Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: Sci Total Environ. 2019 Sep 12;711:134435. doi: 10.1016/j.scitotenv.2019.134435

Removal of Antibiotic Resistance Genes in an Algal-based Wastewater Treatment System Employing Galdieria sulphuraria: A Comparative Study

Xiaoxiao Cheng 1, Himali M K Delanka-Pedige 1, Srimali P Munasinghe-Arachchige 1, Isuru S A Abeysiriwardana-Arachchige 1, Geoffrey B Smith 2, Nagamany Nirmalakhandan 1, Yanyan Zhang 1,*
PMCID: PMC6992497  NIHMSID: NIHMS1545909  PMID: 31810689

Abstract

In this study, we compared removal of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) in two wastewater treatment systems fed with the same primary effluent: a conventional wastewater treatment system (consisting of a trickling filter followed by an activated sludge process) versus an algal-based system, employing an extremophilic alga, Galdieria sulphuraria. Our results demonstrated that the algal system can reduce concentrations of erythromycin- and sulfamethoxazole-resistant bacteria in the effluent more effectively than the conventional treatment system. A decreasing trend of total bacteria and ARGs was observed in both the treatment systems. However, the relative ratio of most ARGs (qnrA, qnrB, qnrS, sul1) and intI1 in the surviving bacteria increased in the conventional system; whereas, the algal system reduced more of the relative abundance of qnrA, qnrS, tetW and intI1 in the surviving bacteria. The role of bacteriophages in horizontal gene transfer (HGT) of ARGs in the two systems was indicated by a positive correlation between ARG absolute abundance in bacteriophage and ARG relative abundance in the bacteria. Four of the five detectable genes (qnrS, tetW, sul1 and intI1) were significantly reduced in the algal system in bacteriophage phase which signified a decrease in phage-mediated ARG transfer in the algal system. Results of this study demonstrate the feasibility of the algal-based wastewater treatment system in decreasing ARGs and ARB and in minimizing the spread of antibiotic resistance to the environment.

Keywords: Algal system, Conventional treatment, Antibiotic resistance genes, Bacteriophage

1. Introduction

Due to the massive use of antibiotics in human and veterinary health applications, domestic and agricultural wastewaters are now recognized as major environmental sources of antibiotic-resistant bacteria and genes (Andersson and Hughes, 2010; Bengtsson-Palme et al., 2017). Since all the domestic wastewaters eventually flow through wastewater treatment plants (WWTPs), they have been identified as large reservoirs for antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) (Rizzo et al., 2013). In the US and in most countries worldwide, WWTPs depend on conventional treatment processes, (e.g. preliminary treatment, primary treatment, and secondary treatment) to remove traditional pollutants (e.g. suspended solids, dissolved organics, and nutrients). The activated sludge (AS) process is the most common secondary treatment processes where, high concentrations of bacteria are utilized to biologically oxidize dissolved organic carbon in wastewaters.

Previous studies have reported that biological processes, such as the AS process, could promote the spread of ARB and ARGs (Rizzo et al., 2013) resulting from sub-inhibitory concentrations of antibiotics in wastewater and the abundance of microorganisms in the AS process. First, the sub-inhibitory concentration of antibiotics in wastewater provides potentially suitable environment for developing antibiotic resistance and spreading in the AS process (Auerbach et al., 2007; Da Silva et al., 2006; Davies et al., 2006). Antibiotics with sub-inhibitory concentrations are considered as a trigger of the SOS response to DNA damage in which DNA repair and mutagenesis are induced. These sub-inhibitory effects may play a crucial role in the spread of antibiotic resistance accelerating the rates of mutation and horizontal gene transfer (HGT) (Baharoglu and Mazel, 2014). According to Andersson and Hughes, sub-inhibitory concentrations of antibiotics may be more relevant to the issue of antibiotic resistance than lethal concentrations of antibiotics (Andersson and Hughes, 2012).

