Download - Fastidious bacteria 1
Fastidious bacteria1
Jan Tkadlec
Dept. of medical microbiology
Winter 2020
S. aureus on columbia blood agar
Fastidious bacteria are weird
Normal bacteria:Grow rapidly (visible colonies in 24 hours)At temperature of human body (37°C)AerobiclyOn common culture media (blood agar)Examples of normal = non-fastidiousbacteria: E. coli, S. aureus, P. aeruginosa
They don´t behave normaly
What is normal is matter of perspective.Lot of microorganisms did not grow in condition that we as human consider normal
Which bacteria are fastidious?
1. Non growing or intracellular• Unknown or complex growth requirements• Obligate intracellular pathogens– unable to grow outside of the cell
(chlamydia, mycoplasma, leprosy)
2. Slow growing• Extremely long cultivation (TBC, Bartonella)• Could be overgrowned by other bacteria
(4. Dead bacteria)• If you start ATB treatment before sample colection, the
chance of positive culture is low – Take sample beforegiving antibiotics!
3. Dormant = non growing, resistant (e.g. Result of ATB treatment)• Spores• Persistors• VBNC – Viable But Not Cultivable Cells
They need specialsignal to becamecultivable
VBNC – Viable But Not Cultivable Cells and Persisters
• In each bacterial population (less than 1% ofcells)
• Imposible to cultivate directly, resuscitationnecessary (temperature shift, ATB removal etc.).
• Dormant – can persist through ATB therapy• VBNC could be induced by the human serum• Common among bacteria, common in chronic
infections
Even non-fastidious bacteria could be in nonculturablestate
SporesDormant stadiumGermination require signal – EtOH or heat shock
Diagnostic methods for fastidious bacteria
Sample
Microscopy Culture SerologyIg detection
Cell components detectionAntigenDNA or RNA (PCR or RT-PCR)Enzymatic activityMetabolite detection
Direct detection Indirect detection
How to culture fastidious bacteria?
If cultured on common media (blood agar etc) fastidious bacteria1. Will not grow2. Will grow slowly3. Will be overgrown by other microbes becase they grow slowly or there is
just few of them in sample
Solutions:Anaerobic or microaerophilic atmosphere when neededProlonged cultivation – up to weeks or months drying out of themedia has to be preventedPre-enrichment in liquid media before plating on agar
Solid media for fastidious media has to be:Enriched – has all growth requirements, rich in nutrientsSelective – ATB and antifungal drugs to limit grow of othermicrobesDiagnostic (chromogenic) – colonies of the microbe has distinctcolour due to utilisation of chromogenic compound in the media
Examples of media for fastidiousbacteria
Brilliance CampyCount Agarchromogenic selective medium for C. jejuni and C. coli
Charcoal Blood Agar w/cephalexinFor Bordetella pertusisEnhanced and selective (ATB)
Schaedler agar forAnaerobesWith lysed horse bloodEnhanced (lysed blood)Selective when ATB added
However, sensitivity of culture and time to result could be an issue, combination of culture with other methods could increase speed and chanceof detection
How to culture fastidious bacteria
Some bacteria could be grown on cell culture or in animal models.For diagnostic purposes it is not standard method (expensive, laborious, etc)
Problem with ATB susceptibility testing• ATB susceptibility testing is ultimate reason why to culture bacteria.• Disc diffusion on Mueller-Hinton agar supplemented with blood or other enhanced
media• Fastidious bacteria are not tested by broth microdilution – did not grow• E-test is possible to use in combination with enriched media (e.g. Schaedler media, or
MH agar with blood) but blood interfere with some ATB (e.g. TMP-SXT)
Molecular genetic methods
End point PCRqPCR – quantitative Real Time PCR
• Detection of DNA or RNA
Izolation of nucleic acids (RNA needs to be transcribed into DNA- Reverse transcription=RT PCR)
DNA Amplification = PCR
Evaluation of results
Fluorescent stain or probe withfluorescing marker
Gel electrophoresisSequencing
PCR
qPCR• DNA binding dye (SYBRGreen)• Or specific fluorescent probe
increase specificity
Probe
Level of fluorescence corresponds to quantity oftargett DNA in reaction
Highly positive samples –fluorescence starts to growearlier – lower cycles (Ct)
• Fluorescence based quantification of pathogen load• Contamination or infection?• Efectivenes of therapy
• Real time detection – faster than end point PCR
Approaches in PCR diagnostic
Pathogen specific assaysBroad - range or panbacterial PCR
Positive result only if DNA of specific bacterium ispresent in a sample
Amplification of sequence shared among all bacteria (e.g. 16S)Species identification requires additional steps after PCR – most often sequencing
Fastidious bacteria - examples
Campylobacter sp.• Microaerophilic gram-negative helical shaped
rods
• Campylobacter jejuni, C. coli
• Reservoir: Gastrointestinal tract of animals and livestock; poultry is the most common. Domesticdogs and cats may also be colonized. zoonosis
• Transmission: Typically foodborne via consumption of undercooked poultry, foodstuffs in contact with raw/undercooked poultry, raw/unpasteurized milk, chicken paté, or fecal-oral transmission from symptomatic individuals.
