Fate of antibiotic resistant E. coli and antibiotic resistance genes during full scale conventional and advanced anaerobic digestion of sewage sludge.

Antibiotic resistant bacteria (ARB) and their genes (ARGs) have become recognised as significant emerging environmental pollutants. ARB and ARGs in sewage sludge can be transmitted back to humans via the food chain when sludge is recycled to agricultural land, making sludge treatment key to control the release of ARB and ARGs to the environment. This study investigated the fate of antibiotic resistant Escherichia coli and a large set of antibiotic resistance genes (ARGs) during full scale anaerobic digestion (AD) of sewage sludge at two U.K. wastewater treatment plants and evaluated the impact of thermal hydrolysis (TH) pre-treatment on their abundance and diversity. Absolute abundance of 13 ARGs and the Class I integron gene intI1 was calculated using single gene quantitative (q) PCR. High through-put qPCR analysis was also used to determine the relative abundance of 370 ARGs and mobile genetic elements (MGEs). Results revealed that TH reduced the absolute abundance of all ARGs tested and intI1 by 10-12,000 fold. After subsequent AD, a rebound effect was seen in many ARGs. The fate of ARGs during AD without pre-treatment was variable. Relative abundance of most ARGs and MGEs decreased or fluctuated, with the exception of macrolide resistance genes, which were enriched at both plants, and tetracyline and glycopeptide resistance genes which were enriched in the plant employing TH. Diversity of ARGs and MGEs decreased in both plants during sludge treatment. Principal coordinates analysis revealed that ARGs are clearly distinguished according to treatment step, whereas MGEs in digested sludge cluster according to site. This study provides a comprehensive within-digestor analysis of the fate of ARGs, MGEs and antibiotic resistant E. coli and highlights the effectiveness of AD, particularly when TH is used as a pre-treatment, at reducing the abundance of most ARGs and MGEs in sludgeand preventing their release into the environment.

Introduction

Under a ‘One Health’ apn>proach, n>an class="Chemical">the environment is increasingly being recognised as an essential part of the transmission cycle for antibiotic resistant bacteria (ARB) and their genes (ARGs) [1]. In recent years, ARBs and ARGs have become recognised as significant emerging environmental pollutants [2]. ARB and ARGs in the environment can find their way back to humans via direct or indirect contact with contaminated water or land, via drinking water abstracted from contaminated rivers, or via the food chain [3].

Wastewater treatment plants (WWTPs) are seen as key potential gateways for controlling n>an class="Chemical">the dissemination and release of ARB and ARGs into the environment [4]. WWTPs bring together vast amounts of waste from a variety of different sources [5], with the resulting accumulation of extremely large numbers of bacteria, a significant quantity of which come from the human gastrointestinal tract. Some of these bacteria will carry resistance genes either in their core genome or on mobile genetic elements (MGEs), and, particularly under selection pressure, these can be transferred from one bacterium to another via horizontal gene transfer [6, 7].

Previous studies have documented the effectiveness of WWTPs at removing n>an class="Chemical">ARB and ARGs from liquid effluent released to the aquatic environment [8-10], although in some WWTPs certain ARB and ARGs appear to be enriched [11-13]. However, it is estimated that 90–95% of ARB and ARGs in WWTPs are associated with the sludge fraction [14]. This makes sewage sludge treatment a key process for preventing ARB/ARG release into the environment. Sewage sludges are typically separated from the liquid fraction during primary and secondary settling, and then potentially combined for further treatment. Depending on the country, sewage sludges can either be incinerated, put to landfill, or treated prior to recycling to agricultural land as a fertiliser/soil conditioner. A common method of sewage sludge treatment is anaerobic digestion (AD), which is effective at stabilising sludge, producing renewable energy, and reducing the pathogen load to levels appropriate for reuse [15]. Post digestion, biosolids are often applied to agricultural land as a soil conditioner, returning valuable nutrients in the form of N, P, and K, as well as organic matter to the soil. However, the reuse of sewage sludge also poses a potential risk of direct transmission of ARB and ARGs back into humans via the food chain, or indirectly via agricultural run-off into aquatic ecosystems [3]. Sewage sludge also contains a number of other emerging contaminants such as antibiotics and other organic contaminants, heavy metals and microplastics which may also act as selection pressures for antibiotic resistance, exacerbating their spread [16].

In contrast to treatment of the aqueous compn>onent of sewage, there is a limited number of studies that examine the fate of ARB and ARGs in full-scale sewage sludge treatment. Of those that do, many of them only focus on a small number of ARB and ARGs. The inefficient removal of these potential contaminants is a common finding in these studies [e.g. 8, 17]. The loading rate of ARGs into the environment from sludge is estimated to be 1000 times than from aqueous sewage effluent [18], making sludge management a critical step in the control of environmental ARG release [19]. With over 13 million tonnes of sludge applied to agricultural land each year in the European Union alone, understanding and reducing the potential public health risks associated with sludge recycling to land is recognised as an urgent research gap [19]. There is very little evidence available to characterise the fate of ARB and ARGS once sludge is applied to land, or the relative importance of this particular environmental transmission route for public health. Until there is a better understanding, quantifying ARB and ARGs in particular environments (including sludge) has been suggested as a proxy for risk [20].

The high prevalence of n>an class="Chemical">ARGs in sludge combined with the relative inefficiency of AD for ARG removal makes characterising the removal efficiency of various modifications to AD a research priority in order to inform industry on the best way to reduce AMR-associated risk of sludge recycling.

Prior to AD, sludge can be pre-treated in a number of ways. n>an class="Disease">Advanced digestion using thermal hydrolysis (TH) as a pre-treatment is increasing in deployment [e.g. 20, 21]. The sludge during the TH process undergoes a temperature and pressure increase followed by a rapid pressure release. This has been found to improve methane yields from digestion [22], to allow an increase in sludge throughput and to more effectively remove pathogens [23, 24]. Despite its growing popularity, only one previous study has investigated the fate of ARGs during full-scale TH-AD with analysis of only 14 ARGs [25]. In addition, culture-dependent methods can provide essential information on direct risk of ARB transmission into the environment, but full-scale studies on the fate of ARB during AD have been rare. Therefore, this study was undertaken with the following objectives:

To evaluate the impact of n>an class="Chemical">thermal hydrolysis pre-treatment of sludge on ARB and ARGs during AD

To investigate the fate of n>an class="Chemical">ARB and ARGs in sewage sludge during AD

To overcome knowledge gaps of other studies, the relative abundance of 370 ARGs and mobile genetic elements (MGEs) and the absolute abundance of 13 ARGs (from a range of antibiotic classes) and intI1 was quantified from two WWTPs using mesophilic AD (MAD) to treat sludge. One of the plants used TH as a pre-treatment, the other did not. Samples were taken before and after TH and after subsequent MAD at the first plant, and before and after MAD at the second plant. The number of Escherichia coli colony forming units (CFUs) from each sampling point was also quantified and the antibiotic susceptibility profiles of 300 E. coli isolates to nine antibiotics was characterised. All E. coli isolates were classified into phylotypes to determine whether sludge treatment resulted in a shift in the E. coli community.

