Resistant Escherichia coli was substantially more prevalent in hospital sewer biofilms than in the wastewater flowing through them, a Swedish study found. The findings identified hospital sewer systems as potential environments for antibiotic resistance selection, with ciprofloxacin and cefotaxime implicated as principal selective agents.
The researchers investigated wastewater from 10 sampling locations at 8 Swedish hospitals and corresponding municipal wastewater treatment systems using laboratory experiments and field sampling. The study included 24-hour composite wastewater samples, experiments assessing resistance selection in bacterial communities, and analyses of sewer biofilms. Antibiotic concentration measurements, metagenomic sequencing, and experiments involving bacteria carrying specific resistance genes were used to investigate the mechanisms underlying selection.
The main analyses examined whether hospital wastewater selected for ciprofloxacin- and cefotaxime-resistant E. coli and whether resistant bacteria were enriched in sewer biofilms relative to wastewater. Additional analyses evaluated antibiotic resistance gene abundance, resistance selection in Klebsiella pneumoniae, and the contributions of individual antibiotics and resistance mechanisms.
In laboratory experiments, wastewater from all 10 hospital sampling locations was used to select for ciprofloxacin-resistant E. coli. Cefotaxime resistance was also selected at the hospital sampling sites except 1, where selection was detected only for ciprofloxacin resistance. No statistically significant selection was detected in the corresponding municipal wastewater samples.
Field sampling showed that hospital sewer biofilms contained a 3.11-fold higher proportion of ciprofloxacin-resistant E. coli and a 3.48-fold higher proportion of cefotaxime-resistant E. coli compared with corresponding hospital wastewater samples. At municipal sites, the respective biofilm-to-wastewater ratios were 1.23 and 1.08, with neither showing statistically significant enrichment. However, the statistical interaction comparing enrichment patterns between hospital and municipal sites was not significant.
Metagenomic sequencing provided additional evidence of resistance enrichment in hospital sewer biofilms. Total antibiotic resistance gene abundance was higher in hospital biofilms relative to wastewater but was lower in municipal biofilms. Differences in bacterial community composition could contribute to these patterns, complicating interpretation of resistance gene enrichment.
Experiments involving E. coli carrying specific resistance genes implicated fluoroquinolones and beta-lactams as major contributors to growth inhibition in hospital wastewater. Introducing the quinolone resistance gene qnrB and the beta-lactam resistance gene blaNDM-1 restored bacterial growth under the tested conditions, supporting the contribution of these antibiotic classes to resistance selection.
Chemical analyses and experiments involving individual antibiotics further identified ciprofloxacin and cefotaxime as important selective agents. In bacterial community experiments, ciprofloxacin exposure selected for resistance to all 8 antibiotics tested, while cefotaxime exposure selected for resistance to 7 of 8 antibiotics. Additional experiments involving K. pneumoniae supported the potential for hospital wastewater to select multidrug-resistant bacteria.
The researchers acknowledged several limitations. All sampling sites were in Sweden, and only 1 biofilm sample was collected per location, limiting assessment of within-site variability and comparisons among hospitals. Freezing wastewater before filtration reduced concentrations of certain beta-lactam antibiotics, weakening the evidence for widespread cefotaxime-driven selection relative to ciprofloxacin. The extent of resistance selection across other bacterial species also remained uncertain. Additionally, the study did not establish whether resistance selected within hospital sewers subsequently contributed to infections in patients.
Researchers noted that antibiotic residues become diluted as hospital wastewater enters municipal systems, potentially reducing the selective pressures detected in laboratory experiments. These findings may help identify where interventions to limit environmental antibiotic resistance could have the greatest impact.
“Given the absence of detectable selection at municipal sites, mitigations of risks in hospital sewers should be prioritized,” wrote lead study author Declan A. Gray, PhD, of the University of Gothenburg, Gothenburg, Sweden, and colleagues.
The study received funding from Swedish research agencies, Swedish state grants, and the Adlerbertska Foundation. Gray, D. G. Joakim Larsson, and MutshieneDeogratiasEkwanzala reported filing a patent application related to antibiotic-contaminated wastewater treatment. The remaining authors declared no competing interests.
Source: Nature Communications