Campylobacter sp.Microbiology:
Sample: Stool in transport media orrectal swab
Sensitive to drying
Survival at room temperature is poor
Microscopy is not commonly used
Growth conditions:
Microaerophilic – CO2 suplementation
Temperature:
optimally 37 to 42 °C
Duration: 48 – 72 hours
Media:
Campy-BA – enriched selective (antibiotics) blood agar
Charcoal based selective medium (Karmali)
Direct detection:
Species specific PCR – enteric panels
Antigen detection in stool by latex aglutination
Clinical significance:
• An acute, febrile, bacterial diarrheal illness characterized by watery diarrhea that frequently becomes bloody after a few days
• 9 000 000 cases per year in EU
• Infection of small intestine – jejunum
• Symptoms: diarrhea (often bloody), fever, and abdominal pain, cramps, tenesms, pseudoappendicitis
• Extraintestinal infection (meningitis, endokarditis etc.) – AIDS patients
• Post-infection complication:• Guillain–Barré syndrome - acute demyelination of
the peripheral nerves
• reactive arthritis Self-limiting disease (7 days)ATB treatment is in most cases not needed
Campylobacter jejuni
Gram stainingCharcoal Selective Medium
Contains:ATB (vancomycin, cefoperazon)Cycloheximid – antifungal agent
ATB susceptibility testingMueller Hinton media supplemented with blood
Stool is better sample than rectal swab, but rectal swab is easier to acquireStool has to be wattery otherwise it is not diarhoea
Helicobacter pylori
• Microaerophilic gram-negative motile helicalshaped rod
• Reservoir: H. pylori colonize human stomach, prevalence up to 50% of the population, in about20% of them is clinical manifestation
• Transmission : oral–oral or fecal–oral route, detailsare not known, spread among people in closecontact (family)
Adaptation to the acidic environment (stomach):• Penetration into the mucus layer using flagella• Adhession to stomach epitelia• Urease production - urea to CO2 and ammonia
(neutralization of acidity)
Helicobacter pylori
Microbiology:
Growth conditions:
Microaerophilic – ↑ CO2, H2, N2
Sample: Stool, gastric biopsies
Temperature: optimally 35 to 37 °C
Duration: up to 1 week
Media:
Campylobacter selective agar
Wilkins Chalgren agar
Microscopy of the biopsy
Urease test
Serology
Antigen detection in stool
Clinical significance:
• Gastritis – most common symptom of H. pylori infection, acute or chronic
• Spontaneous recovery, however H. pylori persist without treatment
• For gastritis:triple-drug therapy clarithromycin, amoxicillin, and a proton-pump inhibitor for 14–21 days
• Peptic ulcers – treated like gastritis
• Cancer• MALT (mucosa-associated lymphoid tissue) lymphoma
• Diffuse large B-cell lymphoma
• Stomach adenocarcinoma
Helicobacter pylori – Diagnostics
Urease detectionDirect from the sample
Biopsy: better to take more samples from different locationsTherapy is started empiricaly, if it fails the microbiological testing is performedTests from feces or biopsies
H. pylori on blood agar
Helicobacter pylori selectivemedium with 10% horse serum, and ATB (Cefsulodin, Vancomycin, Amphotericin)
Note the tiny colonies thatcould be missed due to extensive growth of othermicrobes when using non selective media
Helicobacter pylori – Gram stain
Gardnerella vaginalis• Microaerophilic/facultatively anaerobic gram-