Furthermore, the high abundance of microorganisms in the AS process (~4,000 mg/L) would exacerbate the spread of antibiotic resistance. High nutrient load, normal pH and temperature in the AS process provide favorable environment for cell proliferation which would increase the chance of HGT (Di Cesare et al., 2016; Kim et al., 2014; Marano and Cytryn, 2017). A previous report has noted that secondary sludge contained higher number of resistant bacteria of 107 CFU/g secondary sludge (Gao et al., 2012b). Additionally, abundance of bacteriophage in WWTPs is also extraordinarily high, with over 1000 unique viral genomes identified in the activated sludge (Parsley et al., 2010), which increase the chance of gene transfer via transduction. Most wastewaters carry a bacteriophage content of 105-108 plaque forming units (PFU)/mL (McMinn et al., 2017; Yahya et al., 2015). Although conventional treatment could remove significant number of bacteria, it is not designed to remove bacteriophages in wastewater.

The mechanism of antibiotic resistance development could be explained by intrinsic resistance, acquisition through mutations, or HGT which could be from donor bacteria or bacteriophage. HGT represents gene transfer mediated by mobile genetic elements (MGEs) including plasmid, integrons and bacteriophage (Nwosu, 2001). Genetic exchange through generalized or specialized transduction is achieved by bacteriophage, whereby a fragment of DNA is carried by bacteriophage from a donor bacterial to a recipient cell (Balcazar, 2014). Although it was thought transduction occurs at low frequency, recent single cell studies (Touchon et al., 2017) observed transduction rates close to 1% per plaque forming units when natural communities served as recipients. Metagenomic data also show generalized transduction may contribute significantly to HGT in prokaryotes and these analyses indicated that bacteriophage could be a vital reservoir for ARGs (Schmieder and Edwards, 2012). It was demonstrated that bacteriophages play a vital role in the dissemination of ARGs in aquatic environment (Yang et al., 2018). Some recent studies that used both sequence and function-based metagenomic analyses revealed the occurrence of ARG-like genes in the virome of activated sludge, which could transfer resistance to some host bacteria (Balcazar, 2014).

In this study, the feasibility of controlling ARGs and ARB in a pilot-scale, algal-based wastewater treatment system utilizing Galdieria sulphuraria was investigated. Galdieria sulphuraria is a thermo-acidophilic, unicellular red alga, that can grow mixotrophically on dissolved organics in wastewaters (Gross, 2000; Gross and Schnarrenberger, 1995). Long-term operation of this pilot scale algal wastewater treatment system has been demonstrated previously as an energy-efficient and sustainable technology for removing biochemical oxygen demand (BOD), ammoniacal nitrogen, and phosphates in primary-settled domestic wastewater in a single step. In addition to treating the wastewater to the discharge standards, this algal system can produce energy-rich biomass which could be used as a feedstock for recovering biocrude oil (Henkanatte-Gedera et al., 2015). Superior performance of this algal system in concurrent inactivation of pathogenic bacteria in the primary-settled wastewater has also been demonstrated (Delanka-Pedige et al., 2019). Given the high pathogenic bacterial inactivation and the inclement culture conditions (low pH and high temperature) in the algal system, the current study was designed to test the follow-on idea that the system also reduces the occurrence of antibiotic resistance.

The specific goal of this study was to compare the reductions of ARGs and ARB in an algal system against that in a secondary treatment system at a conventional wastewater treatment plant. Five commonly used groups/classes of antibiotics were chosen to assess the fate of ARGs and ARB in the two systems. Given that Class 1 integrons can acquire and express multiple resistance genes by using site-specific recombination mediated by an integron-integrase (int I) (Hall and Collis, 1995), the abundance of int I1 gene in WWTPs was investigated as well. Additionally, the role of bacteriophages in ARG transfer was also assessed in the two systems.

2. Materials and methods

2.1. Sample collection

Samples were obtained from the algal system deployed outdoors at the local wastewater treatment plant (Las Cruces, New Mexico), running in parallel with the existing plant. The extremophilic algal culture, G. sulphuraria (CCMEE 5587.1) was cultivated in a horizontal, enclosed photobioreactor with a working volume of 700 L, operated in batch mode. This pilot system has been operated at a pH of 4.0 within a temperature range of 27–46 °C (Delanka-Pedige et al., 2019; Henkanatte-Gedera et al., 2015; Tchinda et al., 2019). Details about the algal system and sampling points are provided in the Supplementary Information Section (Figure S1). Samples of mixed liquor (20 L) were collected from the algal system after every batch. Since the algal system could remove COD, BOD, ammoniacal nitrogen, and phosphates to discharge standards in one-step in 3–4 days, the effluent was sampled after 5 days of operation.

