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Polyphasic Taxonomy and the use rpoB Gene as an
Alternative Biomarker for Identification of Clinically
        Important and Ambiguous Bacteria:
               Synthesis and Review




             CESAR M. MENDOZA, JR., RMT, M. Bio. Ed.
Introduction

       • One vital area within the
         practice         of        clinical
         microbiology is the system of
         identification    of    microbial
         isolates.
       • Identification of the etiologic or
         causative agent responsible
         for the disease and correlate
         pathological condition is a
         crucial     part     of    clinical
         microbiology.
Introduction

          • In conventional system,
            accurate morphologic and
            phenotypic description is
            used but with the rapid
            sophistication of
            microbiological assays
            intended for clinical isolates,
            automation and genotypic
            identification became an
            alternative system in some
            cases (Clarridge, 2004).
Introduction
                              Phylogenetic
                              relationships
                             not only a
                              taxonomic concern
                             a salient force in
                              bio-molecular
                              applicability and
                              maximum
                              advancement of
                              scientific constructs
                              especially in clinical
                              laboratory and
                              management.
Gram Positive Organisms
Introduction
• Polyphasic Taxonomy
  a consensus type of taxonomy that aims to maximize all
  available data and come up with a decisive conclusion on
  taxonomical aspect. (Gillis, et. al., 2005).




 BBL Crystal ID (Beckton Dickinson)      Thermo cycler
Introduction

  • On the current setting, phylogenetic
    analyses based on the 16S rRNA gene
    is considered vital, important, and
    universal tool for the validation and
    reconstruction of evolutionary history
    and phylogenetic relationships of
    various bacterial organisms (Woese et
    al. in Kupfer, et; al, 2006).
Objectives

• This paper evaluates the present protocols in the
  identification of clinically isolated bacteria
  including the ambiguous identities using
  phenotypic and genotypic protocols
• Traces the development of various systems used
  in clinical microbiology in the identification of
  bacterial isolates both in routine and reference
  laboratories.
• Identifies alternative biomarker as a potential
  housekeeping gene or sole gene determinant for
  identification of clinically-isolated bacteria
Conventional Method of Bacterial ID –
Present state: On the verge of collapse?


                         • Conventional methods:
                           appropriate culture
                           media and phenotypic
                           characterization -
                           staining techniques
                           such as gram stain,
                           morphological
                           descriptions, cultural
                           requirements, and
                           biochemical reactions.
Conventional Method of Bacterial ID –
Present state: On the verge of collapse?


                    Successful in identification of
                     some bacterial species
                    Limitations on discriminating
                     clinically   relevant      taxa
                     because individual test may
                     not be reproducible and that
                     species metabolic phenotype
                     is not an absolute property
                     and may exhibit variability.
Conventional Methods:
    These techniques allow the identification of most bacterial
isolates with good accuracy but are laborious, tedious and
expensive (Bizzini, A., et. al. 2010).

                                                   Thermotolerant
   Presumptive Test    Confirmatory Test
                                                      Coliform
    -Triple strength   -If positive inoculate
                                                -If positive inoculate
    Lauryl Tryptose      in Brilliant Green
                                                in EC Broth at 44oC
          Broth            Lactose Broth
                                                      24 hours
Conventional Methods:
Staining, Biochemical Reactions, Physiological Requirements
                   Questions on Specificity
                       and Accuracy




    Gram Stain            Spore Stain         Hemolytic Patterns



                                                     Biochemical
                                                      Reaction
Speed and Time for ID              Conventional
                                               Methods:
The speed of microbial identification can      Use of Culture Media
produce critical impact on clinical
management and diagnosis




                                               Preparation of Culture Media

  Isolation of bacteria   Staining Technique
Speed and Time of ID
       1. appropriate antimicrobial agents
       can be identified and initiated
       unnecessary treatment with
       ineffective antibiotics can be
       avoided;
       2. the prognosis of the patients
       can be improved
       3. Antibiotic resistance can be
       avoided; and
        4. expenditure on antimicrobials
       and overall hospital costs can be
       markedly reduced.
Conventional Methods-
Setbacks: (Woo, et. al, )
      1. not applicable for non-cultivable
         and non-culturable organisms.
      2. there are organisms that do not
         conform to patterns of known
         bacterial species in terms of
         biochemical reactions.
      3. there are also bacteria that are
         slow growers and are difficult to
         isolate.
Conventional Methods:
Staining, Biochemical Reactions, Physiological Requirements