negative or gram-variable rod
• cause of bacterial vaginosis, however mostly ispresent as asymptomatic comensal, infection whenoverpopulated
• Diagnosis: Microscopy, culture or PCR
Gardnerella selective agarV agar-enriched blood agar
Mycoplasma sp
• Small facultative anaerobes pleomorphic without cell wall(peptidoglycan) with small genome. Parasites of humanand animals
• Sensitive to environmental factors – limited survival
• Respiratory patogen: Mycoplasma pneumoniae
• STD: Mycoplasma hominis, M. genitalium, Ureaplasmaurealyticum
• Reservoir: primary patogens (M. pneumoniae) oropportunistic (M. hominis, M. genitalium, U. urealyticum) pathogens/comensals
• Culture is possible but other methods are prefered
• Use of microscopy is limited due to small size of the cell
Mycoplasma pneumoniae
• Cause of atypical pneumonia and upper airwaysinfection
• Up to 40% of community acquired pneumonia
• „Walking pneumonia“ – often mild symptoms
• Direct PCR detection or indirect IgA detection
M. hominis, M. genitalium, U. urealyticum
Comon colonizers of urogenital tract – asymptomaticCould cause infection:• Non-Gonococcal Urethritis (NGU)
• M. genitalium or U. urealyticum
• Pelvic inflammatory disease (PID)• M. hominis or M. genitalium
• Diagnosis PCR (quantitative – could differentiatecolonisation and infection)
• Culture detection based on urease and other enzymsactivity
• Therapy tetracyclines, macrolides, fluoroquinolones
Chlamydophila and Chlamydia
• Obligate intracellular pathogens
• Unable to syntetize ATP
• Peptidoglycan is missing
• EB – infection
• RB – reproduction
• Could grow on cell culture• But low sensitivity
Chlamydia trachomatis
• Serotype determines type of infection
• D-K are cause of STD• most common STD:Non-Gonococcal Urethritis (NGU) in men, cervicitis in women,
could cause infertility• in infected newborns cause keratoconjunctivitis or pneumonia
• A-C –trachoma - keratoconjunctivitis• Low income countries, highest in Africa• Infection of the eye could cause blindness• Spread through hand to eye contact or by insect vector
• L1-L3 Lymphogranuloma Venereum• Tropical and subtropical regions• STD: painless papule or shallow ulcer at the site of inoculation, untreated could
progress into perirectal abscesses or lymphedema of the genitals
• Diagnosis PCR, direct immunofluorescence (rapid tests)
• Treatment: macrolides, tetracyclines, fluoroquinolones
Incidence of chlamydia, gonorrhoea, trichomoniasis, and syphilis in women and men aged 15–49 years by WHO region, 2012.
Chlamydophyla pneumoniae
• Common cause of community acquired pneumonia
• Also cause of sinusitis, pharyngitis
• Often mild symptoms
• Diagnosis PCR or serology
• Therapy – tetracyclines, macrolides
Chlamydophyla psittaci -psittacosis
• Reservoir: Birds (zoonosis) – parrots disease
• Transmission: bacteria from the aerosolized feces of certain birds, specifically parrots. Infection in people in contact with birds.
• Clin signif.: atypical pneumonia with severe headache, more common in young and middle-aged adults.
• acute fever, severe headache with photophobia, andnonproductive cough. Hepatosplenomegaly, hepatitis, and disseminated intravascular coagulation (DIC) may also occur
• Diagnosis PCR or serology – contact with birds!