The secondary treatment system in the existing treatment plant is composed of a trickling filter followed by an activated sludge process, operated in continuous flow mode at an average flow rate of 10 million gallons per day with an average temperature of 21°C and pH of 7.2. Both the algal and conventional systems were fed with the same primary-effluent. Duplicated samples of primary effluent (P) and the activated sludge system effluent (S) were collected on October 2017 (Batch 1) and July 2018 (Batch 2) during normal operation. All samples were collected in sterile containers that were placed in ice and were stored at 4°C before DNA extraction.

2.2. Sample processing for DNA extraction

Bacteria in 25 mL, 215 mL, and 100 mL of primary treatment effluent (P), secondary treatment effluent (S) and algal reactor effluent (A) were concentrated by membrane filtration with 0.22 µm pore-size membrane (Millipore, Billerica, MA) before DNA extraction using DNeasy PowerWater Kit (Qiagen, Germany). The volumes mentioned above are the maximum volumes which could be filtered through one membrane filter without any clogging. The maximum volumes were used to obtain enough DNA for downstream analysis.

Bacteriophages in water samples were concentrated prior to DNA extraction following the protocols in previous study (Zhang et al., 2013). First, the same volume of water samples was filtered (0.22 µm membrane filter, Millipore, Billerica, MA) to remove bacteria. Each filtrate was dosed with 2M MgCl2 stock solution to reach a final concentration of 0.1 M Mg2+, followed by pH adjustment to 3.5. The pH adjusted samples were then filtrated (0.45 µm pore-size HATF filter, Millipore, Billerica, MA) for virus adsorption on the membrane. Next, 15 mL of eluate (1.5% beef extract, 0.05M glycine, pH 9.5, sterile) was applied to release bacteriophage from the membrane filter prior to centrifugal filtration (30kDa molecular weight cutoff, Millipore, Amicon Ultra-15, Billerica, MA) at a speed of 2,500 × g for 20 min to reduce the sample volume to ~ 200 µL (If the volume of concentrated samples was less than 200 µL, then it was adjusted to 200 µL with DNase-free water). The concentrated bacteriophage samples were treated with 4U of DNase I (Thermo Scientific) for 1h at 37°C to remove extracellular bacterial DNA (Aw et al., 2014; Cantalupo et al., 2011; Kim et al., 2017; O’Brien et al., 2017). Finally, the bacteriophage nucleic acids were extracted from concentrated samples with a PureLink Virus RNA/DNA Mini Kit (Invitrogen, Thermo Fisher) following the manufacturer’s protocol.

2.4. Quantification for ARGs by quantitative real-time PCR (qPCR)

The following twelve antibiotic resistance genes were investigated through PCR assay: 4 quinolone resistance genes (qnrA, qnrB, qnrC, qnrS), 3 tetracycline resistance genes (tetW, tetM, tetO), 2 sulfonamide resistance genes (sul1, sul2), 1 erythromycin resistance gene (ermB), 1 multi-resistance to β-lactam antibiotics gene (blaTEM) and 1 mobile element class 1 integron gene (int I1). Eight of the above 12 ARGs, namely, tetW, qnrA, qnrB, qnrS, sul1, ermB, int I1, and blaTEM, were detected in bacteria DNA of primary effluent. Hence, those eight genes were quantified for all 12 samples (6 bacterial DNA samples and 6 bacteriophage DNA samples) using SYBR-Green based qPCR in a Bio-Rad CFX real-time PCR detection system (Bio-Rad, California), and as a comparison, 16S rRNA was used to quantify total bacteria copy number using TaqMan probe based qPCR (Sims et al., 2012). qPCR reactions for 8 ARGs were performed in 96-well plates with a system volume of 20 µL, containing 10 µL of 2× SsoAdanved Universal SYBR Green Supermix (Bio-Rad), 1 µL of each primer (10 µM), 2 µL of diluted DNA sample and 6 µL of DNA/RNA free water. qPCR reaction for 16S rRNA was performed in 20 µL system consisting 10 µL of SsoAdvanced Universal Probes Supermix (Bio-Rad), 1 µL of each primer (10 µM), 2 µL of diluted DNA sample, 1 µL of probe (2.5 µM) and 5 µL of DNA/RNA free water. The amplification procedures, primers and probe for all the qPCR reactions are listed in Table S1. Each reaction was performed in triplicate. Standard plasmids containing target genes were constructed using a TOPO TA cloning® kit (Invitrogen, Thermo Fisher Scientific) for quantification. Reactions without the DNA template served as negative controls.

