A Review on Occurrence and Spread of Antibiotic Resistance in Wastewaters and in Wastewater Treatment Plants: Mechanisms and Perspectives

This paper reviews current knowledge on sources, spread and removal mechanisms of antibiotic resistance genes (ARGs) in microbial communities of wastewaters, treatment plants and downstream recipients. Antibiotic is the most important tool to cure bacterial infections in humans and animals. The over- and misuse of antibiotics have played a major role in the development, spread, and prevalence of antibiotic resistance (AR) in the microbiomes of humans and animals, and microbial ecosystems worldwide. AR can be transferred and spread amongst bacteria via intra- and interspecies horizontal gene transfer (HGT). Wastewater treatment plants (WWTPs) receive wastewater containing an enormous variety of pollutants, including antibiotics, and chemicals from different sources. They contain large and diverse communities of microorganisms and provide a favorable environment for the spread and reproduction of AR. Existing WWTPs are not designed to remove micropollutants, antibiotic resistant bacteria (ARB) and ARGs, which therefore remain present in the effluent. Studies have shown that raw and treated wastewaters carry a higher amount of ARB in comparison to surface water, and such reports have led to further studies on more advanced treatment processes. This review summarizes what is known about AR removal efficiencies of different wastewater treatment methods, and it shows the variations among different methods. Results vary, but the trend is that conventional activated sludge treatment, with aerobic and/or anaerobic reactors alone or in series, followed by advanced post treatment methods like UV, ozonation, and oxidation removes considerably more ARGs and ARB than activated sludge treatment alone. In addition to AR levels in treated wastewater, it examines AR levels in biosolids, settled by-product from wastewater treatment, and discusses AR removal efficiency of different biosolids treatment procedures. Finally, it puts forward key-points and suggestions for dealing with and preventing further increase of AR in WWTPs and other aquatic environments, together with a discussion on the use of mathematical models to quantify and simulate the spread of ARGs in WWTPs. Mathematical models already play a role in the analysis and development of WWTPs, but they do not consider AR and challenges remain before models can be used to reliably study the dynamics and reduction of AR in such systems.


Total
copies/g (16S rRNA) tetW copies/g tetO copies/g sulI copies/g 10 11.5 -10 12.1 10 8.0 -10 8.5 10 8.8 -10 9.3 10 6.9 -10 9.2 Activated sludge treatment, biosolids from a mix of primary sludge and activated sludge dewatered by belt press only. Bacteria numbers by heterotrophic plate count on R2A media with antifungal cyclohexamide (200 ug/ml), gene copy numbers from qPCR, numbers per gram of biosolids (wet or dry weight not specified), absolute ranges from n = 3 samples taken at different times of the year.
Biosolids disposed to landfill.
Rotating biological contactors (Romeo) with biosolids from unknown sludge mix. Aeration basin and membrane biological reactor (Traverse city) with biosolids from a mix of primary sludge and activated sludge dewatered by a gravity #: (Munir et al., 2011) PT&TP: 3 * sul resistant CFU/g (50.4 ug/ml Sul) 10 6.7 -10 7.2 sulI copies/g 10 7.6 -10 8.2 belt concentrator before digestion. Bacteria numbers by heterotrophic plate count on R2A media with antifungal cyclohexamide (200 ug/ml), gene copy numbers from qPCR, numbers per gram of biosolids (wet or dry weight not specified), interquartile ranges from n = 7 samples in total taken from different times of the year (unspecified how many samples from each site). Temperature during anaerobic digestion unspecified.
Biosolids used as fertilizer on agricultural land. Activated sludge treatment, biosolids from a mix of primary sludge and mechanically thickened activated sludge. Total coliforms, fecal coliforms, and E. coli numbers by plate count on mEndo-LES medium, mFC medium, and MFC-BCIG medium respectively. Gene copy numbers from qPCR. Numbers per gram dry weight of biosolids. Average numbers from n = 3 samples for bacteria count and n = 4 samples for gene numbers. Temperature during anaerobic digestion unspecified.