Materials and methods

Study location and sample collection

Samples were collected from two centralised WWTPs in the UK bon>an class="Chemical">th treating sewage via an aerobic treatment stage which generates an indigenous secondary sludge. Both plants also import secondary sludges as well as primary sludges from satellite sites. Both plants operate on a mixture of primary and secondary sludges, which then undergoes an anaerobic treatment. WWTP1 employs advanced AD, using TH prior to mesophilic AD (TH-MAD). At this WWTP, samples (approx. five litres) were taken of thickened sludge prior to pre-treatment, after sludge had been thermally hydrolysed (165°C for 30 minutes), and after MAD. From WWTP2 which employs conventional MAD to treat sludge with no pre-treatment, samples were taken of thickened sludge before MAD and after MAD. Fig 1 shows the details of the treatment processes and sample points at each WWTP. WWTP1 treats primary and secondary sludge from its own treatment process (which uses an aerobic sequencing batch reactor SBR) (70% by volume), and also imports sludges from other WWTPs (30% by volume). Mixed sludge is thickened through centrifuges to approximately 16% dry solids (DS) content and thermally hydrolysed. TH sludge is then diluted to approximately 8–9% DS with filtered and UV disinfected final effluent from the SBRs and anaerobically digested (digester retention times vary typically between 12–25 days) at mesophilic temperature (typically 37–42°C) before a short aerobically sparged storage. From here the digestate follows to belt presses for dewatering and whilst the digestate liquors return to the treatment process, the solid digestate (cake) is transported to agricultural land. WWTP2 treats indigenous primary and secondary sludge from activated sludge treatment. Sludge is thickened to approximately 5–7% DS before being anaerobically digested (retention time varied between 17 and 22 days in the sampling period) at mesophilic temperatures (32.6–38°C over the sampling period). The digestate is then centrifuged, with the liquor returned to the treatment process and the digestate cake transported to agricultural land. In total, four sets of samples were collected from WWTP1, and five sets of samples were collected from WWTP2, between early Spring and mid-Autumn (span of 9 months).

Fig 1

Showing the sludge treatment processes and sampling points for each WWTP.

SBR = sequencing batch reactor. Final SBR effluent post filtration (300 μm) is used for dilution of the sludge post TH whilst TH sludge stream is still hot (>80°C).

SBR = sequencing batch reactor. Final SBR effluent post filtration (300 μm) is used for dilution of the sludge post n>an class="Chemical">TH whilst TH sludge stream is still hot (>80°C).

Processing of samples for enumeration of E. coli and total heterotrophs

All samples were processed within four hours of collection. To ensure sampn>les were repn>resentative, each sampn>le (apn>prox. five litres) was thoroughly stirred to resuspend settled sludge. 20 mL of the stirred sludge was then taken and saved for further analysis. A serial dilution of each sample was prepared in triplicate and plated onto Membrane Lactose Glucuronidase Agar (MLGA) (Oxoid) for isolation of E. coli, and nutrient agar for the enumeration of total heterotrophs. MLGA plates were incubated at 37°C for 24 hours, and nutrient agar plates were incubated at 30°C for 48 hours. Colony forming units (CFUs) of E. coli and total heterotrophs per gram of dry weight (g-1 dry wt) were calculated.

Antibiotic susceptibility testing of E. coli

A set of 300 isolated colonies of E. coli were randomly selected for antibiotic resistance profiling against clinically relevant antibiotics. n>an class="Chemical">These included 100 from untreated sludge from each WWTP (sample points 1 and 4) and 100 from after MAD at WWTP two (sample point 5). All antibiotic testing was carried out according to CLSI disk diffusion guidelines (Clinical Laboratory Standards Institute, 2017). Selected isolates were first re-streaked from MLGA onto nutrient agar and incubated for 24 hours at 37°C. The isolate was then resuspended in phosphate buffered saline (PBS) to an OD600 of 0.5 using a MacFarland standard, plated onto Müller-Hinton agar (Oxoid) and incubated at 35°C for 18–20 hours. Zones of inhibition were measured to the nearest millimetre.

The antibiotics used were n>an class="Chemical">ciprofloxacin (5 μg), gentamicin (10 μg), meropenem, (10 μg), ampicillin (10 μg) (used to test for amoxicillin resistance (Clinical Laboratory Standards Institute, 2017)), trimethoprim (5 μg), and four different third generation cephalosporins—ceftazidime (30 μg), cefoperazone (75 μg), ceftiofur (30 μg) and cefpodoxine (30 μg) (all antibiotics were from Oxoid., Basingstoke, UK).

Phylotyping of E. coli isolates

Phylotyping of the 300 n>an class="Species">E. coli isolates was carried out according to the Clermont method [26]. Briefly, a quadruplex PCR reaction was set up for the E. coli genes/genome regions chuA, yjaA, TspE4.C2, arpA. The resulting banding pattern allows the determination of the specific E. coli phylotype. To distinguish between phylotype A and C or D and E, a further confirmatory PCR was carried out using primers specific for trpA or arpA, respectively.

Quantification of ARGs and intI1 using single gene and high-throughput quantitative PCR

Two methon>an class="Chemical">ds of quantitative (q) PCR were used in this study. Single gene qPCR gives absolute abundance measures which are important for quantitative environmental monitoring [27], and it is a more sensitive technique. High throughput qPCR provides a more comprehensive picture of the fate of ARGs within the sludge treatment plant and does not limit researchers to pre-selected genes. However, it is generally a less sensitive technique than single gene qPCR and only relative abundance can be quantified.

DNA was extracted from samples of centrifuged sludge (250 milligram) using the QIAGEN DNeasy PowerSoil Kit (QIAGEN, Manchester, UK). Extracted DNA was stored at -20°C until furn>an class="Chemical">ther use. The quantity of extracted DNA was measured using Qubit, and extractions were considered good enough quality for downstream analysis with a Qubit reading of 30 ng/μL or more.

For single gene qPCR, the absolute abundance of 14 different genes were analysed using a n>an class="Chemical">SYBR Green (QIAGEN) qPCR assay. These genes conferred resistance to a range of different antibiotic families including the tetracylines (tetM), the β-lactamases (bla-CTX-M families 1 and 9 and blaIMP), the fluoroquinolones (qnrS), the aminoglycosides (aac(3)-1), trimethoprim (dfrA1, dfrA5, dfrA7 and dfrA12), MLSB antibiotics (ermF) and sulphonamides (sul1). The class one integron gene (intl1) and the 16S rRNA gene were also analysed. Primers, annealing temperatures and the reaction matrix are described in S1 and S2 Tables.

A rotogene series Q (QIAGEN) was used for the qPCR assay. Sampn>les were processed in tripn>licate. Each run consisted of an initial denaturation stepn> of 94 °C for 5 minutes followed by 35–60 cycles of 95 °C for 10 seconn>an class="Chemical">ds then 60 °C for 10 seconds. Total amounts of the gene of interest were calculated from a standard curve created using dilutions of a small section of the gene (idtDNA, Leuven, Belgium), this was used to calculate copy number (g-1 dry wt) of sludge sample for absolute abundance. Melt curves were analysed for quality control, and selected PCR products were sequenced to ensure specific amplification of the target gene.