2.5. Enumeration of ARB by culture method

Heterotrophic plate count (HPC) method was used to quantify the population of ARB in each sample. The following five types of antibiotics were chosen for enumerating ARB: ampicillin-32 mg/L; tetracycline-16 mg/L; ciprofloxacin-4 mg/L; erythromycin-8 mg/L; sulfamethoxazole-50.4 mg/L. Each antibiotic represents a class of broad-spectrum antibiotics referred to as penicillin class; tetracycline class; quinolone class; macrolide class; and gantanol class, respectively. R2A agar (BD, Difco) was used as culture medium with the above antibiotics with clinically relevant concentrations while 200 mg/L cyclohexamide was added to prevent the growth of fungi. Samples were diluted by serial ten-fold dilutions and spread on the plates containing the specific concentration of antibiotics. All the analyses were conducted in duplicate. Plates were incubated for 48 h at 37 °C and then incubated for 72 h at room temperature. The colony counts between 20–300 CFU in each plate were recorded for ARB concentration in water samples. The log reduction between primary effluent and the effluent of conventional or algal system was used to show the performance of two systems in ARB and ARG removal.

3. Results and discussion

3.1. Occurrence of total bacteria in wastewater treatment processes

The qPCR results for batch 1 showed total bacterial numbers (copies/mL) of (4.33 ± 0.06) × 108 in the primary effluent; (1.18 ± 0.05) × 107 in the activated sludge effluent; and (5.00 ± 0.10) × 107 in the algal effluent. Corresponding results in batch 2 were (7.04 ± 1.05) × 107, (9.09 ± 1.09) × 105, and (2.53 ± 0.65) × 106. In both batches, bacterial concentration in the primary effluent was the highest and that in activated sludge effluent was the lowest. In the conventional system, bacterial concentration decreased along the treatment train with a total bacterial removal of 1.5–2 log after the activated sludge process, which is consistent with our previous report (Delanka-Pedige et al., 2019). Activated sludge with good settling capability could aggregate most bacteria in mixed liquor to settle in the sedimentation tank while predators such as ciliates (protozoa) and rotifers (metazoan) could consume the dispersed bacteria resulting in low bacterial concentration in the effluent.

The bacterial concentration in the algal effluent was higher than that in the conventional system, resulting in a lower removal of 88.5%. It is worth mentioning that the algal system was collected from the mixed liquor rather than the supernatant due to the absence of settling tank, which may account for the higher total bacterial concentration in the effluent of the algal system compared with the conventional system. Given that the algal reactor was operated at pH of 4 for the cultivation of acidophilic G. sulphuraria, the reduction of total bacteria in the algal system is attributed, in part, to the low cultivation pH of 4.0 (Henkanatte-Gedera et al., 2015; Munasinghe-Arachchige et al., 2019). 16S rRNA Illumina sequencing data from our previous study revealed that Acidobacteria phylum was the most dominant bacterial phylum in the algal reactor with a relative abundance of 98% (Delanka-Pedige et al., 2019). Also, thermophilic treatment might decrease the abundance of mesophilic bacteria including Bacteroidetes and Proteobacteria.

3.2. Concentration of ARB in the conventional system vs. algal system

Similar to its performance in reducing total bacteria, the conventional system showed high reductions of ARB as well. In the case of ampicillin-resistant bacteria, 3.5 log and 1.5 log removals from the primary effluent were recorded in batch 1 and batch 2, respectively. In the case of ciprofloxacin- and tetracycline-resistant bacteria, about 1.5 log removal was observed, which resulted in the negotiable ARB number (around 1 CFU/mL) in its effluent. Lower removals (0.5 −1.5 log) of erythromycin- and sulfamethoxazole-resistant bacteria were observed in the conventional system as substantial amount of erythromycin- and sulfamethoxazole-resistant bacteria were still present in its effluent. It can be inferred that both initial bacteria number and drug-resistant class had influence in the ARB removal performance of the conventional system. Additionally, compared to the 1.5–2 log removal of total bacteria, removals of erythromycin- and sulfamethoxazole-resistant bacteria were lower, suggesting that antibiotic resistance of surviving bacteria increased after activated sludge treatment.