Mycobacteria
• Strictly aerobic acid fast rods
• free-living (water, soil) but also obligate parasites
• cell wall is thicker than in many other bacteria, being hydrophobic, waxy, and rich in mycolicacids/mycolates – resistant to physical or chemicalfactors including ATB – hard to treat
• Slow growth
• Could not be stain by gram staining
• Ziehl-Neelsen staining
Mycobacteria stain (Ziehl-Neelsen)
Tuberculosis
Mycobacterium tuberculosisClinical significance:
M. tuberculosis – tuberculosis (TB) – consumption
Airborne transmission
Active TB: fever, malaise, fatigue, night sweats, weight loss, cough, dyspnea, pleuritic chest pain, and hemoptysis
In imunocompromised TB couldspread to meninges, lymphaticsystem, genitourinary (GU) system, and the bones causingextrapulmonary TB
Latent TB is not contagious, whereas active TB is
Vaccination
Long term ATB: isoniazid, rifampin, ethambutol, streptomycin, and pyrazinamide
Problem with antibiotic resistence of TB
M. bovis – cattle is reservoir , infection through contaminatedmilk (zoonosis). Similar symptomsto TB
Koch´s Bacill = TBC
Robert Koch
Mycobacterium tuberculosisMicrobiology:
Sample: pulmonary samples, tissue biopsies
Growth conditions: ↑ CO2, dark, high humidity
During long cultivation media should not dry out
Temperature: 35 °C
Duration: up to 8 weeks (doubling time once per 21 hours)
Media:
egg-based solid media such as Lowenstein-Jensen
Middlebrook
Cultivation is inevitable to detect antibiotic resistance
But performed only in specialised laboratories
PCR
Microscopy – Ziehl-Neelsen (acid-fast) or fluorochrome staining
PPD skin test – Tuberculin (contains TB antigens) is injected intradermaly- ifimmune reaction appear in up to 72 h – person has been exposed to TB
Serology
PPD measured as a diameter ofinduration
Mycobacterium leprae• Leprosy = Hansen disease
• Reservoir: Armadilos
• India, Brazil, and Indonesia have the highest incidence.
• M. leprae reproduces at cooler temperatures, thus disease is limited to the skin and cutaneous nerves in humans
• Despite long-held historical beliefs, leprosy is not very contagious.
Mycobacterium lepraeClinical significance:leprosy• infection of the skin, nasal
mucosa, and cutaneous nerves - intracellular bacteria
• Symptoms: Cutaneous skin lesions, sensory loss (burns orother injuries). Hypopigmentedskin nasal perforation, saddle nose, and corneal scaring leading to blindness can occur.
• severe disease, auto-amputation of digits, peroneal, tibial, and ulnar neuropathy may be seen.
Microbiology:
Intracellular pathogen - not possible to culture on solid media
• In vivo mouse foot pads model to growM. leprae - research not diagnostic
• extreme doubling time – 12-14 days
Sample: tissue biopsies
Microscopy of the biopsy
PCR
Symptomatic diagnosis
Treatment:6-12 months – Dapsone, rifampicin,
Mycobacterium leprae
Face deformation
New cases of leprosy in 2016
Leprosy tissue biopsy
Other mycobacteria
• Atypical mycobacteria – oportunic human patogens
• Reservoir: water or soil
• M. avium-intracellulare - infection of lymphaticnodes or respiratory TBC like infection
• M. abscessum – fast growing, soft tissue and wound infection, chronic lung infection in immunocompromised patients (cystic fibrosis)
• M. marinum – fish patogen, wound infection in aquarists
Nocardia
• Gram positive branching rods
• Strictly aerobic
• cell wall contains mycolic acids –weakly stained by gram
• Environmental bacteria (soil)
• Acid fast
• Nocardiosis – slowly progressingpneumonia, could lead to systemicspread
• Encephalitis and brain abscesses
• Culture on common media 3-5 days but up to 3 weeks
Nocardia on chocolate agar
Clostridium difficile
• Spore forming gram-positive anaerobic bacterium
• Difficult to culture - difficille
• Disease cause only toxin producing strains
• Colonizer of gatrointestinal tract human (5%) and animals, common in water and soil
• Post antibiotic diarhoea (clindamycin, fluoroquinolones, 2nd and higher gen. ofcephalosporines), toxic megacolon, pseudomembranous colitis
• Nosocomial infection
• Sample: watery stool (=diarhoea)
• Antigen and toxin detection or PCR detection of respektive genes
• Culture: Ethanol or heat treatment of the sample – fyzicalor chemical shock promotes germination of spores. Sample is mixed 1:1 with ethanol and inoculated on agar plate
• Selective media with ATB, strictly anaerobic atmosphereduration 2-5 days
C. difficile
C. difficile
Antigen and toxin production detection
Selective cultureSchaedler agar
Anaerobic culture methods
Chemical reaction remove O2Anaerobic atmosphere is made by gas supplement
C. difficileTreatment:
• Metronidazol, vancomycin, fidaxomicin
• Fecal transplantation
Prevention
• Hand washing
• !Alcohol desinfection does not work – alcohol promotesspore germination!