For high throughpn>ut qPCR, n>an class="Chemical">ARGs and MGEs in the samples were detected and quantified using SmartChip Real-time PCR system (Takara, Japan), a high-throughput quantitative PCR system. Primer set 2.0 consisting of 312 ARG-targeted and 58 MGE-targeted primers was employed for the analysis [28]. PCR reaction, data collection, CT value calculation, normalization and abundance calculation of ARGs and MGEs were conducted as previously described [29, 30].

Data analysis

Statistical analysis of data was carried out using SPSS version 23. Chi squared goodness of fit analysis was used to determine any significant differences between the percentages of resistant n>an class="Species">E. coli in each sample. A Mann Whitney U test was used to determine whether any of the observed differences in absolute abundance of resistance genes between samples was significant. Significance was determined at a P>0.05. The composition and abundance of ARGs and MGEs at each sample were used for the principal coordinates analysis (PCoA) based on Bray-Curtis dissimilarity matrix. The analysis and plotting were conducted using a PAST program [31].

Results

Fate of antibiotic resistant E. coli and total heterotrophs during MAD and TH-MAD

E. coli CFUs from WWTP1 ranged from 3.8 x 105 to 5.7 x 105 (mean 5.1 x 105 ± 7.1 x 103 SD) g-1 dry wt in untreated sludge. After n>an class="Chemical">TH there was no detectable growth of E. coli on MLGA (i.e. less than 1 CFU per gram), and there was no resurgence of E. coli detected after subsequent MAD. E. coli CFUs from WWTP 2 ranged from 4.6 x 105–8.3 x 105(mean 5.8 x 105 ± 1.1 x 105 SD) CFUs g-1 dry wt of untreated sludge. After MAD, the number of detectable E. coli CFUs on MLGA was between 2.7 x 103–9.6 x 103 (mean 3.7 x 103 ± 2.9 x 103 SD) CFUs per gram dry weight, a decrease of more than 99% (Fig 2A).

Fig 2

A) E. coli CFUs in sludge from WWTP 1 and WWTP2. This shows that there was no detectable E. coli after thermal hydrolysis at WWTP 1, and no resurgence in detectable E. coli numbers after subsequent MAD. MAD achieved a 2–3 log10 reduction in E. coli CFUs. B) Prevalence of antibiotic resistance in E. coli isolated from sewage sludge. Graph shows E. coli resistance rates to six antibiotics at WWTP 1 before TH-MAD, and at WWTP 2 before and after MAD. The four third generation cephalosporins (3GCs) tested have been grouped together. No E. coli were tested for resistance after TH or after TH-MAD because no E. coli were detected.

A) E. coli CFUs in sludge from WWTP 1 and WWTP2. n>an class="Chemical">This shows that there was no detectable E. coli after thermal hydrolysis at WWTP 1, and no resurgence in detectable E. coli numbers after subsequent MAD. MAD achieved a 2–3 log10 reduction in E. coli CFUs. B) Prevalence of antibiotic resistance in E. coli isolated from sewage sludge. Graph shows E. coli resistance rates to six antibiotics at WWTP 1 before TH-MAD, and at WWTP 2 before and after MAD. The four third generation cephalosporins (3GCs) tested have been grouped together. No E. coli were tested for resistance after TH or after TH-MAD because no E. coli were detected.

Resistance to amoxicillin, n>an class="Chemical">trimethoprim, ciprofloxacin and gentamicin was found in E. coli isolates in untreated sludge at WWTP1, and before and after MAD at WWTP2 (Fig 2B). Resistance to third generation cephalosporins was also found before and after MAD at WWTP2 but was not detected in WWTP1. Resistance to meropenem was not detected at either WWTP throughout this study. Prevalence of amoxicillin resistance was highest, with 42% and 46% of isolates from untreated sludge at WWTP1 and WWTP2 showing resistance, respectively. Trimethoprim resistance increased significantly from 7% to 16% during MAD at WWTP2 (P = 0.046).

One multi-drug resistant (MDR) isolate (resistant to pan class="Chemical">three or more antibiotic families) was found in untreated sludge and two MDR isolates were found after Mn>an class="Disease">AD at WWTP2. Four MDR isolates were found in untreated sludge from WWTP1.

The pattern of resistance found in bon>an class="Chemical">th WWTPs combined is comparable to the pattern of clinical levels of resistance in Wales, with the percentage of E. coli isolates from WWTPs resistant to each antibiotic tracking between 8.3 and 29% below the percentage of E. coli isolates from the clinic resistant to each antibiotic [32] (S1 Fig).

The phylotypn>e composition of n>an class="Species">E. coli isolates from before and after MAD at WWTP2 was compared to determine whether there was any shift in phylotypes during AD. Phylotype A and B2 were the most dominant phylotypes (each 24% before MAD and 30% (A) and 22% (B2) after MAD), and no significant changes were observed (S2 Fig).

Fate of ARGS and MGEs during TH-MAD and MAD

Fig 3 shows the absolute abundance of n>an class="Chemical">ARGs in sewage sludge. All 14 genes tested for were detected in untreated sludge samples from both WWTPs. The most abundant ARG in untreated sludge was sul1 (1.9 x 107–1.3x108 copies g-1 dry wt). After thermal hydrolysis at WWTP 1, there was a significant reduction in absolute abundance of all genes tested. The largest reduction was in bla-Imp, which showed greater than a 12,000 fold decrease. The smallest reductions were seen in CTX-M-9 which exhibited less than a 10 fold decrease (Fig 3A).

Fig 3

Absolute abundance of ARGs in sewage sludge.

A) Absolute abundance (copy number g-1 dry wt) of all 13 resistance genes tested, and intI1, during sludge treatment at WWTP 1. Statistically significant changes between samples is indicated by a * and statistically significant changes between feed sludge and digestate is indicated by a °. B) Absolute abundance (copy number g-1 dry wt) of all 13 resistance genes tested, and intI1 during sludge treatment at WWTP 2. Statistically significant changes from before to after MAD are indicated by a *.

A) Absolute abundance (copy number g-1 dry wt) of all 13 resistance genes tested, and intI1, during sludge treatment at WWTP 1. Statistically significant changes between samples is indicated by a * and statistically significant changes between feed sludge and digestate is indicated by a °. B) Absolute abundance (copn>y number g-1 dry wt) of all 13 resistance genes tested, and n>an class="Gene">intI1 during sludge treatment at WWTP 2. Statistically significant changes from before to after MAD are indicated by a *.

The absolute abundance of many genes n>an class="Chemical">then rebounded after MAD at WWTP1 (Fig 3A). Significant increases during MAD were observed in ermF, CTX-M-9, CTX-M-1, dfrA12, tetM, sul1 and intI1. No resistance gene tested showed a significant decrease. The biggest increase was seen in ermF, from 1.2 x 104 to 1.2 x 108 copies (g-1 dry wt). In the case of most genes, the rebound during MAD was not sufficient to increase abundance beyond the level of untreated sludge, meaning that an overall reduction in resistance gene load was seen during treatment (between samples 1 and 3). The exceptions were ermF and tetM which both increased about 10-fold from before to after TH-MAD.

