B-SMART™: Biological System for Molecular Antibiotic ... · B-SMART™ is a fully automatable...
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B-SMART™: Biological System for Molecular Antibiotic Resistance Testing B-SMART™ is a fully automatable nucleic acid-based system that measures bacterial viability and drug resistance. By combining the accuracy of functional drug susceptibility assays with the speed and sensitivity of molecular diagnostics, B-SMART™ can accurately and simultane- ously report microbial identification and drug susceptibility in a matter of hours or days, depending upon the bacterial species. B-SMART™ is the first rapid nulceic acid-based test that can detect drug reistence without any prior knowledge of the genes or mutations that confer drug resistence. How B-SMART™ works Viruses rely on the protein and nucleic acid synthesis machinery of their host cell to manufacture their progeny because they don’t carry these processes in their genes. The extent to which a virus can produce new viral components depends on the metabolic activity of the cell it infects. B-SMART™ uses bacteriophage (viruses that infect bacteria) to assess the metabolic capacity of bacteria in a sample exposed to one or more antimicrobial antibiotics. B-SMART™ phages are designed to create a new and novel nucleic acid, the Surrogate Marker Locus (SML). Because phage infection requires a metabolically active cell, synthesis of the SML is compromised in drug-susceptible bacteria exposed to an effective antibiotic. If the organism is drug-resistant, the antibiotic has no effect on bacterial metabolism and the SML is synthesized efficiently. SML is the bridge between two general strategies for determining cellular viability or detecting drug resistance: functional assays (to assess the ability of bacteria to grow in the absence or presence of antibiotics) and molecular assays (to amplify and detect pathogen-specific nucleic acid sequences or resistance-conferring mutations). B-SMART™ can be adapted to determine antibiotic susceptibility of any microorganism infected by a bacteriophage. B-SMART™ is especially useful for pathogens that are difficult to culture, for which the gene(s) that encode drug resistance are unknown, or for which drug resistance is controlled by multiple genes. The SML-generation module used in B-SMART™ functions in diverse bacterial species, including Mycobacteria and E. coli, as well as in eukaryotic cells. Our initial efforts focus on the development of a uniquely simple device that can be used to detect resistance to rifampicin, a marker for multidrug-resistant tuberculosis (MDR-TB), in Mycobacterium tuberculosis (Mtb) obtained directly from sputum samples. We are developing a product that can be used in clinics and local laboratories, since currently available rapid Mtb AST are expensive, complicated, and available only in central laboratories. B-SMART™ could greatly decrease time from TB diagnosis to appropriate treatment, leading to a decrease in transmission of MDR-TB and XDR-TB. This development path will lay the groundwork for technology expansion to detection of Mtb AST for other antitubercular drugs, as well as detection of drug resistance in other pathogens The ability of SML-phage to detect RIF-resistant Mtb is shown on the right. In (A), one clinical strain of Mtb is resistant to RIF and the other is susceptible. The bacteria were left untreated or were treated with RIF and then infected with SML-phage for 4 hours. For the RIF-resistant strain, no reduction in SML occurred after treatment with RIF (RIF-treated bacteria were not killed). This demonstrates that RIF resistance is easily detected by SML-phage. (B) RIF treatment time of only 3 hours allowed B-SMART™ to detect drug susceptibility. The entire test could take place in under 10 hours. Proof of Principle SML-Phage Technology. Upon infection of a viable, metabolically active bacterium, a new nucleic acid is created (the SML) which can be detected by any nucleic acid detection technology.
Transcript of B-SMART™: Biological System for Molecular Antibiotic ... · B-SMART™ is a fully automatable...
B-SMART™: Biological System for Molecular Antibiotic Resistance Testing
B-SMART™ is a fully automatable nucleic acid-based system that measures bacterial viability and drug resistance. By combining the accuracy of functional drug susceptibility assays with the speed and sensitivity of molecular diagnostics, B-SMART™ can accurately and simultane-ously report microbial identi�cation and drug susceptibility in a matter of hours or days, depending upon the bacterial species. B-SMART™ is the �rst rapid nulceic acid-based test that can detect drug reistence without any prior knowledge of the genes or mutations that confer drug resistence.
How B-SMART™ works
Viruses rely on the protein and nucleic acid synthesis machinery of their host cell to manufacture their progeny because they don’t carry these processes in their genes. The extent to which a virus can produce new viral components depends on the metabolic activity of the cell it infects.
B-SMART™ uses bacteriophage (viruses that infect bacteria) to assess the metabolic capacity of bacteria in a sample exposed to one or more antimicrobial antibiotics. B-SMART™ phages are designed to create a new and novel nucleic acid, the Surrogate Marker Locus (SML). Because phage infection requires a metabolically active cell, synthesis of the SML is compromised in drug-susceptible bacteria exposed to an e�ective antibiotic. If the organism is drug-resistant, the antibiotic has no e�ect on bacterial metabolism and the SML is synthesized e�ciently. SML is the bridge between two general strategies for determining cellular viability or detecting drug resistance: functional assays (to assess the ability of bacteria to grow in the absence or presence of antibiotics) and molecular assays (to amplify and detect pathogen-speci�c nucleic acid sequences or resistance-conferring mutations).
