Method to find antibiotic-resistant genes shows limits of 'snapshot' samples, chlorination
From: Rice University
December 19, 2022 -- Testing
the contents of a simple sample of wastewater can reveal a lot about what it
carries, but fails to tell the whole story, according to Rice University
engineers.
Their new study shows
that composite samples taken over 24 hours at an urban wastewater plant give a
much more accurate representation of the level of antibiotic-resistant genes
(ARGs) in the water. According to the Centers for Disease Control and
Prevention (CDC), antibiotic resistance is a global health threat responsible
for millions of deaths worldwide.
In the process, the
researchers discovered that while secondary wastewater treatment significantly
reduces the amount of target ARG, chlorine disinfectants often used in later
stages of treatment can, in some situations, have a negative impact on water
released back into the environment.
The lab of Lauren
Stadler at Rice's George R. Brown School of Engineering reported seeing levels
of antibiotic-resistant RNA concentrations 10 times higher in composite samples
than what they see in "grabs," snapshots collected when flow through
a wastewater plant is at a minimum.
Stadler and lead
authors Esther Lou and Priyanka Ali, both graduate students in her lab,
reported their results in the American Chemical Society journal Environmental
Science & Technology: Water.
The results could lead
to better protocols for treating wastewater to lower the prevalence of
antibiotic-resistant genes in bacteria that propagate at plants and can transfer
those genes to other organisms in the environment.
The issue is critical
because antibiotic resistance is a killer, causing an estimated 2.8 million
infections in the U.S. every year, leading to more than 35,000 deaths, said
Stadler, an assistant professor of civil and environmental engineering and a
pioneer in the ongoing analysis of wastewater for signs of the SARS-CoV-2 virus
responsible for COVID-19.
Those statistics have
made it a long-standing focus of efforts at Rice that led to the foundation of
a new center, Houston Wastewater Epidemiology, a partnership with the Houston
Health Department and Houston Public Works. The center is one of two designated
by the CDC announced this year to develop tools and train other state and local
health departments in the sciences of monitoring wastewater-borne diseases.
The takeaway for
testers is that snapshots can lead to unintended biases in their results,
Stadler said.
"I think it's
intuitive that grabbing a single sample of wastewater is not representative of
what flows across the entire day," said Stadler, who is also a faculty
member of the Rice-based, National Science Foundation-supported Nanotechnology
Enabled Water Treatment (NEWT) Center. "Wastewater flows and loads vary
across the day, due to patterns of water use. While we know this to be true, no
one had shown the degree to which antibiotic-resistant genes vary throughout
the day."
For the study, the Rice
team took both grab and composite samples in two 24-hour campaigns, one during
the summer and another during winter, at a Houston-area plant that routinely
disinfects wastewater.
They took samples every
two hours from various stages of the wastewater treatment process and ran PCR
tests in the lab to quantify several clinically relevant genes that confer
resistance to fluoroquinolone, carbapenem, ESBL and colistin, as well as a
class 1 integron-integrase gene known as a mobile genetic element (MGE) for its
ability to move within a genome or transfer from one species to another.
The samples they
collected allowed them to determine the concentration of ARGs and loads across
a typical weekday, the variability in removal rates at plants based on the grab
samples and the impact of secondary treatment and chlorine disinfection on the
removal of ARGs, as well as the ability to compare grabs and composites.
The team found that the
vast majority of ARG removal occurred due to biological processes as opposed to
chemical disinfection. In fact, they observed that chlorination, used as the
final disinfectant before the treated wastewater is discharged into the
environment, may have selected for antibiotic-resistant organisms.
Because the results
from snapshots can vary significantly during any given day, they had to be
collected at a steady pace over 24 hours. That required Lou and Ali to spend
several long shifts at the City of West University Place wastewater treatment
plant. "They camped out," Stadler said. "They set up their cots
and ordered takeout."
Such commitment will
not be necessary if real-time wastewater monitoring becomes a reality. Stadler
is part of a Rice collaboration developing living bacterial sensors that would
detect the presence of ARGs and pathogens, including SARS-CoV-2, without pause
at different locations within a wastewater system. The project underway at Rice
to build bacterial sensors that emit an immediate electrical signal upon
sensing a target was the subject of a study in Nature in November.
"Living sensors
can enable continuous monitoring as opposed to relying on expensive equipment
to collect composite samples that need to be brought back to the lab to
analyze," she said. "I think the future is these living sensors that
can be placed anywhere in the wastewater system and report on what they see in
real time. We're working towards that."
Rice undergraduate
Karen Lu and Prashant Kalvapalle, a graduate student in the Systems, Synthetic
and Physical Biology Ph. D. program, are co-authors of the study.
The National Science
Foundation (2029025, 1805901, 1932000) and a Johnson & Johnson WiSTEM2D
award supported the research.
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