SBIR-STTR Award

The primary goal of this Phase I STTR proposal is to develop a prototype of a rapid molecular diagnostics test to improve prediction of the antibiotic resistance of uropathogenic. Escherichia coli by determining their clonal identity (clonotype). The clonotype determination will be done by detection of the presence/absence of a limited number (d10) of single nucleotide polymorphisms (SNPs) that will be selected from 8 genetic loci used for the clonal typing of E. coli - seven loci from the standard multi-locus sequence typing (MLST) scheme for E. coli and fast-evolving gene for the type 1 fimbrial adhesin, fimH. The SNPs binary interrogation could be performed in either thermocycler or isothermal DNA amplification protocols using a simple single-plex configuration adapted to the standard 8-/12-tube PCR strips and common instrumentation platforms. These tests will be designed to perform in near-patient settings such as clinical laboratories in emergency rooms, urgent care clinics and/or hospitals. The test will be done using patients' urine in a cost-effective and timely manner (<30 min), with the goal to improve the empirical (pre-antibiogram) choice of antibiotic treatment, reducing potential 'drug-bug' mismatch and overuse of last-line broad-spectrum antimicrobials. To achieve the goals, we will determine MLST/fimH and antibiogram profiles of a comprehensive number of clinical isolates and establish the association between resistance to commonly used antibiotics and the clonal identity of pathogens. Phylogenetically-unlinked (homoplasic) SNPs that are distributed across the clonotypes in a relatively random and even fashion will be identified. Alternate combinations of 6 to 10 SNPs will be analyzed for the ability to discriminate up to 100 of the most critical (size- and resistance-wise) clonotypes of E. coli. Oligonucleotide primers capable of robust interrogation of the SNP combinations will be designed and validated for different amplification protocols. The proposed studies will be performed as collaboration between ID Genomics, Inc, the University of Washington's start-up company specializing in sequence-based typing of microbial pathogens, and the University of Washington, Seattle, laboratory of Evgeni Sokurenko (the lead PI).

Public Health Relevance Statement:


Public Health Relevance:
We are proposing to develop a molecular diagnostic technology for a test kit to predict antibiotic resistance of bacteria causing urinary tract infectons in humans, based on the genetic family (clonal type) they belong to. The kits will be designed for use in emergency or urgent care clinics and hospitals to perform directly on urine and in a timely (<30 min) manner.

Project Terms:
Accident and Emergency department; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; antimicrobial; Bacteria; base; Base Sequence; Clinical; clinical research site; Clinics and Hospitals; Collaborations; commercialization; cost effective; Cystitis; Data Set; design; Detection; Devices; Diagnostic; DNA amplification; Emergency Care; Escherichia coli; family genetics; Fimbrial Adhesins; Foundations; Future; Genes; Genetic; Genomics; Goals; Human; improved; instrumentation; Laboratories; Lead; Legal patent; microbial; Microfluidics; Molecular; Molecular Diagnostic Testing; Oligonucleotide Primers; Outcome; pathogen; Patients; Performance; Pharmaceutical Preparations; Phase; phase 1 study; phase 2 study; point of care; Predisposition; Preparation; Protocols documentation; prototype; public health relevance; Pyelonephritis; Reaction; repository; Resistance; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Scheme; Single Nucleotide Polymorphism; Small Business Technology Transfer Research; Specimen; Technetium Tc 99m ciprofloxacin; Technology; Testing; Time; Tube; Universities; urgent care; Urinary tract; Urinary tract infection; Urine; Uropathogenic E. coli; Validation; Washington

The primary goal of this proposal is to develop and finalize configuration of a rapid molecular diagnostics test to predict antibiotic resistance of th major human uropathogens - Escherichia coli, Klebsiella pneumoniae, Enterobacter spp and P. mirabilis - by determining the sub-species clonal identity (clonotype) of the infecting bacteria directly from urine samples. Currently, the empirical (pre- antibiogram) choice of antibiotic treatment is guided by an educated guess about the suspected species of pathogen and its cumulative antibiogram from data compiled locally every year. We have shown, in the Phase I, studies that a significant improvement in antibiotic choice against uropathogenic E. coli could be achieved if the antibiotic treatment choice would be guided by cumulative antibiogram of the clonotype to which the infecting E. coli strain belongs. We also have shown that the clonal identity of E. coli can be determined directly in urine using qPCR. The basic idea for the Phase II studies is to refine the E. coli clonotyping test and add to the test clonal and/or species markers for non- E. coli uropathogens, with a goal of covering 90-95% of pathogens, and reduce the potential drug-bug mismatch for common antibiotics at least 3-folg. Clonotype determination is done by detection of the presence/absence of a limited number (e.g. six to ten) of polymorphic gene markers - mosaically- distributed genes and/or nucleotide polymorphisms - that will be selected genome-wide. Interrogation of the markers will be performed by qPCR using a simple, adaptable configuration of standard PCR tube strips and existing FDA-cleared instrumentation platforms. To achieve these goals, we will determine genome sequences and cumulative antibiograms of a comprehensive number of E. coli and non-E. coli clinical isolates, and establish the association between the resistance to common antibiotics and clonal identity of the pathogens. Polymorphic clonal markers, that are suitable for binary differentiation, will be identified across the genome of each species. Oligonucleotide primers, capable of robust interrogation of the binary loci, will be designed and validated using clinical isolates from different geographic regions of the USA over 3 years. The proposed studies will be performed as a collaboration between ID Genomics, Inc., a Seattle-based start-up company, specializing in sequence-based typing of microbial pathogens, and the University of Washington, Seattle, the laboratory of Evgeni Sokurenko, and a consortium of eight different clinical microbiology laboratories.

Public Health Relevance Statement:


Public Health Relevance:
We are proposing to develop a molecular diagnostics test to predict antibiotic susceptibility of bacteria that cause urinary tract infections, in humans, by identifying to which species and genetic (clonal) family the pathogen belongs. The test will be designed to be performed directly from urine in a timely (<30 min) and cost-effective manner. This will provide a tool for physicians to make a more informed choice of antibiotic treatment in the point of care setting.

Project Terms:
Antibiotic Resistance; Antibiotic susceptibility; Antibiotic Therapy; Antibiotics; antimicrobial; Bacteria; Bacterial Typing; base; Base Sequence; Biological Assay; Clinical; Clinical Microbiology; Clinical Trials; Collaborations; cost effective; Data; design; Detection; Development; Diagnostic; Enterobacter; Escherichia coli; Family; Genes; Genetic; Genetic Polymorphism; Genome; genome sequencing; genome-wide; Genomics; Geographic Locations; Goals; Health Personnel; Human; improved; instrumentation; Klebsiella pneumonia bacterium; Laboratories; Legal patent; Location; Marketing; microbial; Mirabilis; Molecular Diagnostic Testing; Nucleotides; Oligonucleotide Primers; Open Reading Frames; pathogen; Patient-Focused Outcomes; Patients; Performance; performance tests; Pharmaceutical Preparations; Phase; phase 1 study; phase 2 study; Physicians; point of care; Predisposition; product development; public health relevance; R Factors; research clinical testing; Resistance; Resolution; Sampling; Sensitivity and Specificity; Single Nucleotide Polymorphism; Small Business Innovation Research Grant; Small Business Technology Transfer Research; Specimen; Staging; Study Section; success; Technology; Testing; Time; tool; Tube; Universities; Urinary tract infection; Urine; Uropathogen; Uropathogenic E. coli; Washington; Work