                        • Studies indicated that compared
                          to phenotypic tests, gene
                          sequence-based identification
                          schemes are superior in the
                          identification of strains
                          considered ambiguous.
                        • “Ambiguous” - Atypical
                          biochemical, profiles, slow-
                          growing bacteria, rarely
                          encountered bacterial species,
                          and non-cultivable strains. (Woo,
                          et. al., 2003)
Current Trends on Microbial Identification – Clinical
Setting


                              • Microbial identification in
     • Conventional             clinical setting both in
       Methods                  routine and reference
                                laboratory has evolved
                                in the past few decades.
     • Rapid Automated
       ID systems             • Current trends include
                                highly automated
                                identification systems that
     • Molecular                have been introduced in
       Techniques               many medium- to high-
                                throughput clinical
                                microbiology laboratories
                                worldwide.
Current Trends on Microbial Identification – Clinical
Setting


               • Phenotypic        characterization       and
                 automated strips were not enough to
                 identify accurately some clinical isolates
               • Gene sequence can discriminate far more
                 finely among strains of bacteria than
                 phenotypic methods
                 (poorly described, rarely isolated, or
                 phenotypically aberrant strains)
               • This is an area in which 16S rRNA gene
                 sequence identification might have an
                 immediate impact on patient care
                 (Clarridge, 2004).
Molecular Marker – Is Newer
                        Better?
Vital Criteria:             • Molecular diagnostic
 presence in a wide          methods based on the
  distribution among          detection of bacterial
  bacteria                    nucleic acids from
 uniqueness in the           clinical samples hold
  genomic structure           the promise of rapid
                              detection and
 size of phylogenetic        identification of the
  information that can be     etiologic agent of the
  derived                     disease (Pingle, M., et.
 diverse sequence            al. 2007).
  among related species.
The search for a new biomarker:
The “rpoB gene”

  DNA-dependent RNA polymerase is a
  multi-subunit enzyme that consist of two α
  subunits encoded by the rpoA gene, one β
  subunit (rpoB) and one β’ subunit (rpoC)
  (Kupfer, et. al. 2006).
The search for a new biomarker:
 • Comparison of rpoB sequences has been
   used as a basis for phylogenetic analyses
   among some archaea and bacteria
 • The protein is approximately 150kD in size
   and is a subunit of RNA polymerase with
   the beta subunit involved in the synthesis
   and elongation of the RNA chain
   (Donnabella, 1994)
The search for a new biomarker:
• The      rpoB      gene     universal distribution,
  contains           many      highly conserved
  characteristics     that    presence of conserved
  have made the SSU            and hypervariable regions
  rRNA gene important          of the gene.
  in    the    field    of    in contrast to SSU rRNA,
  taxonomic        studies     rpoB has not been
  and          molecular       detected in multiple
  research since its           copies in any complete
  introduction     as    a     sequenced of the
  universal                    prokaryotic gene (Walsh,
  phylogenetic marker.         D., et. al., 2004).
Usage of rpoB gene in Bacterial
ID of Clinical Isolates
• In a study on the comparative phylogenies
  of the housekeeping gene that contains
  rpoB compared with 16S RNA for the
  phylogenetic study of Pasteurellaceae
  (Christensen, 2004),
   concluded that:
   ……..analysis of housekeeping gene
  like rpoB is a promising approach for
  the revision and study on the taxonomy
  of    various    organisms     such    as
  Pasteurellaceae.
Usage of rpoB gene in Bacterial
                  ID of Clinical Isolates
• In Comparative genetic relationships of Aeromonas strains
  using 16S rRNA, gyrB, and rpoB gene sequences were done
  with gyrB evaluated against rpoB gene and the 16S rRNa
  sequencing as the reference:
The study revealed: both gyrB and rpoB can be use to clarify
  the taxonomical and evolutionary relationships among
  strains of human, animal (carriers or patients) and
  environmental species of Aeromonas in contrast with 16S
  rRNA gene sequencing which is useful only in identifying
  the organism in genus level only…
  ……. rpoB sequences are more preserved in the genus
  Aeromonas…
  ……. using rpoB gene sequencing, controversial and
  issue-laden taxa of Aeromonas are better understood….
Usage of rpoB gene in Bacterial
                  ID of Clinical Isolates
• A study on the potential utilization of rpoB - fast and direct
  assay on the presence of Mycobacteria spp. (various
  respiratory specimens) and compared it with concentration
  technique using flurochrome staining.
• Study revealed: A high percentage yield for sensitivity
  (92.3%) of rpoB-PCR was noted as compared to CF
  (concentration fluorochrome) staining (88.4%)
• 94.5% of resistant isolates exhibited mutations in the
  hot-spot variable region of the gene while no mutation
  within the 305-bp region for any of the rifampin-sensitive
  strains tested and isolated - sequencing of the DNA of
  the rpoB gene may result in the early detection of strains
  that may later on exhibit resistance to rifampin (Hirano,
  K., et. al., 1999).
Other Related Studies - rpoB
• Partial utilization of rpoB gene sequence
  also proved to be critical for identification
  of emerging Acinetobacter species
• Partial rpoB was also utilized in
  comparative analysis of Pasteurella
  pneumotropica isolates from laboratory
  rats and mice.
Conclusion:

• phenotypic and genotypic characterization
  for the polyphasic taxonomy clearly
  indicated that proper identification of the
  bacterial isolates in clinical setting is a
  critical matter needed to be resolved.
• The available systems in placed in routine
  and reference laboratories are still the
  rapid and automated system
• it still cannot address fully the issues on
  proper taxonomy and relationships of
  ambiguous profiles
Conclusion:

• Time and speed of the conventional method is a major
  drawback
• Immense need for an alternative biomarker gene to
  augment the usage of 16S rRNA and definitely the
  utilization of rpoB can be an alternative biomarker for the
  accurate identification of bacteria in clinical setting
  including the ambiguous ones.
• As a conclusion, the rpoB gene is an alternative
  biomarker as it encodes the β-subunit of RNA
  polymerase and common to all bacteria as it exist as a
  single copy in the genome with highly conserved and
  variable sequence.
Conclusion:

• It is not only of primary importance in the
  identification of rifampin-resistant
  Mycobacterium spp. but also of various
  genera such as Pasteurellaceace,
  Aeromonas, Acinetobacter, and
  halobacteriales previously identified to
  produce inaccuracy using the 16S rRNA
  gene.
Thank you very
   much!!!

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Cesar Mendoza ppt

  • 1. Polyphasic Taxonomy and the use rpoB Gene as an Alternative Biomarker for Identification of Clinically Important and Ambiguous Bacteria: Synthesis and Review CESAR M. MENDOZA, JR., RMT, M. Bio. Ed.
  • 2. Introduction • One vital area within the practice of clinical microbiology is the system of identification of microbial isolates. • Identification of the etiologic or causative agent responsible for the disease and correlate pathological condition is a crucial part of clinical microbiology.
  • 3. Introduction • In conventional system, accurate morphologic and phenotypic description is used but with the rapid sophistication of microbiological assays intended for clinical isolates, automation and genotypic identification became an alternative system in some cases (Clarridge, 2004).
  • 4. Introduction Phylogenetic relationships  not only a taxonomic concern  a salient force in bio-molecular applicability and maximum advancement of scientific constructs especially in clinical laboratory and management. Gram Positive Organisms
  • 5. Introduction • Polyphasic Taxonomy a consensus type of taxonomy that aims to maximize all available data and come up with a decisive conclusion on taxonomical aspect. (Gillis, et. al., 2005). BBL Crystal ID (Beckton Dickinson) Thermo cycler
  • 6. Introduction • On the current setting, phylogenetic analyses based on the 16S rRNA gene is considered vital, important, and universal tool for the validation and reconstruction of evolutionary history and phylogenetic relationships of various bacterial organisms (Woese et al. in Kupfer, et; al, 2006).
  • 7. Objectives • This paper evaluates the present protocols in the identification of clinically isolated bacteria including the ambiguous identities using phenotypic and genotypic protocols • Traces the development of various systems used in clinical microbiology in the identification of bacterial isolates both in routine and reference laboratories. • Identifies alternative biomarker as a potential housekeeping gene or sole gene determinant for identification of clinically-isolated bacteria
  • 8. Conventional Method of Bacterial ID – Present state: On the verge of collapse? • Conventional methods: appropriate culture media and phenotypic characterization - staining techniques such as gram stain, morphological descriptions, cultural requirements, and biochemical reactions.
  • 9. Conventional Method of Bacterial ID – Present state: On the verge of collapse?  Successful in identification of some bacterial species  Limitations on discriminating clinically relevant taxa because individual test may not be reproducible and that species metabolic phenotype is not an absolute property and may exhibit variability.
