SP 3: Evolution and selection of resistant bacteria in the presence of subinhibitory concentrations of micropollutants
Background
Irrigation with untreated wastewater in the past resulted in an accumulation of chemical pollutants like pharmaceutical residues and disinfectants in soils. These pollutants might be released into soil water when untreated wastewater is replaced with treated wastewater for irrigation.
Hypotheses
We hypothesize that this shift will transiently
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trigger the release of legacy pollutants from soil back into soil water
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lead to environmental concentrations of antibiotics and disinfectants that are large enough to promote the selection of antibiotic resistance, with
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release and concentrations of antibiotics depending on soil type.
Methods
Determination of single and combined Minimal Selective Concentrations (MSCs) with fluorescently labeled susceptible and resistant A. baylyi BD413 strains.
Competition experiments within colloidal and truly dissolved fractions obtained from the joint incubation experiment (provided by SP1) with fluorescently labeled susceptible and resistant A. baylyi BD413 strains as well as within wastewater- including rebuilding of antibiotic concentrations (SP1) measured within the fractions.
Characterization of antibacterial resistance of strains isolated in all joint experiments.
Growth experiments to test effect of soil colloids on antibiotic bioavailability. Growth experiments were performed for simulations.
Results
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Combinations of antibiotics lead to lower MSCs.
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Most truly dissolved and colloidal fractions (produced by SP1) of the incubation experiment showed selective effects on the most sensitive A. baylyi BD413 652 strain. This strain carries a chromosomal deletion that removes the gene of the repressor of a rather unspecific efflux pump. However, there were no significant differences between spiked and unspiked fractions, soil extract incubated with treated and untreated wastewater, or different soil types. As an exception, 4-day extracts from one out of four leptosol samples (leptosol 4) showed stronger selective effects which were lost after 4 weeks of incubation.
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In contrast, experiments with media that contained measured concentrations (data from SP1, Bonn) of sulfamethoxazole, ciprofloxacin, erythromycin, trimethoprim, azithromycin and clindamycin did not select A. baylyi BD413 652, indicating that additional antibiotic or other pollutants are involved in selection of this indicator strain.
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A. baylyi BD413 strains with the resistance plasmid pHHV216, harbouring sul2, strA, strB, floR, and tetH, were not selected by the fractions from the incubation experiment.
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Raw spiked wastewater selected both resistant strains (A. baylyi BD413 652 and 777) with upregulated efflux pumps. Unspiked wastewater showed a weak effect, which was stronger in treated wastewater.
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Ciprofloxacin was sequestered by montmorillonite (cooperation SP1 and SP2).
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Carbapenemase producing E. coli (18) isolates were isolated by SP6 (Gießen) during the field experiment on alfalfa leaf remnants irrigated with untreated and mixed wastewater, on cilantro leaf remnants irrigated with mixed wastewater as well as in the untreated and mixed wastewater directly. 3MRGN (resistant to ciprofloxacin, piperacillin-taxobactam, cefotaxim (3MRGN) E. coli isolates from SP6 (Gießen) were found in untreated and mixed wastewater and on leaf remnants (cilantro and alfalfa) irrigated with groundwater, mixed and untreated wastewater
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Four vancomycin resistant E. faecium isolates (SP6) were found during the field experiment in mixed wastewater and on alfalfa leaf remnants irrigated with mixed wastewater
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3MRGN E. coli isolates (Gießen) from the incubation experiment were found within all wastewater types (untreated/treated, unspiked/spiked)
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A system for checkerboard MSCs with two or three antibiotics was developed and growth curves for simulations (SP7, Tübingen).
Team
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Katharina Axtmann (PhD) |
Gabriele Bierbaum (PI) |
Theses:
Silas Brinkmann, Untersuchung des Einflusses von Bodenkolloiden auf bakterielles Wachstum unter subinhibitorischen Antibiotikakonzentrationen; M.Sc. Microbiology, Bonn, June 2023
Katharina Axtmann, Bakterielle Systeme zur Feststellung der minimalen selektiven Konzentrationen, M.Sc. Microbiology, Bonn, April 2022