Polyphasic Taxonomy and the use rpoB Gene as anAlternative 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 bacterialisolates with good accuracy but are laborious, tedious andexpensive (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 Mediaproduce critical impact on clinicalmanagement 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 – ClinicalSetting • 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 – ClinicalSetting • 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 BacterialID 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.