Conventional wastewater treatment facilities using activated sludge, secondary clarifiers, and chlorine disinfection comprise a large portion of urban wastewater treatment practices in the United States. While highly adept at removing chemical, physical, and numerous biological contaminants, these treatment methods are ineffective at removing contaminants of emerging concern, such as antibiotics and antibiotic resistance genes (ARGs) from wastewater. As a result, antibiotics and ARGs, as well as any antibiotic-resistant bacteria (ARB) not inactivated during the disinfection process, can be discharged through biosolids or treated wastewater effluent (TWE) into receiving environments and pose a threat to human health. We investigated how abundances of 9 ARGs and one class 1 integron (an indicator gene for multiple resistance and horizontal gene transfer) changed in TWE over time by collecting weekly samples from February to April 2019. Analysis revealed that these genes were highly prevalent in TWE, with relative abundances (ARG copies per mL normalized to 16S rRNA copies per mL) ranging from 2.3 × 10-5 to 4.2 × 10-1 in TWE. No samples contained relative abundances of ARGs that were outliers compared to the study average. Additionally, multiple of the individual ARGs showed correlation with each other and with the class 1 integron (intI1), suggesting multi-drug resistance is a relevant concern.
In addition to environmental discharge or landfill disposal, reuse of TWE and biosolids is a current practice to combat global issues of water and fertilizer shortage. To understand the prevalence of ARGs in TWE, a second study was completed investigating the persistence of a more direct threat to human health: potentially human pathogenic ARB. While decay rates of total fecal bacterial indicators in soil have been frequently reported, very few studies have been completed comparing persistence of the antibiotic-resistant counterparts. Additionally, little is known about how the multi-drug resistance of ARB changes over time after biosolids amendment or TWE irrigation. In this study, germinated carrot seeds were planted in soils that received biosolids amendment and/or TWE irrigation. Total and antibiotic-resistant quantities of two fecal indicator bacteria (Escherichia coli and enterococci) were measured in soil weekly until harvest 77 days after planting. E. coli and enterococci were detectable in the biosolids amended soil, and E. coli was present on carrots from amended soil at harvest. No statistical difference was found between TWE irrigation and irrigation with DI water regarding concentrations in soil or on harvested carrots (p > 0.05) Concentrations of antibiotic-resistant E. coli and enterococci declined at faster rates than their total abundances. Isolated colonies of these bacteria were collected at days 0, 35, and 77 to investigate changes in multi-drug resistance. Analysis revealed that percentages of multi-drug resistant (MDR) E. coli declined significantly over time (p < 0.001), while percentages of MDR enterococci did not significantly change (p > 0.05). These results allow for cautious optimism about reuse of TWE in terms of its impact on ARB in soil, however, the continued presence of fecal indicator bacteria in soil and on harvested carrots further demonstrates the risks associated with biosolids amendment. Difference of multi-drug resistance patterns between E. coli and enterococci isolates encourages the need for additional studies on the topic.