  • 10. Conventional Methods: These techniques allow the identification of most bacterial isolates with good accuracy but are laborious, tedious and expensive (Bizzini, A., et. al. 2010). Thermotolerant Presumptive Test Confirmatory Test Coliform -Triple strength -If positive inoculate -If positive inoculate Lauryl Tryptose in Brilliant Green in EC Broth at 44oC Broth Lactose Broth 24 hours
  • 11. Conventional Methods: Staining, Biochemical Reactions, Physiological Requirements Questions on Specificity and Accuracy Gram Stain Spore Stain Hemolytic Patterns Biochemical Reaction
  • 12. Speed and Time for ID Conventional Methods: The speed of microbial identification can Use of Culture Media produce critical impact on clinical management and diagnosis Preparation of Culture Media Isolation of bacteria Staining Technique
  • 13. Speed and Time of ID 1. appropriate antimicrobial agents can be identified and initiated unnecessary treatment with ineffective antibiotics can be avoided; 2. the prognosis of the patients can be improved 3. Antibiotic resistance can be avoided; and 4. expenditure on antimicrobials and overall hospital costs can be markedly reduced.
  • 14. Conventional Methods- Setbacks: (Woo, et. al, ) 1. not applicable for non-cultivable and non-culturable organisms. 2. there are organisms that do not conform to patterns of known bacterial species in terms of biochemical reactions. 3. there are also bacteria that are slow growers and are difficult to isolate.
  • 15. Conventional Methods: Staining, Biochemical Reactions, Physiological Requirements • Studies indicated that compared to phenotypic tests, gene sequence-based identification schemes are superior in the identification of strains considered ambiguous. • “Ambiguous” - Atypical biochemical, profiles, slow- growing bacteria, rarely encountered bacterial species, and non-cultivable strains. (Woo, et. al., 2003)
  • 16. Current Trends on Microbial Identification – Clinical Setting • Microbial identification in • Conventional clinical setting both in Methods routine and reference laboratory has evolved in the past few decades. • Rapid Automated ID systems • Current trends include highly automated identification systems that • Molecular have been introduced in Techniques many medium- to high- throughput clinical microbiology laboratories worldwide.
  • 17. Current Trends on Microbial Identification – Clinical Setting • Phenotypic characterization and automated strips were not enough to identify accurately some clinical isolates • Gene sequence can discriminate far more finely among strains of bacteria than phenotypic methods (poorly described, rarely isolated, or phenotypically aberrant strains) • This is an area in which 16S rRNA gene sequence identification might have an immediate impact on patient care (Clarridge, 2004).
  • 18. Molecular Marker – Is Newer Better? Vital Criteria: • Molecular diagnostic  presence in a wide methods based on the distribution among detection of bacterial bacteria nucleic acids from  uniqueness in the clinical samples hold genomic structure the promise of rapid detection and  size of phylogenetic identification of the information that can be etiologic agent of the derived disease (Pingle, M., et.  diverse sequence al. 2007). among related species.
  • 19. The search for a new biomarker: The “rpoB gene” DNA-dependent RNA polymerase is a multi-subunit enzyme that consist of two α subunits encoded by the rpoA gene, one β subunit (rpoB) and one β’ subunit (rpoC) (Kupfer, et. al. 2006).
  • 20. The search for a new biomarker: • Comparison of rpoB sequences has been used as a basis for phylogenetic analyses among some archaea and bacteria • The protein is approximately 150kD in size and is a subunit of RNA polymerase with the beta subunit involved in the synthesis and elongation of the RNA chain (Donnabella, 1994)