B-SMART™ can be adapted to determine antibiotic susceptibility of any microorganism infected by a bacteriophage. B-SMART™ is especially useful for pathogens that are di�cult to culture, for which the gene(s) that encode drug resistance are unknown, or for which drug resistance is controlled by multiple genes. The SML-generation module used in B-SMART™ functions in diverse bacterial species, including Mycobacteria and E. coli, as well as in eukaryotic cells.
Our initial e�orts focus on the development of a uniquely simple device that can be used to detect resistance to rifampicin, a marker for multidrug-resistant tuberculosis (MDR-TB), in Mycobacterium tuberculosis (Mtb) obtained directly from sputum samples. We are developing a product that can be used in clinics and local laboratories, since currently available rapid Mtb AST are expensive, complicated, and available only in central laboratories. B-SMART™ could greatly decrease time from TB diagnosis to appropriate treatment, leading to a decrease in transmission of MDR-TB and XDR-TB. This development path will lay the groundwork for technology expansion to detection of Mtb AST for other antitubercular drugs, as well as detection of drug resistance in other pathogens
The ability of SML-phage to detect RIF-resistant Mtb is shown on the right. In (A), one clinical strain of Mtb is resistant to RIF and the other is susceptible. The bacteria were left untreated or were treated with RIF and then infected with SML-phage for 4 hours. For the RIF-resistant strain, no reduction in SML occurred after treatment with RIF (RIF-treated bacteria were not killed). This demonstrates that RIF resistance is easily detected by SML-phage. (B) RIF treatment time of only 3 hours allowed B-SMART™ to detect drug susceptibility. The entire test could take place in under 10 hours.
Proof of Principle
SML-Phage Technology. Upon infection of a viable, metabolically active bacterium, a new nucleic acid is created (the SML) which can be detected by any nucleic acid detection technology.
Product Design
Module 2. User-friendly SMART-stick.
We designed inexpensive and simple-to-use devices for a semi-automated Mtb RIF resistance assay directly from sputum in <10 hours. This assay will consist of �ve modules with the following desirable characteristics unique to B-SMART™:
1. Sputum Processing. B-SMART™ will not require a decontamination step, which should increase sensitivity in the detection of viable Mtb. In addition, B-SMART™ will not require bacterial concentration, since the SML will be concentrated in Module 3.
2. Drug Exposure and SML-phage infection. Minimally processed sputum from Module 1 is added to the “SMART-stick,” a low-cost device designed for B-SMART: drug exposure and phage infection is by “push-button” release of preloaded reagents. The device has two reaction chambers: untreated control and Rif treatment. Each chamber is pre-packaged with SML phage that encode distinct SML sequence for downstream detection.
3. SML RNA Puri�cation. The SML is engineered to bind with high a�nity inexpensive paramagnetic beads, without the need for denaturation. This facilitates simple and e�cient nucleic acid puri�cation from large volumes. We designed a user-friendly, inexpensive nucleic acid capture vessel containing preloaded beads that can be trapped with a magnet in a built-in removable tube for straightforward washing and transfer of SML-bound beads to Module 4.
4. NASBA. NASBA is a nucleic acid ampli�cation method that isothermally ampli�es nucleic acid, obviating the need for an expensive thermocycler. Lyophilized reagents for NASBA are commercially available, stored at room temperature, and do not require a cold chain.
5. Nucleic Acid Lateral Flow Assay (NLFA). NLFAs are inexpensive and user-friendly devices for point-of-care detection of nucleic acids. We are working with experts in the development of multiplex NLFA, who will design an assay that can distinguish SML ampli�cation products from a control.
Module 3. Simple purification of SML RNA using prepackaged paramagnetic beads.
Market Opportunity for Infectious Disease Nucleic Acid Testing
Market Opportunity for Infectious Disease Nucleic Acid Testing Market Opportunity for Infectious Disease Nucleic Acid Testing
Nucleic acid-based diagnostics are well established in laboratory medicine, and many are used to screen blood supplies for a variety of infectious diseases. In the U.S., the market for hospital and laboratory-based infectious disease nucleic acid tests is a $1.1 billion industry. This market can quickly expand to include:• Antibiotic susceptibility testing• Replacement/augmentation of sputum smear or culture for diagnosis of TB• Potable water screens• Food pathogen screens (dairy products, meat)
Market Opportunity
Time toDetection (TTD)
Keypath™ Phenotype Lateral Flow No Methicillin 2 days
FastPlaque PhenotypePlaque Formation
No Rifampin 2-3 days
Rifampin+Isoniazid
FluorescentProbes (FP)FluorescentMetabolitesLateral Flow, FP,and others
System Type Detection Automation Drug(s)
1 day
GeneXpert –TB Nucleic Acid Yes Rifampin 1 day
Genotype MTBDRplus
Nucleic Acid Line Probe Assay Automatable
B-SMART™Phenotype + Nucleic Acid
Automatable Any Hours to <2 days
BACTEC Phenotype Yes Any Culture 2-4 wk
Compared to existing TB diagnostics, B-SMART™ TB is di�erentiated by the following attributes:
