Targeted next-generation sequencing for the detection of ciprofloxacin resistance markers using molecular inversion probes

Abstract

Antibiotic resistance (AR) is an epidemic of increasing magnitude requiring rapid identification and profiling for appropriate and timely therapeutic measures and containment strategies. In this context, ciprofloxacin is part of the first-line of countermeasures against numerous high consequence bacteria. Significant resistance can occur via single nucleotide polymorphisms (SNP) and deletions within ciprofloxacin targeted genes. Ideally, use of ciprofloxacin would be prefaced with AR determination to avoid overuse or misuse of the antibiotic. Here, we describe the development and evaluation of a panel of 44 single-stranded molecular inversion probes (MIPs) coupled to next-generation sequencing (NGS) for the detection of genetic variants known to confer ciprofloxacin resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Sequencing results demonstrate MIPs capture and amplify targeted regions of interest at significant levels of coverage. Depending on the genetic variant, limits of detection (LOD) for high-throughput pooled sequencing ranged from approximately 300–1800 input genome copies. LODs increased 10-fold in the presence of contaminating human genome DNA. In addition, we show that MIPs can be used as an enrichment step with high resolution melt (HRM) real-time PCR which is a sensitive assay with a rapid time-to-answer. Overall, this technology is a multiplexable upfront enrichment applicable with multiple downstream molecular assays for the detection of targeted genetic regions.

Clinical metagenomics

“…Although this enrichment method has been shown to increase the sensitivity of metagenomic detec tion in research settings, especially for viruses, it has yet to be used routinely for clinical diagnosis. A promising appli cation of this approach may be the enrichment of clinical samples for characterization of antibiotic resistance 23 , a considerable problem in hospitals and the primary focus of the US National Action Plan for Combating Antibiotic Resistant Bacteria 53 . However, drawbacks of capture probe enrichment, compared with untargeted approaches for infectious disease diagnosis, include a bias towards tar geted microorganisms, added steps, increased costs and long hybridization times (24-48 hours) as a result of the additional processing needed for maximal efficiency.…”

“…21 ) and for surveillance of antimicrobial resistance in the food supply by bacterial whole genome sequencing 22 . Increasingly, big data provided by mNGS is being leveraged for clinical purposes, including charac terization of antibiotic resistance directly from clinical samples 23 and analysis of human host response (tran scriptomic) data to predict causes of infection and evalu ate disease risk 24,25 . Thus, mNGS can be a key driver for precision diagnosis of infectious diseases, advancing precision medicine efforts to personalize patient care in this field.…”

A Survey of Antimicrobial Resistance Determinants in Category A Select Agents, Exempt Strains, and Near-Neighbor Species

“…Provided suitable sensitivity can be achieved and data processing simplified, this is where rapidly evolving technologies such as next generation sequencing (NGS) may find tremendous application [56]. Several excellent studies have recently demonstrated use of targeted pyrosequencing and next generation sequencing approaches to rapid detect and identify both horizontally and vertically transmitted AMR determinants within Select Agent strains [57][58][59]. While these recent studies emphasized a limited number of AMR genes, we hope that these technologies will prove useful in performing similarly broad-based surveys of Select Agents and other pathogenic species.…”

FDA-ARGOS: A Public Quality-Controlled Genome Database Resource for Infectious Disease Sequencing Diagnostics and Regulatory Science Research

“…For example, isolate shotgun sequencing information uncovered unexpected transmission routes during multi-drug resistant nosocomial organism outbreaks (Snitkin, Zelazny et al 2012, Roach, Burton et al 2015, Snitkin, Won et al 2017). Other studies showed use of targeted sequencing to group E. coli clonotypes from patient’s direct urine samples (Tchesnokova, Billig et al 2013), or to detect ciprofloxacin resistance markers (Stefan, Koehler et al 2016), resulting in antimicrobial susceptibility data and improvement in clinical outcome prediction. Finally, agnostic (unbiased, metagenomic) sequencing shows promise as a diagnostic of last resort where no other diagnostic can determine the infectious microorganism, such as the successful ID-NGS diagnosis of leptospira infection with resulting positive outcome for the patient (Wilson, Naccache et al 2014).…”