Hipo Template - PowerPoint

							Rapid detection and Identification of
Campylobacter and Arcobacter species




         Marwan Abu-Halaweh
                           Phylogeny
• Campylobacter and Arcobacter are microaerophiles and member of
  the order Campylobacterales , Class Epsilonproteobacteria phylum
  Protobacteria
• Natural inhabitants of intestinal tracts of poultry and worm blooded
  domestic animals
          Campylobacter history

• In 1886 Esherich observed organism resembling
  Campylobacters in stool samples of children with
  diarrhoea
• In 1913 Campylobacter were mis-identified as Vibrio.
• King In 1957 reported that a thermophilic Vibrio-like
  bacterium associated with human acute enteritis.

• In 1973 Campylobacter have been identified as new genus
  by Veron and Chatelain.
• C. jejuni the first species to be identified
Campylobacter Phenotypic Characterization

      Members are
•   Thermotolerant bacteria.
•   Curved, spiral or S-shaped, Gram-ve
•   Cells measure 1.5-6.0 m x 0.9 m
•   Motile by monopolar flagella
•   Microaerophilic
•   Grow aerobically or anaerobically between 15-42 oC.
•   Intolerate to freezing and drying
    Campylobacter Molecular characterization



•   Genome size is 1.6-1.7 Mb, except C. upsaliensis 2.0 Mb
•   GC contents around 30%
•   Multiple copies of 16S rDNA have been described
•   Campylobacter jejuni 81116 genome had been sequenced.
•   LPS as most G-ve bacteria is the pathogenic factor.
• LPS had been sequenced and express in E. coli
   Campylobacter Infection


• Campylobacter associated with human and animal diseases
• incubation periods of illness vary from 1 day to seven
  days.
• Guillain-Barré Syndrome(GBS)
• AIDS and Immunocomprmised patients are at high risk of
  Campylobacter infection.
• Most of the Campylobacter related illness caused by C.
  jejuni.
Epidemiology of gastrointestinal pathogens




                               Campylobacter is the most
                               common bacterial disease
                               Causing 46% of diarrhoeal
                               illness reported by Centre
                               for Disease control and
                               prevention (CDC)/USA
Arcobacter phenotypic characteristics

 Members are:
 • Gram -ve, curved or slightly curved, S-shaped, or helical
 • Non spore former
 • Motile by monopolar flagella
 • Microaerophilic
 •   Intolerate to freezing and drying..
       Arcobacter Infection


• Arcobacter Associated with animal disease (Abortion,
  Diarrhoea and mastitis)
• Arcobacter isolated from patient with diarrhoea.
Campylobacter And Arcobacter Detection methods

   • Selective media, Biochemical test, DNA-DNA
     hybridization, gene sequencing, PCR and Maldi mass
     spectrometry.
   • The appearance of colonies in Campylobacter selective
     media at 42 º C indicate the presence of Campylobacter.

   • Biochemical test such as catalase and cytochrome oxidase
     tests which are positive for C. jejuni, C. coli and C. lari.
   • Further biochemical test to identify the species (eg.
     Hippurarte hydrolysis)
 Campylobacter and Arcobacter biochemical
           detection problems


• Isolation and Identification of Campylobacter is time
  consuming require up to 4 days.
• Biochemical test depend on biochemical pathways and
  their disruption can lead to product failure leading to false
  result.
 Advantage of Molecular technique detection


• Denis et al observed that biochemical test provide only
  34% efficiency compared to 100% with the PCR.
• PCR tests have been developed for the detection and
  identification directly from pathological and food sample.
    Aim and advantage of this project

• 67 biochemical test and molecular technique have been
  devised for Campylobacter identification
• Not all these test are suitable for routine testing in
  microbiological laboratory.
• Project aim is to Develop a new rapid, easy to use,
  sensitive, accurate and low cost molecular methods for the
  detection, identification and quantitation of Campylobacter
  and Arcobacter species from enriched and isolated culture
  or directly from environmental and /or clinical sample.
Choice of Targets and Sensitivity
Moleculare Techniques
  Real Time PCR