After MAD at WWTP2, n>an class="Chemical">the absolute abundance of many genes (dfrA1, dfrA5, dfrA7, aac(3)-1, CTX-M-1, bla-Imp, and qnrS) decreased significantly, while other genes (CTX-M-9, tetM, sul1, and intl1) showed no overall decrease (Fig 3B). Two genes (ermF and dfrA12), showed an increase in absolute abundance after MAD (from 2.3 x 107 to 2.4 x 108 copies (g-1 dry wt), and from 3.9 x 105 to 1.4 x 106 copies (g-1 dry wt), respectively). However, the increase in dfrA12 was not significant.

To gain a more complete understanding of the dynamics of resistance during n>an class="Disease">AD, the relative abundance of 372 genes (including ARGs and MGEs) in each sample was analysed using SmartChip qPCR. Figs 4 and 5 show the results.

Fig 4

Relative abundance of ARGs (normalised to 16S rRNA gene) during sludge treatment.

ARGs are grouped according to class. Samples 1–9 are from WWTP 1. Samples 1, 4, 7 are influent sludge, samples 2, 5, 8 are post-TH sludge, samples 3, 6, 9 are post-MAD sludge. Each sample set (1–3, 4–6, 7–9) is from an independent sampling event. Samples 10–15 are from WWTP 2. Samples 10, 12, 14 are influent sludge, samples 11,13,15 are post-MAD sludge. Each sample set (10 and 11, 12 and 13, 14 and 15) is from an independent sampling event.

Fig 5

Relative abundance of MGEs (normalised to 16S rRNA gene) during sludge treatment.

Samples 1–9 are from WWTP 1. Samples 1, 4, 7 are influent sludge, samples 2, 5, 8 are post-TH sludge, samples 3, 6, 9 are post-MAD sludge. Each sample set (1–3, 4–6, 7–9) is from an independent sampling event. Samples 10–15 are from WWTP 2. Samples 10, 12, 14 are influent sludge, samples 11,13,15 are post-MAD sludge. Each sample set (10 and 11, 12 and 13, 14 and 15) is from an independent sampling event.

ARGs are groupn>ed according to class. Samples 1–9 are from WWTP 1. Samples 1, 4, 7 are influent sludge, samples 2, 5, 8 are post-n>an class="Chemical">TH sludge, samples 3, 6, 9 are post-MAD sludge. Each sample set (1–3, 4–6, 7–9) is from an independent sampling event. Samples 10–15 are from WWTP 2. Samples 10, 12, 14 are influent sludge, samples 11,13,15 are post-MAD sludge. Each sample set (10 and 11, 12 and 13, 14 and 15) is from an independent sampling event.

Samples 1–9 are from WWTP 1. Samples 1, 4, 7 are influent sludge, samples 2, 5, 8 are post-TH sludge, samples 3, 6, 9 are post-Mn>an class="Disease">AD sludge. Each sample set (1–3, 4–6, 7–9) is from an independent sampling event. Samples 10–15 are from WWTP 2. Samples 10, 12, 14 are influent sludge, samples 11,13,15 are post-MAD sludge. Each sample set (10 and 11, 12 and 13, 14 and 15) is from an independent sampling event.

Relative abundance of most of the 312 n>an class="Disease">ARGs decreased or fluctuated during TH-MAD or MAD (Fig 4). However, several macrolide and aminoglycoside resistance genes were significantly enriched during both processes. In addition, several tetracycline resistance genes and two glycopeptide resistance genes were enriched specifically during TH-MAD.

The abundance of MGEs dramatically decreased during treatment in bon>an class="Chemical">th plants (Fig 5). Diversity of all ARG classes and MGEs decreased during treatment, especially during TH, but there was a rebound after AD (Fig 6). In most cases, this rebound did not return ARG diversity to influent sludge levels. Glycopeptide resistance genes were however consistently more diverse in digested sludge than influent sludge. Principal coordinate analysis (PCoA) showed that ARGs were clearly distinguished according to the treatment steps (influent sludge, after TH, after AD) (Fig 7), and that no local or seasonal variation was observed. However, for MGEs, influent sludge clustered together regardless of WWTP, but after AD samples were clearly distinguished according to WWTP.

Fig 6

Diversity of ARGs (A) and MGEs (B) during sludge treatment. Samples 1–9 are from WWTP 1.

Samples 1, 4, 7 are influent sludge, samples 2, 5, 8 are post-TH sludge, samples 3,6,9 are post-MAD sludge. Each sample set (1–3, 4–6, 7–9) is from an independent sampling event. Samples 10–15 are from WWTP 2. Samples 10, 12, 14 are influent sludge, samples 11,13,15 are post-MAD sludge. Each sample set (10 and 11, 12 and 13, 14 and 15) is from an independent sampling event.

Fig 7

A. PCoA of ARGs within sludge samples. B. PCoA of MGEs within sludge samples. Each colour shows an independent sampling event. Samples taken from the same point in the treatment process are indicated using the same shapes.

Samples 1, 4, 7 are influent sludge, samples 2, 5, 8 are post-TH sludge, samples 3,6,9 are post-Mn>an class="Disease">AD sludge. Each sample set (1–3, 4–6, 7–9) is from an independent sampling event. Samples 10–15 are from WWTP 2. Samples 10, 12, 14 are influent sludge, samples 11,13,15 are post-MAD sludge. Each sample set (10 and 11, 12 and 13, 14 and 15) is from an independent sampling event.

Discussion

This study highlights the importance of sewage sludge treatment in the control of ARB and ARG release to the environment. Untreated sewage sludge contains a large number of ARGs, with over a billion copies per gram of sludge for some genes and hundreds of thousands of antibiotic resistant E. coli per gram of sludge. This demonstrates that ARB and ARGs are either surviving the aerobic sewage treatment process and/or being diverted from the aqueous phase to sewage sludge in large numbers, and that untreated sewage sludge represents a significant source of ARB and ARGs.

Fate of antibiotic resistant and virulent E. coli during MAD and TH-MAD

Sewage sludge treatment using TH-MAD reduces n>an class="Species">E. coli to undetectable levels, as well as all coliforms and Gram-negatives (S3 Fig). Therefore, the direct risk of resistant potential pathogens being applied to agricultural land is greatly reduced. Although MAD reduces E. coli CFUs by more than 99%, they are still present in treated sludge in quantities ranging from 1.2 x 103 to 4.3 x 103 g-1 dry wt. Overall, 45% of the E. coli tested showed resistance to one or more antibiotic, meaning that significant numbers of antibiotic resistant E. coli are being added to UK agricultural soil which may pose a direct risk to humans via the food chain, or an indirect risk if these resistant E. coli or their genes spread in the environment. A key future research priority is to determine the fate of ARB and ARGs once sludge is applied to agricultural land.

