# Environmental reservoirs account for high levels of carbapenem resistance genes in wastewater

## TL;DR

Wastewater contains high levels of carbapenem resistance genes, likely from environmental bacteria, complicating their use for tracking public health threats.

## Contribution

The study identifies environmental reservoirs as a major source of carbapenem resistance genes in wastewater, challenging their use for clinical surveillance.

## Key findings

- blaKPC, blaOXA-24/40, and blaOXA-48 were found at very high concentrations in wastewater.
- Sequence types matched clinical strains, ruling out non-specific amplification as a cause.
- Environmental bacteria like Aeromonas and Acinetobacter baumannii were linked to ARGs.

## Abstract

Wastewater surveillance has moved to the forefront as a practical and informative public health tool for assessing viral and bacterial pathogens in the human population; however, the usefulness for tracking antimicrobial-resistance genes (ARGs) of clinical concern is not straightforward given the large reservoir of free-living bacteria within sewer systems. In this study, we examined six high-priority carbapenem resistance gene targets and common host organisms in wastewater collected from two treatment plants. Polymerase chain reaction (PCR)-based assays demonstrated an extremely high abundance of blaKPC, blaOXA-24/40, and blaOXA-48 with mean concentrations as high as 3.55E+06 copy number (cn)/L for blaKPC. In addition, blaVIM and blaIMP were detectable in ~99% of samples at lower levels of ~1.5E+05 cn/L and 1.0E+04 cn/L, respectively. The target blaNDM was readily detected but quantifiable in only 49% of samples with levels of ~1.0E+03 cn/L. Each PCR target showed one to two dominant sequence types that matched exactly to reported clinical strains, indicating that high levels could not be attributed to non-specific amplification. Select ARG targets showed moderate-to-weak correlations to human fecal markers and Klebsiella pneumoniae or Escherichia coli, whereas blaOXA-24/40 most closely correlated to Acinetobacter baumannii. The two treatment plants showed different dynamics, suggesting that the inherent characteristics of the individual conveyance systems influence concentrations. Culture on selective media revealed an abundance of Aeromonas in addition to sporadic Enterobacteriaceae carrying blaKPC. Understanding the ecological dynamics of bacteria harboring ARGs will be important for understanding reservoirs and interpreting wastewater surveillance data.

Wastewater surveillance for carbapenem-resistant bacteria has been proposed as a potentially valuable tool for assessing the human burden, but their distribution in environmental reservoirs is not well understood. Untreated sewage contains a high density of resident organisms mixed with human inputs, which are transported through the complex environment of sewer conveyance systems. In addition to resident microbial community members, organisms seeded into the system from the human microbiome have the potential to grow. This work shows that environmental bacteria may be a significant source of carbapenem resistance genes collected from sewer systems, making wastewater surveillance data difficult to interpret or use for public health actions. More knowledge is needed to unravel the ecology of these systems and identify targets for surveillance that are meaningful to clinicians. This work sheds light on the complex dynamics and confounding factors for antimicrobial-resistance gene wastewater surveillance, which will improve the interpretation of wastewater surveillance data.