In the algal system, substantial removals of all the five types of ARB were recorded. Specifically, 4 log removal of ampicillin-resistant bacteria was observed, and no ARB harboring ampicillin-resistant bacteria was detected. Removal of erythromycin-, sulfamethoxazole-, ciprofloxacin- and tetracycline-resistant bacteria in the algal system were 2.5 log, 2 log, 1 log and 1 log respectively in batch 1; and 1 log, 2.5 log, 1 log and 1 log, respectively, in batch 2 (Figure 1).

Fig 1.

Fig 1.

Open in a new tab

The concentration of Antibiotic resistance bacteria (ARB) in primary treatment effluent (Primary), secondary treatment effluent (Secondary), algal reactor effluent (Algal).

Compared to the conventional system, higher concentrations of ciprofloxacin- and tetracycline-resistant bacteria were observed in the algal system effluent. However, the algal system can reduce the concentration of erythromycin- and sulfamethoxazole-resistant bacteria in the effluent more effectively. In spite of the low removal of total bacteria (< 1 log) in the algal system, significant ARB reduction was noted, indicating reduced antibiotic resistance in the surviving bacteria in its effluent. Overall, removal of ARB in the algal system was superior to that of the conventional system which may be attributed to low cultivation pH and thermophilic treatment in the algal system.

3.3. Abundance of ARGs in bacteria in conventional system vs. algal system

Eight of the twelve genes (blaTEM, int I1, tetW, qnrA, qnrB, qnrS, sul1, ermB) considered in this study covering five classes of antibiotic resistance were detected in the primary effluent by qPCR, indicating the widespread occurrence of ARGs.

Similar to the decrease of total bacteria number in the conventional system, ARG concentration also decreased after the activated sludge process (Figure 2). Although the total bacterial number in the algal system was higher compared with activated sludge effluent, many ARGs in algal effluent had comparable (blaTEM, qnrA and IntI1), or even lower (tetW) concentration, suggesting the potential for this algal system in ARG control.

Fig 2.

Fig 2.

Open in a new tab

The concentration of ARGs in bacteria fraction in primary treatment effluent (Primary), secondary treatment effluent (Secondary), algal reactor (Algal).

The relative abundance of ARGs in residual bacteria of each treatment process is presented by the ratio of copy numbers of ARGs and 16S rRNA (McCann et al., 2019), which can be used as an indicator of ARG transfer (Figure 3). Increasing trends were observed for relative abundance of most ARGs in the conventional system; compared to the primary effluent, higher relative abundance of qnrA, qnrB, qnrS, sul1 and intI1 gene were observed in its effluent. For tetW, contrarily, the lower relative abundance of the genes occurred in the activated sludge effluent which was 3.04 × 10−3 in batch 1; and 0.108 in batch 2. There was no significant difference in the relative abundance of blaTEM and ermB gene between the primary effluent and the effluent of the conventional system. Even though considerable bacterial reduction occurred along the conventional system, the relative ratio of most ARGs and intI1 in the surviving bacteria increased. A similar pattern was observed in a previous study (Rodriguez-Mozaz et al., 2015), which reported the relative abundance of tetW and ermB gene decreased in activated sludge effluent while qnrS, blaTEM and sul1 gene had a higher relative abundance in the effluent compared to the influent. There are some studies indicating that wastewater treatment systems, especially those that rely on biological processes, may promote the spread of ARGs through horizontal gene transfer (Davies, 2012; Kruse and Sørum, 1994; Mach and Grimes, 1982; Poté et al., 2003). High resistance was observed in the final effluent after all conventional treatment processes (Munir et al., 2011).

Fig 3.

Fig 3.

Open in a new tab

The relative abundance of ARGs in bacteria fraction (normalize the abundance of ARGs with abundance of 16S rRNA) in primary treatment effluent (Primary), secondary treatment effluent (Secondary), algal reactor (Algal).