MAD apn>pears to enrich for n>an class="Chemical">trimethoprim resistant E. coli. Specific enrichment of certain ARB have been reported previously in the literature [33]. The conditions driving the selection of ARB within treatment plants need to be characterised and avoided where possible. No significant change in the abundance of different phylotypes was detected but interestingly, a high prevalence of E. coli type B2, responsible for 72% of E. coli bloodstream infections [34], was observed.

Fate of ARGs and MGEs during TH-MAD and MAD

AD decreases the abundance of most ARGs, but macrolide, lincosamide, streptogramin B (MLSB) resistance genes are consistently enriched, in both plants. An increase in MLSB resistance genes has also been reported during anaerobic digestion of sludge in other studies [8, 35]. This apparent enrichment for MLSB resistance genes could be due to carriage in surviving gut bacteria, although this is unlikely as enrichment is also seen after TH, which appears to remove almost all gut bacteria from sludge. MLSB resistance genes could be spreading during AD through horizontal gene transfer, although this seems unlikely due to dramatic decreases in abundance and diversity of MGEs in both plants. MLSB resistance genes could be inherent in a member of the AD microbial community. Interestingly, the only other study to examine the impact of TH on ARGs also found MLSB resistance gene enrichment [25]. One of the enriched genes, ermF, showed an increase in absolute abundance during treatment so that treated sludge had a higher abundance than untreated sludge, in both WWTPs. Sequencing of the ermF amplicon revealed 100% identity to a plasmid-encoded gene from a clinical isolate (S4 Fig), highlighting the need for further investigation into the fate of these enriched genes in treated sludge. Several aminoglycoside resistance genes are also enriched during AD.

In the n>an class="Chemical">TH-MAD plant, tetracycline and glycopeptide resistance genes are also enriched, as are the MGEs tnpA (IS6 family) and IS613. IS613 was also enriched during the temperature decreasing stage of composting [36] and has been found to be associated with ARGs [37, 38].

TH reduced n>an class="Chemical">the abundance of all genes, but some of these genes rebounded during subsequent MAD. The effectiveness of TH at removing ARGs and antibiotics at pilot scale has previously been reported [39]. Interestingly, an ARG rebound after TH was also observed in that study, which, showed that subsequent thermophilic AD reduced this increase.

The dramatic decrease in diversity and abundance of MGEs during sludge treatment would suggest n>an class="Chemical">that anaerobic digesters are not a hotspot for HGT, as has previously been suggested [40, 41]. However, the influence of the small number of MGEs which do increase in abundance; MGEs not included in the assay conducted; or other methods of HGT (transformation and transduction) which were not accounted for in this study cannot be ruled out.

It is reassuring to note that n>an class="Chemical">ARG abundance results from both methods of qPCR show similar patterns, providing further evidence for the usefulness of select ARGs such as sul1, tetM and ermF as candidate marker genes to assess overall resistance gene load [27, 42, 43].

PCoA showed that n>an class="Chemical">ARGs are clustered according to treatment step with no local or seasonal differences, but MGEs in treated sludge cluster by site. This suggests that conditions within each specific AD plant will influence the abundance and diversity of MGEs, but that this change does not then fundamentally alter ARG abundance and diversity of effluent sludge. However, these differences in the MGE community between different digesters might be responsible for the specific increases in certain genes that were seen and shows that MGEs (and therefore HGT potential) can be very different in different digesters. Tong et al. (2019) found that, while MGEs were a strong influence on total ARG abundance, the bacterial and archaeal biomass and community largely influenced the fate of ARGs.

Conclusions and recommendations

This study is an important stepn> towarn>an class="Chemical">ds filling the research gap on the fate of ARB and ARGs during full scale sludge treatment. The high abundance of ARB and ARGs in raw and treated sludge highlights the importance of sewage sludge as a transmission route for AMR into the environment. ARB and ARGs are applied to agricultural land in vast numbers, which is a potential public health risk.

Measuring the public healn>an class="Chemical">th risks associated with AMR in the environment is inherently difficult, and many have suggested using the ‘precautionary principle’ to reduce ARB and ARG release rather than doing nothing. Although AD generally reduces the amount of ARGs and MGEs in sewage sludge, it is a relatively inefficient method for ARG removal. Therefore any ‘quick wins’ to reduce ARB and ARGs in sludge would be an excellent way of reducing risk, according to the precautionary principle. Thermal hydrolysis is an increasingly common pre-treatment with additional benefits including an increase in methane yield. This research shows that it effectively reduces ARB and most ARGs in sewage sludge, which provides important evidence for the sludge treatment industry considering the installation of new AD plants or the upgrade of existing AD plants. However, the rebound effect shown by some ARGs, and their increase in absolute abundance, deserves further investigation. The increased prevalence of trimethoprim resistant E. coli in digested MAD sludge compared to raw sludge is direct evidence of the selection for a resistant potential pathogen within the digestor and may have public health implications when this sludge is applied to agricultural land.

Primers and annealing temperatures used in this study.

Table shows primer name and sequence, length (in base pairs) of amplicon, annealing temperature, sources, and (where relevant) which antibiotic n>an class="Chemical">the target gene confers resistance to.

(DOCX)

Click here for pan class="Disease">additional data file.

Reaction matrix used for qPCR of resistance genes, int1, and the 16S rRNA gene.

(DOCX)

Click here for pan class="Disease">additional data file.

Comparison of resistance rates in clinical isolates with resistance rates in isolates from WWTP sludge.

The percentage of n>an class="Species">E. coli clinical and WWTP isolates resistant to each antibiotic tested in this study is shown. Clinical data from Public Health Wales (2017).

(DOCX)

Click here for pan class="Disease">additional data file.

E.coli phylotypes.

A. Before AD, WWTP 2. B. After n>an class="Disease">AD, WWTP 2. No significant phylotype shift during AD was observed.

(DOCX)

Click here for pan class="Disease">additional data file.

An alignment between the ermF sequence obtained from sewage sludge with a plasmid form of ermF from a clinical isolate of Bacteroides fragilis BLAST sequence ID M14730.1.

There is 100% similarity between n>an class="Chemical">the sewage sludge sequence and the plasmid version of ermF.

(DOCX)

Click here for pan class="Disease">additional data file.

E. coli, other coliforms and non-coliform gram negatives CFUs.

Graph shows before TH, after n>an class="Chemical">TH and after TH-MAD at WWTP1, as well as before and after MAD at WWTP2.

(DOCX)

Click here for pan class="Disease">additional data file.