• What is REAL TIME PCR?
• Continues monitoring of fluorescent signals derived from
  fluorescent resonance energy transfer (FRET).
• FRET PCR (ABI PRISM and the LightCycler TM) was
  described in 1996. Since then, there have been major
  innovations in both probe technology and instrumentation
  design.
Fluorophore probe innovations
Real Time PCR instrumentation innovations
Real Time PCR instruments used in this project


  LightCycler
                                      iCycler
Fluroprobe mechanism

      Hybrdization     Taqman probe
      probe
                             Project Outline


                                                       Real time PCR                                                       T-RFLP




                             Adjacent hybridization probes
                                                                                                       Multiplex Chapter


              One tube assay                                 Two tube assay
                                                                                             C. coli          C. jejuni    Other
                                                                                                                           Campyloba
                                                                                                                           cter
        Arcobacter (Chapter 5)
                                                               Campylobacter (Chapter 3)
          Adjacent probes                                           Adjacent probes

                                                                   C. coli and C. jejuni

A. butzleri   A. skirrowii     A. nitrofigilis
                                                                                             SYBR Green I         (Chapter 4)



                         Multi FAM probe (Chapter 6)
                                                                                           C. coli                   C.jejuni
Campylobacter Sequencing
Campylobacter coli and C. jejuni detection




                                                                        Agarose gel electrophoresis of PCR products from
                                                                        C. coli (Lane 1), C. jejuni (Lane 2), and C.
                                                                        hyointestinalis (Lane 3), C. upsaliensis (Lane 4), E.
 Increase in flouresence during specifciehybridisation of probes -
                                                                        coli (Lane 5) and no DNA template control (Lane
 Jejuni-coli FITC and Universal- CY5 to the target site in the 16S      6). Only C. coli and C. jejuni but none of the others
 rDNA of (--) C. jejuni, (--) C. coli suring PCR as measured in the   produce the amplicons of 683 bp as expected.
 LightCycler. Purified DNA was prepared using CTAB method. (-×-)
 C. hyointestinalis, (--) C. upsaliansis, (--) E. coli and (-+-) No
 template were used as negative controls.
DNA melting curve
                            DNA quantification




                                                   681 bp



                                                   Primer
                                                   dimer


Real-time detection of C. je juni CTAB-purifie d
DNA at different concentrations. 1920 ng (--
),192 ng (-19.2 ng (--), 1.92 ng (-+-), 0. 192
                                         -
ng (-×-), 0.0192 ng (- -), 0.00192 ng (-ž) and

0.000192 ng (--)
                     Colony serial dilution




Real time PCR of different dilution from one colony. The numbers of cells in each
dilution was determined by plating the dilutions onto BA plates. 50000 cells (--), 5000
cells (-×-), 500 cells (--), 125 cells (--), 50 cells (-о-) and Negative (--)
              Different growth phase




Real time PCR of different culture incubation
time of C coli 24 hours incubation (--), 8 hours   Real time PCR of different culture incubation time
(--), 6 hours (--), 4 hours (--), 2 hours (-•
                                               -)   of C jejuni 24 hours incubation (--), 8 hours (--),
and 0hours(--)                                                                             -)
                                                    6 hours (--), 4 hours (--), 2 hours (-•and 0hours(-
                                                    -)
         Hippuricase gene (HipO)

• HipO code for hippuricase enzyme.
• Catalyses the hydrolysis of N-benzoyleglycin (Hippuric
  acid) to glycine and benzoic acid.
• HipO gene present only in C. jejuni but not in any other
  Campylobacter spp.
     Hippuricase detection using the LightCycler



                                                       1        2         3    4     5




                                                                                           270 bp

                                                                                           primer
                                                                                           dimer


