, C.coli – involved in human infections

• Microscopy: – Gram negative bacilli, encurved/S-shaped, motile (single polar

flagellus)

• Cultivation: – special media + microaerophilia (anaerobic jar/candle

jar/anaerobic kits)

Campylobacter – Gram staining

• Gram negative bacilli, spiralated/S-shaped/encurved/”seagull wings”

Campy selective medium: chocolate agar + antibiotics

• normal fecal flora inhibited (trimethoprim, vancomycin, and polymyxin B)

• prepared, stored, dispensed and packaged under oxygen-free conditions

• Small (1 mm), non-hemolytic colonies, white/grey, Oxydase positive

• Gram stained smears from colonies: Gram negative, spiralated/S-shaped bacilli

• Antimicrobial sensitivity must be tested

Genus Helicobacter

• Clinical significance: – gastro-duodenal ulcer, gastritis (Helicobacter pylori)

• Collection of specimens: – gastric mucosa fragments (endoscopy, gastric lavage)– transport media (e.g. ”Portagerm pylori”, BioMerieux), at +4°C

• Microscopy: – Gram stained smear: Gram negative bacilli, encurved/spiraled,

4-6 polar flagella

• Cultivation: – selective, blood containing media + antibiotics e.g. Helicobacter

pylori agar; at least 3 days incubation!!– Microaerophilic incubation* (see next slide)

*Definition of terms: bacterial growth in relation with respiratory

processes (use of O2, CO2)

• Obligate aerobes need oxygen because they cannot ferment or respire anaerobically (e.g. Mycobacterium tuberculosis)

• Obligate anaerobes are poisoned by oxygen (e.g. Clostridium tetani, Clostridium difficile)

• Facultative anaerobes can grow with or without oxygen (e.g. Staphylococcus, Streptococcus, E.coli)

• Microaerophiles need some amount of oxygen but are poisoned by high concentrations of oxygen (e.g. Campylobacter, Helicobacter, Neisseria gonorrhoeae)

Helicobacter pylori – Gram stained smears

Helicobacter pylori on blood agar

• non-hemolytic, small (1-2 mm) colonies after 3 days of microaerophilic incubation

• Oxydase-positive

Genus Pseudomonas

• Gram negative bacilli, obligate aerobic, motile, nonsporulating, do not ferment glucose, oxydase positive

• Pseudomonas aeruginosa a.k.a. pyocyanic bacillus• Habitat:

– water, soil, air, human skin;

– humid conditions - biofilm e.g. in hospitals (toilets, humidifiers, respirators, plants, etc); human carriers

Pseudomonas aeruginosa

• Clinical significance:– Comensal, facultatively pathogen; colonizes mucous

membranes e.g. mere isolation in throat swab in the absence of relevant clinical context does not mean etiologic diagnosis but rather positive selection after antibiotic treatment

– opportunistic pathogen = infects impaired tissues & organs (tissues with lesions, patients with decreased immunity, etc); involved in hospital acquired infections e.g. eye infections, infection of burns/surgical wounds, UTI, lung infections, meningitis (by iatrogenic inoculation), sepsis

Pseudomonas aeruginosa infections: surgical wound, eye, burns

Pseudomonas aeruginosa- Bacteriological diagnosis -

• Collection of specimens: – depending on site of infection (pus, wound secretion, CSF, etc)

• Microscopy: – Gram stained smear: only relevant for naturally sterile

specimens (e.g. CSF): high number of PMNs + Gram negative bacilli

• Cultivation: – nonfastidious germ; grows on any medium– Naturally sterile specimens (CSF): blood agar– Faeces: selective media for enterobacteria

Pseudomonas aeruginosa

• Green colonies on agar (secretion of pigments: e.g. pyocyanin)

• There are also achromogenic strains!

Pseudomonas aeruginosa on blood agar

• metallic sheen

Pseudomonas aeruginosa- Biochemical tests -

• API 20NE – identification system for non-fastidious, non-enteric, Gram negative bacilli

OR• API 20E

Pseudomonas aeruginosa- Antimicrobial susceptibility testing -

• Required due to rapid acquisition of antibiotic resistance (especially in case of ”hospital strains” – sometimes highly resistant)