19 Aug 2020

pan class="Chemical">PONE-D-20-22627

An investigation into the fate of antibiotic resistant n>an class="Species">E. coli and antibiotic resistance genes during full scale conventional and advanced anaerobic digestion of sewage sludge

PLOS ONE

Dear Dr. Emma Hayhurst,

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Energy and Environment (NRN-LCEE), from a Welsh Government and European Regional

Development Fund supported under pan class="Chemical">the Smart Expn>ertise Programme SMART CIRCLE project, and by

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to pan class="Chemical">the Aupan class="Chemical">thor

1. Is the manuscripn>t technically sound, and do n>an class="Chemical">the data support the conclusions?

The manuscripn>t must describe a technically sound piece of scientific research win>an class="Chemical">th data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has pan class="Chemical">the statistical analysis been performed apn>propriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

**********

3. Have the aun>an class="Chemical">thors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is pan class="Chemical">the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so pan class="Chemical">the language in submitted articles must be clear, correct, and unambiguous. Any typn>ograpn>hical or grammatical errors should be corrected at revision, so please note any spn>ecific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to pan class="Chemical">the Aupan class="Chemical">thor

Please use the spn>ace provided to expn>lain your answers to n>an class="Chemical">the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Review

The authors present information on the fate of antibiotic resistant E. coli and antibiotic resistance genes in municipal anaerobic digesters with and without thermal hydrolysis pretreatment. The fact that a very large number of ARGs and MGEs were targeted using SmartChip system was interesting to see and a strength of the work. The authors should consider adding more information on the implications of their results and/or future work that may be beneficial based on their results. The following comments are made in a sincere effort to help the authors improve the manuscript.

General comments:

1. I think there are some very practical things that could come out of the results presented. But the manuscript lacks a good, cohesive description of what the results tell us and how they might be used to improve public health and the environment. The authors should add information regarding the implications and usefulness of their results. To describe the usefulness of the results, for example, the authors could also provide a description of future research that would be important to do based on their results. The asuthors should describe in discussion and conclusions sections how the results now help fill the gap described in the introduction.

2. The results lack a descripn>tion of data precision. n>an class="Chemical">Throughout the manuscript, a description of measurement precision is needed along with the range or mean values. What were the detection limits for the genes and E. Coli enumeration? Were values before and after treatment statistically different?

Specific comments:

3.pan class="Chemical">The titles is long and cumbersome. Could “An investigation into pan class="Chemical">the…” be deleted?

4. Line 31, by how much were the 13 n>an class="Chemical">ARG abundance values reduced by TH?

5. Line 38, line 198 and pan class="Chemical">throughout n>an class="Chemical">the manuscript, was it PCA or PCoA employed?

6. Line 40, I disagree pan class="Chemical">that n>an class="Chemical">the results are comprehensive. Two full-scale WWTPs are not a comprehensive sampling of WWTPs.

7. line 78, sewage sludge reuse also poses a risk from opan class="Chemical">ther emerging contaminants such as EDCs.

8. line 87, is it an urgent research gap? Describe and justify the urgency. You should describe in discussion and conclusions sections how your results now help fill n>an class="Chemical">the gap you describe in the introduction. For example, can your results be used in quantitative risk assessment?

9. Section 2.1, what was the exact temperature and holding time of n>an class="Chemical">the digesters? Since pathogen inactivation is a function of these, it is important to know the exact values.

10. line 123, “Sludge is thickened…” which sludge? n>an class="Chemical">The mix of primary and secondary? Just the secondary?

11. Line 123, “…SBR)(70%)…” is it percent by volume or mass of solipan class="Chemical">ds?

12. Line 126, What is “filter treated effluent?”? Describe.

13. Line 142, why “decanted”? Why take only the supn>ernatant? Does taking n>an class="Chemical">the supernatant and not the entire mix influence the results? How?

14. Line 153, briefly describe pan class="Chemical">the CLSI mepan class="Chemical">thod.

15. line 164, briefly describe pan class="Chemical">the Clermont mepan class="Chemical">thod.

16. Page 18, pan class="Chemical">there is no section 2.6.

17. Sections 2.5 and 2.7, It is confusing to the ren>an class="Disease">ader why two different methods for ARG enumeration were used – SYBR green (QIAGEN) and SmartChip. Please let the reader know up front that two methods were used, why two methods were used, and not just one, why a gene was chosen to be measured by one method and not by another, etc. Why not just use SmartChip?

18. Line 202, “378,000”; and other CFU/g dry weight values in the manuscript. It would be more appropriate to report the log of the results. Also, with three significant figures, you imply your measurements are accurate to +/- 1%. Is this true? What is the standard deviation of the values or log of the values?

19. Line 228, What does “resistance levels” mean? Be more specific.

20, Line 256, change “sample 1 and 3” to “samples 1 and 3.”

21. Line 321, “a high abundance” compared to what?

22. Line 326, “entirely removes”. What was the detection limit of n>an class="Species">E coli? Is it zero? Can you be sure that it is entirely removed to zero?

23. line 340, in opan class="Chemical">ther studies of what? Anaerobic digesters? n>an class="Species">Activated sludge? Receiving streams?

24. Line 357, “pan class="Chemical">their study”. Who are pan class="Chemical">they?

Reviewer #2: The manuscripn>t, “An investigation into n>an class="Chemical">the fate of antibiotic resistant E. coli and antibiotic resistance genes during full scale conventional and advanced anerobic digestion of sewage sludge,” describe work in which the authors examined the fate of resistant bacteria and multiple resistance genes over the length of two treatment trains. This work is highly relevant to global-scale questions regarding antibiotic resistance and WWTPs.

However, I do not feel as if this papn>er is a good fit for n>an class="Chemical">this target journal. As you will see below, I feel that this work doesn’t do enough to separate itself from a wide body of existing literature. PLOS One reports novel and cutting-edge work and, although this is an interesting and well-designed study, it is similar to work that has already been reported.

Specific comments:

Line 23 – a very effective way to start off the abstract is a “big picture” statement. Why is this study relevant in the big picture? The authors do this well in lines 52-56 – I would suggest adding some of the information in lines 52-56 to the beginning of the abstract. This compels the reader to continue reading.

Line 38 – I believe that n>an class="Chemical">the authors mean “principal” (rather than principle). I think.

Throughout – n>an class="Chemical">the authors use ARBs as an acronym for antibiotic resistant bacteria. This acronym should not be plural.

Line 54 should repan class="Disease">ad, “…as significant emerging environmental pollutants.”

Lines 80-81 – actually, there have been more n>an class="Chemical">than 1,500 publications since 2019 in the peer-reviewed literature that focus on sewage sludge (specifically, anaerobic digestion of sewage sludge) and antibiotic resistance. In addition, more than 100 publications have focused on thermal hydrolysis and antibiotic resistance in the same time period. What does your publication lend to this body of literature? You are targeting a high-impact, widely-read journal – it is extremely important here to set your paper apart from this large body of existing literature.

Line 144 – there are literature repn>orts that find low, but measurable false positive rates on MLGA used to identify E. coli. Was any additional testing performed on these isolates to confirm their identity as E. coli? This may have been realized through the phylotyping – but there is no description of what “the Clermont method” is. Perhaps it would be helpful to insert a sentence or two describing this method.

Lines 169-170 – here, you are describing DNA quantity, NOT quality.

**********

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Reviewer #1: Yes: Daniel H. Zitomer

Reviewer #2: No

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29 Oct 2020

Response to Reviewers

Reviewer #1: Review

The authors present information on the fate of antibiotic resistant E. coli and antibiotic resistance genes in municipal anaerobic digesters with and without thermal hydrolysis pretreatment. The fact that a very large number of ARGs and MGEs were targeted using SmartChip system was interesting to see and a strength of the work. The authors should consider adding more information on the implications of their results and/or future work that may be beneficial based on their results. The following comments are made in a sincere effort to help the authors improve the manuscript.