                                                           Agarose gel electrophoresis of the PCR products
Real time SYBR Green 1 assay with CTAB-purified            from Real Time SYBR Green 1 assay showing the
DNA from C. jejuni ATCC 940565(-  -), C. coli             expected 270 bp specific product for C. jejuni
NCTC 11366 (--), C. upsaliensis strain 99M126 (--)       (Lane 1), and much lower sized non-specific
C. hyointestinalis strain 99M2318 (-  -) for the          products from C. coli (Lane 2), C. hyointestinalis
detection of hippuricase gene (hipO).                      (Lane 3), C. upsaliensis (Lane 4). and No
                                                           Template (Lane 5)
Melting curve of the HipO PCR product




        A




        B
Hippuricase detection using the iCycler



                                1   2     3   4




                                                  292 bp
                                                  Primer
                                                  dimer
                      Arcobacter detection




The increase in fluorescence during specific             Agarose gel electrophoresis of PCR
hybridisation of probes probe Butz, probe Skir-Cry       products from no DNA template (Lane 1),
and Universal CY5 to the target site in the 16S          A. butzleri (Lane 2), A. skirrowii (Lane 3),
rDNA of A. butzleri (--), A. skirrowii (--), A.        A. nitrofigilis (Lane 4), C. coli (Lane 5),
nitrofigilis (-+-), C. coli (--), C. jejuni (--) and   and C. jejuni (Lane 6).
no template (--) suring PCR as measured in the
LightCycler. Purified DNA was prepared using
CTAB method. (--) C. jejuni, (-●-) C. coli. No
templates (-+-) were used as negative controls.
DNA melting curve



A




B
              Colony serial Dilution and Growth Time




Real time PCR of different dilution from one
colony of A. butzleri. The numbers of cells in       culture incubation time required by Arcobacter
each dilution was determined by plating the          species before it could be detected byLightCyclerTM
dilutions onto BA plates. 50000 cells (--),         were: (--) 0 hours, (--) 2 hours, (--) 4 hours, (--)
5000 cells (--), 500 cells (--), 125 cells (--)   6 hours, (-○-) 8 hours, and (--□) 24 hours
and Negative (- -)
                      Arcobacter DNA Quantification




                                                                                        315 bp

                                                                                        Primer
                                                                                        dimer



                                                               Agarsoe gel electrophoresis of
                                                               different DNA concentration 113
                                                               ng ((Lane 1), 11.3 ng (Lane 2),
Real-time detection of A. butzleri CTAB-purified DNA at        1.13 ng (Lane 3), 0.113 ng (Lane
different concentrations. 113 ng (--), 11.3 ng (--), 1.13    4), 0.0113 ng (Lane 5), 0.00113
ng (--), 0.113 ng (--), 0.0113 ng (-+-), 0.00113 ng (--),   ng (Lane 6), 0.000113ng (Lane 7),
and 0.000113 ng (-□)                                          No template (Lane 8)
           Multi-FAM detection to differentiate between
            C. coli, c. jejuni, A. butzleri and A. skirrowii




                                                         Agarose gel electrophoresis of PCR products
Real time detection of Campylobacter species and         from no DNA template (Lane 1), A. butzleri
Arcobacter species using probe Skir-Cry, probe Butz      (ATCC1248) (Lane 2), A. skirrowii (ATCC 12713)
and probe Jejuni-coli, C. jejuni (00M2260) (--), C.     (Lane 3), A butzleri (99M3958) (Lane 4), C. coli
coli (P287/96) (--), A. butzleri (ATCC1248) (-*-), A.   (P287/96) (Lane 5), and C. jejuni (00M2260) (Lane
skirrowii (ATCC 12713) (-^-), A butzleri (99M3958) (-    6)
-) and NO template (--).
DNA melting curve
         Conclusion and Future Direction
• Rapid Methods for both C. coli and C. jejuni have been
  developed.
• Distinguish between the closely related species C. coli and
  C. jejuni.
• Time required for Campylobacter and Arcobacter
  detection reduced tow minutes instead of days
• Application of the methods to environmental and food
  samples.
• PCR multiplex for rapid detection and identification in one
  step
• T-RFLP for rapid detection and identification of
  Campylobacter and Arcobacter.
• Determine the pathogenisity gene that present in C. coli
  and A. butzleri