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!