Thankyou Reviewer #1 for n>an class="Chemical">the positive comments above and we really appreciate the effort to help us improve the manuscript. Our response to each individual point raised is detailed below:

1.I think there are some very practical things that could come out of the results presented. But the manuscript lacks a good, cohesive description of what the results tell us and how they might be used to improve public health and the environment. The authors should add information regarding the implications and usefulness of their results. To describe the usefulness of the results, for example, the authors could also provide a description of future research that would be important to do based on their results. The authors should describe in discussion and conclusions sections how the results now help fill the gap described in the introduction.

We were very aware that we did not want to over interpn>ret our results and were careful to present n>an class="Chemical">them without speculation. However, we have now amended our discussion and conclusions section to include potential implications of this research, future research directions and recommendations. We hope this helps give a clearer picture of the importance of our findings.

2. The results lack a descripn>tion of data precision. n>an class="Chemical">Throughout the manuscript, a description of measurement precision is needed along with the range or mean values. What were the detection limits for the genes and E. Coli enumeration? Were values before and after treatment statistically different?

We have now put mean values and SD into our E.coli values. Our n>an class="Chemical">ARG values already show this information. Detection limits for each gene varied for the high throughput qPCR, for single gene qPCR every gene was detected in every sample and therefore we did not reach the detection limit. For E.coli, detection limit was approximately 1 cell per gram.

3.pan class="Chemical">The titles is long and cumbersome. Could “An investigation into pan class="Chemical">the…” be deleted?

Done.

4. Line 31, by how much were the 13 n>an class="Chemical">ARG abundance values reduced by TH?

Amounts pan class="Disease">added (fold change)

5. Line 38, line 198 and pan class="Chemical">throughout n>an class="Chemical">the manuscript, was it PCA or PCoA employed?

Apologies – it was PCoA - pan class="Chemical">this has been corrected pan class="Chemical">throughout.

6. Line 40, I disagree pan class="Chemical">that n>an class="Chemical">the results are comprehensive. Two full-scale WWTPs are not a comprehensive sampling of WWTPs.

What we meant was that our results are comprehensive win>an class="Chemical">thin these two specific WWTPs – we have changed the wording to reflect this.

7. line 78, sewage sludge reuse also poses a risk from opan class="Chemical">ther emerging contaminants such as EDCs.

We have pan class="Disease">added in a line about opan class="Chemical">ther contaminants.

8. line 87, is it an urgent research gap?Describe and justify the urgency. You should describe in discussion and conclusions sections how your results now help fill n>an class="Chemical">the gap you describe in the introduction. For example, can your results be used in quantitative risk assessment?

It is an urgent research gap. Measuring the public healn>an class="Chemical">th risks associated with AMR in the environment is inherently difficult, and many have suggested using the ‘precautionary principle’ to reduce ARB and ARG release rather than doing nothing until the research quantifies public health risk. We know from other studies that although AD generally reduces the amount of ARGs in sewage sludge, it can be a relatively inefficient method for ARG removal. Therefore any ‘quick wins’ to reduce ARB and ARGs in sludge would be an excellent way of reducing risk, according to the precautionary principle. Thermal hydrolysis is an increasingly common pre-treatment with additional benefits including an increase in methane yield. Investigating its impact on ARB and ARGs provides important evidence for the sludge treatment industry who may be considering the installation of new AD plants or the upgrade of existing AD plants. This has been added to the manuscript. We have also changed the order of our objectives to reflect the importance of this piece of research.

9. Section 2.1, what was the exact temperature and holding time of n>an class="Chemical">the digesters? Since pathogen inactivation is a function of these, it is important to know the exact values.

WWTP 2 ranged in temperature from 32.6°C to 38°C over the sampn>ling period. Retention time ranged from 17 to 22 days. Tempn>erature detail, and more spn>ecific retention times, have been added to the manuscript. Unfortunately detailed information was not available for WWTP1, however, as stated in the paper we know that the retention time varies between and 12 and 25 days, and we have added in the typical temperature range for mesophlic digestion.

10. line 123, “Sludge is thickened…” which sludge? n>an class="Chemical">The mix of primary and secondary? Just the secondary?

The mix of primary and secondary sludge is n>an class="Chemical">thickened – this has now been clarified in the manuscript.

11. Line 123, “…SBR)(70%)…” is it percent by volume or mass of solipan class="Chemical">ds?

It is by volume – pan class="Chemical">this has been clarified in n>an class="Chemical">the manuscript.

12. Line 126, What is “filter treated effluent?”? Describe.

Apologies – this is an error. n>an class="Chemical">The plant uses UV treated and filtered final effluent to dilute sludge. This has been amended in the manuscript.

13. Line 142, why “decanted”? Why take only the supn>ernatant? Does taking n>an class="Chemical">the supernatant and not the entire mix influence the results? How?

Apologies – ‘decanted’ was the wrong choice of word. n>an class="Chemical">The five litre sample was mixed thoroughly to resuspend settled sludge and then 20 ml was taken for further analysis. So each sample was the entire mix, not just the supernatant. This has been amended and clarified in the manuscript.

14. Line 153, briefly describe pan class="Chemical">the CLSI mepan class="Chemical">thod.

The descripn>tion in Section 2.3 is n>an class="Chemical">the CLSI method so this was already included. We have amended the order of the text to make this clearer.

15. line 164, briefly describe pan class="Chemical">the Clermont mepan class="Chemical">thod.

pan class="Chemical">This has now been pan class="Disease">added.

16. Page 18, pan class="Chemical">there is no section 2.6.

Apologies – pan class="Chemical">this has now been fixed.

17. Sections 2.5 and 2.7, It is confusing to the ren>an class="Disease">ader why two different methods for ARG enumeration were used – SYBR green (QIAGEN) and SmartChip. Please let the reader know up front that two methods were used, why two methods were used, and not just one, why a gene was chosen to be measured by one method and not by another, etc. Why not just use SmartChip?

Single gene qPCR gives absolute abundance measures, which are important for quantitative risk analysis, and it is a more sensitive technique. High throughpn>ut qPCR provides a more compn>rehensive picture of the fate of ARGs within the sludge treatment plant and does not limit researchers to pre-selected genes. However, it is a less sensitive technique than single gene qPCR and only relative abundance can be quantified, making it less suitable for quantitative risk analysis. Therefore we chose to use a combination of both, selecting 14 genes we felt were representative, commonly found in treatment plants and of clinical interest, but supplementing this with SmartChip analysis which allowed for a much more comprehensive analysis of ARG fate. This explanation has now been added to the manuscript.

18. Line 202, “378,000”; and other CFU/g dry weight values in the manuscript. It would be more appropriate to report the log of the results. Also, with three significant figures, you imply your measurements are accurate to +/- 1%. Is this true? What is the standard deviation of the values or log of the values?

For consistency we have now changed these to expn>onential values – we hopn>e n>an class="Chemical">this is acceptable. We have also added SD values and changed to 2 significant figures.