  • 21. The search for a new biomarker: • The rpoB gene  universal distribution, contains many highly conserved characteristics that  presence of conserved have made the SSU and hypervariable regions rRNA gene important of the gene. in the field of  in contrast to SSU rRNA, taxonomic studies rpoB has not been and molecular detected in multiple research since its copies in any complete introduction as a sequenced of the universal prokaryotic gene (Walsh, phylogenetic marker. D., et. al., 2004).
  • 22. Usage of rpoB gene in Bacterial ID of Clinical Isolates • In a study on the comparative phylogenies of the housekeeping gene that contains rpoB compared with 16S RNA for the phylogenetic study of Pasteurellaceae (Christensen, 2004), concluded that: ……..analysis of housekeeping gene like rpoB is a promising approach for the revision and study on the taxonomy of various organisms such as Pasteurellaceae.
  • 23. Usage of rpoB gene in Bacterial ID of Clinical Isolates • In Comparative genetic relationships of Aeromonas strains using 16S rRNA, gyrB, and rpoB gene sequences were done with gyrB evaluated against rpoB gene and the 16S rRNa sequencing as the reference: The study revealed: both gyrB and rpoB can be use to clarify the taxonomical and evolutionary relationships among strains of human, animal (carriers or patients) and environmental species of Aeromonas in contrast with 16S rRNA gene sequencing which is useful only in identifying the organism in genus level only… ……. rpoB sequences are more preserved in the genus Aeromonas… ……. using rpoB gene sequencing, controversial and issue-laden taxa of Aeromonas are better understood….
  • 24. Usage of rpoB gene in Bacterial ID of Clinical Isolates • A study on the potential utilization of rpoB - fast and direct assay on the presence of Mycobacteria spp. (various respiratory specimens) and compared it with concentration technique using flurochrome staining. • Study revealed: A high percentage yield for sensitivity (92.3%) of rpoB-PCR was noted as compared to CF (concentration fluorochrome) staining (88.4%) • 94.5% of resistant isolates exhibited mutations in the hot-spot variable region of the gene while no mutation within the 305-bp region for any of the rifampin-sensitive strains tested and isolated - sequencing of the DNA of the rpoB gene may result in the early detection of strains that may later on exhibit resistance to rifampin (Hirano, K., et. al., 1999).
  • 25. Other Related Studies - rpoB • Partial utilization of rpoB gene sequence also proved to be critical for identification of emerging Acinetobacter species • Partial rpoB was also utilized in comparative analysis of Pasteurella pneumotropica isolates from laboratory rats and mice.
  • 26. Conclusion: • phenotypic and genotypic characterization for the polyphasic taxonomy clearly indicated that proper identification of the bacterial isolates in clinical setting is a critical matter needed to be resolved. • The available systems in placed in routine and reference laboratories are still the rapid and automated system • it still cannot address fully the issues on proper taxonomy and relationships of ambiguous profiles
  • 27. Conclusion: • Time and speed of the conventional method is a major drawback • Immense need for an alternative biomarker gene to augment the usage of 16S rRNA and definitely the utilization of rpoB can be an alternative biomarker for the accurate identification of bacteria in clinical setting including the ambiguous ones. • As a conclusion, the rpoB gene is an alternative biomarker as it encodes the β-subunit of RNA polymerase and common to all bacteria as it exist as a single copy in the genome with highly conserved and variable sequence.
  • 28. Conclusion: • It is not only of primary importance in the identification of rifampin-resistant Mycobacterium spp. but also of various genera such as Pasteurellaceace, Aeromonas, Acinetobacter, and halobacteriales previously identified to produce inaccuracy using the 16S rRNA gene.
  • 29. Thank you very much!!!