Cesar Mendoza ppt

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.

• 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).

Introduction

• 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

Objectives

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.

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 Method of Bacterial ID –

Present state: On the verge of collapse?

Conventional Methods:

Presumptive Test

-Triple strength Lauryl Tryptose

Broth

Confirmatory Test

-If positive inoculate in Brilliant Green

Lactose Broth

Thermotolerant Coliform

-If positive inoculate in EC Broth at 44oC

24 hours

These techniques allow the identification of most bacterial

isolates with good accuracy but are laborious, tedious and

expensive (Bizzini, A., et. al. 2010).

Questions on Specificity

and Accuracy


Staining, Biochemical Reactions, Physiological Requirements

Gram Stain Spore Stain Hemolytic Patterns

Biochemical

Reaction

Conventional

Methods:

Use of Culture Media

Preparation of Culture Media

Speed and Time for ID

The speed of microbial identification can

produce critical impact on clinical

management and diagnosis

Staining Technique Isolation of bacteria

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.

• 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)


Staining, Biochemical Reactions, Physiological Requirements

Current Trends on Microbial Identification – Clinical

Setting

• Microbial identification in

clinical setting both in

routine and reference

laboratory has evolved

in the past few decades.

• Current trends include

highly automated

identification systems that

have been introduced in

many medium- to high-

throughput clinical

microbiology laboratories

worldwide.

• Conventional Methods

• Rapid Automated ID systems

• Molecular Techniques

• 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).

Current Trends on Microbial Identification – Clinical

Setting

Molecular Marker – Is Newer

Better?

Vital Criteria:

presence in a wide

distribution among

bacteria

uniqueness in the

genomic structure

size of phylogenetic

information that can be

derived

diverse sequence

among related species.

• Molecular diagnostic

methods based on the

detection of bacterial

nucleic acids from

clinical samples hold

the promise of rapid

detection and

identification of the

etiologic agent of the

disease (Pingle, M., et.

al. 2007).

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).

• 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 rpoB gene

contains many

characteristics that

have made the SSU

rRNA gene important

in the field of

taxonomic studies

and molecular

research since its

introduction as a

universal

phylogenetic marker.

universal distribution,

highly conserved

presence of conserved

and hypervariable regions

of the gene.

in contrast to SSU rRNA,

rpoB has not been

detected in multiple

copies in any complete

sequenced of the

prokaryotic gene (Walsh,

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.

• 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….



• 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

• 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.

Conclusion:

Thank you very

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Cesar Mendoza ppt

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