19. Line 228, What does “resistance levels” mean? Be more specific.

We mean the percentage of n>an class="Species">E.coli isolates resistant to each antibiotic – this has been amended.

20, Line 256, change “sample 1 and 3” to “samples 1 and 3.”

Done.

21. Line 321, “a high abundance” compared to what?

pan class="Chemical">This section has been amended to make our point clearer and pan class="Chemical">this term removed.

22. Line 326, “entirely removes”. What was the detection limit of n>an class="Species">E coli? Is it zero? Can you be sure that it is entirely removed to zero?

No, we cannot and pan class="Chemical">this phrase has been changed to ‘reduces to undetectable levels’.

23. line 340, in opan class="Chemical">ther studies of what? Anaerobic digesters? n>an class="Species">Activated sludge? Receiving streams?

MLS genes increase during anaerobic digestion in opan class="Chemical">ther studies – n>an class="Chemical">this has been clarified.

24. Line 357, “pan class="Chemical">their study”. Who are pan class="Chemical">they?

Reference 38. pan class="Chemical">This has now been changed to ‘n>an class="Chemical">that study’.

Reviewer #2: The manuscripn>t, “An investigation into n>an class="Chemical">the fate of antibiotic resistant E. coli and antibiotic resistance genes during full scale conventional and advanced anerobic digestion of sewage sludge,” describe work in which the authors examined the fate of resistant bacteria and multiple resistance genes over the length of two treatment trains. This work is highly relevant to global-scale questions regarding antibiotic resistance and WWTPs.

However, I do not feel as if this papn>er is a good fit for n>an class="Chemical">this target journal. As you will see below, I feel that this work doesn’t do enough to separate itself from a wide body of existing literature. PLOS One reports novel and cutting-edge work and, although this is an interesting and well-designed study, it is similar to work that has already been reported.

Thankyou Reviewer #2 for recognising the global relevance of our research. We feel that our amended manuscript makes clearer how our work is different from existing literature. We have also addressed this comment in more depth under the relevant specific comment below. We believe that this work will be highly relevant to academics, industry representatives and policy makers and will therefore attract a wide readership. For example, in the UK, reports on the topic of AMR and sludge treatment have been commissioned by both public sector (DEFRA) and private sector (UKWIR) organisations, and in both reports the lack of full scale studies was identified as a research gap. By attempting to fill this research gap, our work is novel and cutting-edge.

Specific comments:

Line 23 – a very effective way to start off the abstract is a “big picture” statement. Why is this study relevant in the big picture? The authors do this well in lines 52-56 – I would suggest adding some of the information in lines 52-56 to the beginning of the abstract. This compels the reader to continue reading.

This has been n>an class="Disease">added to the abstract.

Line 38 – I believe that n>an class="Chemical">the authors mean “principal” (rather than principle). I think

Apologies – changed pan class="Chemical">throughout.

Throughout – n>an class="Chemical">the authors use ARBs as an acronym for antibiotic resistant bacteria. This acronym should not be plural.

Apologies - changed pan class="Chemical">throughout.

Line 54 should repan class="Disease">ad, “…as significant emerging environmental pollutants.”

Changed as suggested

Lines 80-81 – actually, there have been more n>an class="Chemical">than 1,500 publications since 2019 in the peer-reviewed literature that focus on sewage sludge (specifically, anaerobic digestion of sewage sludge) and antibiotic resistance. In addition, more than 100 publications have focused on thermal hydrolysis and antibiotic resistance in the same time period. What does your publication lend to this body of literature? You are targeting a high-impact, widely-read journal – it is extremely important here to set your paper apart from this large body of existing literature.

We cannot return the numbers of studies quoted by Reviewer #2 when we search using the same keywords. Regardless, there are still very few full scale studies carried out which specifically investigate the fate of ARB and ARGs in during sludge treatment. The vast majority of published works on antibiotic resistance and sludge treatment are run on lab or pilot scale digestors. While these can provide important insights into the behaviour of particular genes or bacteria under idealised conditions with easily controlled variables, they are simply not representative of systems at full scale. To truly understand the effectiveness of sludge treatment then full scale system studies must be carried out. In the few full scale studies that do exist, comprehensive high-throughput qPCR analysis is rare with most studies focusing on just a few ARGs which are not necessarily representative of the whole picture. Few studies also include culture dependent work, but this is a more direct measure of risk which is of relevance to the water industry who are responsible for the safety of sewage sludge. Finally, almost all studies have been carried out in China where treatment processes and climatic conditions are very different to elsewhere. A recent DEFRA-funded report summarised the literature on AMR and AD and highlighted the lack of UK based studies (as an example) as an urgent research gap (Atkins, 2018). Our study gives academic insight into the fate of ARB and ARGs but also gives practical recommendations of relevance to the water industry. This work has been presented at UK water industry events and has received attention and interest. We therefore think that it adds an important piece of work to an under-researched highly complex topic area and is worthy of publication.

Line 144 – there are literature repn>orts that find low, but measurable false positive rates on MLGA used to identify E. coli. Was any additional testing performed on these isolates to confirm their identity as E. coli? This may have been realized through the phylotyping – but there is no description of what “the Clermont method” is. Perhaps it would be helpful to insert a sentence or two describing this method.

The phylotypn>ing men>an class="Chemical">thod confirms isolates as E.coli. A description of this method has been added to the paper. We did find a few isolates which were other Escherichia species and these were removed from further analysis – the results presented here are confirmed E.coli isolates.

Lines 169-170 – here, you are describing DNA quantity, NOT quality

Changed as suggested.

Submitted filename: response to reviewers.docx

Click here for pan class="Disease">additional data file.

11 Nov 2020

Fate of antibiotic resistant pan class="Species">E. coli and antibiotic resistance genes during full scale conventional and n>an class="Disease">advanced anaerobic digestion of sewage sludge

pan class="Chemical">PONE-D-20-22627R1

Dear Dr. Hayhurst,

We’re pleased to inform you pan class="Chemical">that your manuscripn>t has been judged scientifically suitable for publication and will be formally accepn>ted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing n>an class="Chemical">the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal accepn>tance. To ensure an efficient process, please log into Editorial Manager at httpn>://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upn>coming papn>er to help maximize its impn>act. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regarpan class="Chemical">ds,

Tim Hülsen

Acpan class="Disease">ademic Editor

PLOS ONE

pan class="Disease">Additional Editor Comments (optional):

Reviewers' comments:

16 Nov 2020

pan class="Chemical">PONE-D-20-22627R1

Fate of antibiotic resistant pan class="Species">E. coli and antibiotic resistance genes during full scale conventional and n>an class="Disease">advanced anaerobic digestion of sewage sludge

Dear Dr. Hayhurst:

I'm pleased to inform you that your manuscripn>t has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscripn>t is now win>an class="Chemical">th our production department.

If your institution or institutions have a press office, please let them know about your upn>coming papn>er now to help maximize its impn>act. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help wipan class="Chemical">th anyn>an class="Chemical">thing else, please email us at plosone@plos.org.

pan class="Chemical">Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regarpan class="Chemical">ds,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tim Hülsen

Acpan class="Disease">ademic Editor

PLOS ONE