Increased levels of bacterial resistance to antibiotics noted in recent decades poses a significant obstacle to the effective treatment and prevention of disease. Although overuse of antibiotics in agriculture and medicine is partially responsible, environmental exposure to heavy metals may also contribute to antibiotic resistance, even in the absence of antibiotics themselves. In this study, a series of eight lab-scale activated-sludge reactors were amended with Zn and/or a suite of three antibiotics (oxytetracycline, ciprofloxacin, and tylosin), in parallel with unamended controls. Classical spread-plating methods were used to assess resistance to these compounds in culturable bacteria over 21 weeks. After seven weeks of general acclimation and development of baseline resistance levels (phase 1), 5.0 mg/L Zn was added to half of the reactors, which were then operated for an additional 7 weeks (phase 2). For the final seven weeks (phase 3), two of the Zn-amended reactors and two of the control reactors were amended with all three antibiotics, each at 0.2 mg/L. Zn amendment alone did not significantly change resistance levels at the 95% confidence level in phase 2. However, tylosin resistance increased significantly during phase 3 in the Zn-only reactors and resistance to all three antibiotics significantly increased as a consequence of combined Zn+antibiotic amendments. Ambient dissolved Zn levels in the reactors were only 12% of added levels, indicating substantial Zn removal by adsorption and/or precipitation. These results show that sub-toxic levels of Zn can cause increased antibiotic resistance in waste treatment microbial communities at comparatively low antibiotic levels, probably due to developed cross-resistance resulting from pre-exposure to Zn.
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