Electrochemical Sensors to Monitor Multi-Drug Resistant Organisms in the Gut Microbiome

Abstract

Infections caused by multi-drug resistant organisms (MDROs) are responsible for over 2 million of hospitalization and 23,000 deaths annually in the United States, with direct financial impact of $20 billion a year. Microbiome dysbiosis (imbalance) is considered to be the main cause for MDRO colonization and the resulting infections. MDRO outbreaks need to be quickly detected and monitored for effective intervention to provide relief to the stressed patient community and healthcare facility. Unfortunately, current diagnostic methods for MDRO detection are slow and costly. Polymerase chain reaction (PCR) and enzyme immunoassay (EIA) techniques or the next generation sequencing (NGS) methods do not provide the low cost and rapid platform necessary for such MDRO pathology for a patient care facility. These methods often require several days to complete, use many reagents, and need skilled technicians in a resource intensive laboratory. In this work, we report a rapid, accurate and cost-effective electrochemical sensor capable of MDRO detection down to 104 colony forming units (CFU)/g in mice and human stool samples. To achieve this, we utilize disposable screen printed electrodes (SPEs) modified with highly specific oligonucleotide probes capable of hybridizing (duplex formation) select target gene sequences associated with MDROs. In the presence of a redox mediator, the hybridized probe/target complex generates an electrochemical current signal that differs from a non-hybridized probe molecule. The signal is highly sensitive and selective of target gene sequences and can detect as little as 3.5 picomols of target DNA. Furthermore, the SPEs can be pre-functionalized, stored, and used when needed, reducing individual sample analysis time to less than an hour. Several target sequences from two exemplary genes (AmpC and AcrB found in MDRO E. coli) have been identified and successfully detected in clinical stool samples with results comparable to the standard quantitative PCR method. Additional target genes associated with drug resistance (TEM-1, VanA, and SHV) have also been successfully detected in vitro and are ready to be tested in clinical samples. Our gut microbiome sensor is currently being developed for multiplexed operation to simultaneously detect up to eight MDROs genes, including the ones that are responsible for breaking down b-lactam containing antibiotics. This novel sensor platform will be an economical point-of-care device with little requirement of reagent handling or technical training. Figure 1