Methods All phenotypically carbapenem resistant bacteria underwent CarbaNP (R) testing and PCR, followed by NGS. Commercial (Newbler and Geneious), on-line (ResFinder), and opensource software (Btrim, FLASh, Bowtie2, an Samtools) were used for assembly, SNP detection and clustering. Laboratory capacity, throughput, and response time were assessed. Results From 2009 through 2015, 27,000 multidrug-resistant Gram-negative isolates were submitted. 225 contained carbapenemase-encoding genes (most commonly bla(KPC), bla(NDM), and bla(OXA23)). These were found in 15 species from 146 inpatients in 19 facilities. Genetically related CPB were found in more than one hospital. Other clusters or outbreaks were not clonal and involved genetically related plasmids, while some involved several unrelated plasmids. Relatedness depended on the clustering algorithm used. Transmission patterns of plasmids and other mobile genetic elements could not be determined without ultra-long read, single-molecule real-time sequencing. 80% of carbapenem-resistant phenotypes retained susceptibility to aminoglycosides, and 70% retained susceptibility to fluoroquinolones. However, among the CPB-confirmed genotypes, fewer than 25% retained susceptibility to aminoglycosides or fluoroquinolones. Conclusion Although NGS is increasingly acclaimed to revolutionize clinical practice, resource-constrained environments, large or geographically dispersed healthcare networks, and military or government-funded public health laboratories are likely to encounter constraints and challenges as they implement NGS across their health systems. These include lack of standardized definitions and quality control metrics, limitations of short-read sequencing, insufficient bandwidth, and the current limited availability of very expensive and scarcely available sequencing platforms. Possible solutions and mitigations are also proposed.
