Comparative Analysis of Culture- and Ddpcr-Based Wastewater Surveillance for Carbapenem-Resistant Bacteria

With the widespread use of last-resort antibiotics, carbapenems, clinical reports of infections associated with carbapenem-resistant Enterobacterales (CRE) have increased. Clinical surveillance for CRE involves susceptibility testing and/or whole genome sequencing of resistant isolates, which is laborious, resource intensive, and requires expertise. Wastewater surveillance can potentially complement clinical surveillance of CRE, and population-level antibiotic resistance (AR) surveillance more broadly. In this study, we quantitatively and qualitatively compared two widely used methods for AR wastewater surveillance: (1) a culture-based approach for quantifying carbapenem-resistant bacteria and (2) a digital droplet PCR (ddPCR) assay targeting five major carbapenemase-encoding genes. We developed a new multiplexed ddPCR assay to detect five carbapenemase-encoding genes and applied it to wastewater samples from three sites over 12 weeks. In parallel, we quantified carbapenem resistant bacteria and carbapenemase-producing bacteria using culture-based methods. We assessed associations between the concentrations of carbapenemase-encoding genes and resistant bacteria. Although both approaches showed similar trends in the overall abundance of dominant carbapenem-resistant bacteria and genes, there were weak correlations between the quantitative levels of resistance. Nanopore sequencing of the resistome of the carbapenem-resistant bacteria revealed that discrepancies arose from differences in the sensitivity and specificity of the methods. This study highlights tradeoffs between methods: culture-based methods offer detailed phenotypic data on carbapenem-resistant bacteria but have longer turnaround times and lower throughput, whereas ddPCR offers rapid, sensitive detection but may miss some resistance mechanisms. Integrating these methods with sequencing provides sensitive, quantitative AR information and their clinical relevance. This study compares culture-based and ddPCR methods for quantifying carbapenem resistance in wastewater, revealing weak correlations. Nanopore sequencing clarifies resistance mechanisms, emphasizing method selection and integrated AR surveillance.