Biosolids used as fertilizer on agricultural land and for remediation of mine tailing ponds from mining industry. Landfill as contingency option.
Biosolids used as fertilizer on agricultural land and for remediation of mine tailing ponds from mining industry. Landfill as contingency option. Heat-dried biosolids from a blend of wasted activated sludge and anaerobically digested primary sludge from an unspecified municipal WWTP. Gene copy numbers from qPCR. Numbers are reported per gram dry weight. Temperature during heat drying unspecified.
Use of biosolids unspecified.
The table includes levels of resistant bacteria and resistance genes reported in the literature for resistance against four selected antibiotics: ampicillin (amp), tetracycline (tet), sulfonamide (sul), and streptomycin (str). In addition to the numbers reported from the scientific literature (references marked with #), we have also gathered extended information on plant type, treatment processes, sludge sources, and the normal use of biosolids from each plant from public reports and webpages from municipalities and plant operators (references marked with PT&TP). We have designated sludge/biosolids with solids content below 12% as slurry, sludge/biosolids with solids content from 12-55% as cake, and biosolids with solids content above 55% as dry.
Note that the more advanced methods are not used instead of the simple methods listed at the top of the table, but in addition. E.g., thickened/dewatered sludge is used as input for aerobic or anaerobic digestion, and heat drying is done with sludge that has already been digested and/or thickened/dewatered.
Numbers originally reported as ranges are given as ranges with a note detailing whether the range is absolute (min-max), quartiles, or in any other format. Numbers given as averages are reported as a single average number, and variance numbers are disregarded as there is inconsistency on how these are reported (e.g., as variance, as standard deviation (SD), or even as standard error of the mean (SEM)) and from how many samples they are calculated. All numbers are rounded to one decimal digit in the exponent. All gene numbers are total, most studies do not distinguish between cellular DNA and external DNA.
* These plants showed a significant reduction of resistance genes (copies/100ml) and a significant reduction of resistant bacteria (CFU/100ml) from the raw influent sewage to the treated water effluent, but no significant reduction of resistance genes nor resistant bacteria from the raw influent sewage to the sludge biosolids (Munir et al., 2011).
** It is unknown whether the sludge is aerobically digested in a digestor, or if the aerobic digestion happens during or as part of an air-drying process (the sludge is aerobically digested according to (Gondim-Porto et al., 2016), but no specific information about an aerobic digestor is found on the plant fact sheet from the operator (Canal de Isabel II S.A., 2017)).
*** Note that the gene copy numbers from this study indicate that the aerobic biosolids contains a roughly 3 times higher number of sul1 genes than rrnS genes which were used as a reference for total amount of bacteria. The authors of the study do not comment on this anomality. However, in their Supplementary Material they report that 0.0018 copies of sul1 genes was detected for each copy of the 16S rRNA gene (reference for total amount of bacteria) in a sample of aerobic slurry analyzed by a different method (next generation sequencing) (Lau et al., 2017).
**** The density of living bacteria in the pelletized biosolids from Windsor are below the detection limits in this study, but the number of genes still seems relatively high. However, the same study reports that the abundance of the same gene targets quantified in agricultural soil directly (2-hours, + 24-48 hours storage at 4° C) after application of the pelletized material was remarkably low compared to digested slurry or cake biosolids from other plants. The authors hypothesize that the DNA are carried in non-viable cells or as free DNA segments with lower than normal stability (due to the heat treatment) in the pellets and that it is rapidly destroyed following contact with soil (Lau et al., 2017).
*5 The biosolids from the Windsor Biosolids Pelletizing Facility was tested in both 2014 (Lau et al., 2017) and 2017 (Murray et al., 2019). The plant was built in 1999 and operated from 1999-2019 by American Water (formerly Prism-Berlie) (City of Windsor, 2020). The treatments and processing involved to create the pelletized biosolids have to the authors' knowledge not changed from 2014 to 2017 (Lau et al., 2017;Murray et al., 2019;City of Windsor, 2020).