In both batches of samples from the conventional system, an increase was noted for five of the eight genes (qnrA, qnrB, qnrS, sul1 and IntI1) including all the quinolone resistance genes (Figure 3). Quinolone resistance could be formed by mutation of chromosomal genes (Yoshida et al., 1990), which prevents the drug accumulation in the cell. Plasmid-mediated quinolone resistance have been also discovered, which is usually associated with mobilizing or transposable elements on plasmids (Tran and Jacoby, 2002). Qnr genes investigated in this study (qnrA, qnrB and qnrS) is a series of plasmid-encoded quinolone resistance genes which protect bacteria DNA gyrase and topoisomerase ΙV from the quinolone class of antibiotics. These genes have been found in variety of species, especially C. freundii, K. pneumoniae, S. enterica, and the most common mobile element ISCR (insertion sequences and common regions) which associate with qnrA gene could promote resistance gene expression (Jacoby, 2017). The mobile plasmids with qnr genes may spread in biological treatment processes through conjugation, resulting the high abundance of qnr genes in the effluent of the activated sludge system. Also, qnr genes are found in multi-drug resistance plasmid contained in other resistance genes such as β-lactamase genes (Xu et al., 2019). Sulfonamide resistance gene encodes dihydropteroate synthase as the drug target replacement that are not inhibited by the drug. Sulfonamide resistance gene sul1 is linked to int I1 gene which belongs to class 1 integrons. According to Antunes et al. (2005), 77% of isolates which are resistant to sulfonamides carried class 1 intgrons, and 98% of isolates which contained sul1 gene carried class 1 integrons. All class 1 integrons possess int I1, a gene that encodes a site-specific recombinase (Int I1), responsible for the insertion and excision of exogenous gene cassettes at the integron-associated recombination site, which may be a good indicator of antibiotic resistance acquisition. Class 1 integrons have been reported broadly in Gram negative bacteria which were responsible for the spread and increase of antibiotic resistance all over the world (Canal et al., 2016; Koczura et al., 2013). In this study, the trends for the abundance of sul1 and intl1 gene are similar in the conventional system, which suggests the critical role of horizontal gene transfer in dissemination of ARGs in wastewater treatment processes. Unlike the genes mentioned above, the tetW gene encodes the ribosomal protection protein that results in resistance to tetracycline (Chee-Sanford et al., 2001). In this study, significant removal of tetW gene was observed in activated sludge process, that may suggest low HGT frequency of tetW gene.

In contrast to the increase of relative abundance of ARG in the conventional system, a remarkable reduction of relative abundance of qnrA, tetW, qnrS and int I1 was noted in the algal system, ranging 0.62 to 3 log in batch 1; and 0.5 to 2.1 log in batch 2. Reduction of relative abundance of blaTEM and ermB also was noted in batch 2. The relative abundance of tetW in the algal effluent was one or two orders of magnitude lower than that in the conventional system. The risk of spread of antibiotic resistance is less in the algal system because much lower abundance of int I1 gene was observed than that in the secondary effluent. Also, int I1 gene in algal system was lower than that in the primary effluent which indicates that the algal system could reduce the spread of resistance. The sul1 gene was the only gene showing the increasing relative abundance after algal treatment, which may be harbored by the acidophilic bacteria in algal system.

Some previous reports indicated that the diversity of bacteria community can shape the resistance in WWTPs (Ju et al., 2019). The lower dissemination of antibiotic resistance noted in the algal system may be mainly due to the simpler bacterial community in the algal system compared to the highly diverse community in the conventional system (Delanka-Pedige et al., 2019). Low pH in the algal system resulted in the dominance of Acidobacteria with relative abundance of 98% (Delanka-Pedige et al., 2019). There is only limited study revealing that the antibiotic resistance in Acidobacteria although the prevalence of mobile element-associated genes in Acidobacteria was demonstrated (Bouhajja et al., 2016; Challacombe and Kuske, 2012; Quentmeier and Friedrich, 1994). Conjugal transfer of broad-host range antibiotic resistance plasmids from neutrophilic donors into acidophiles like Thiobacillus acidophilus at a low rate has been described previously (Davidson and Summers, 1983). The wide disparity of pH ranges tolerated by bacteria in the two systems may be the major contributor for their distinct performance in antibiotic resistance control. In our previous study, possible factors affecting fecal coliform removal in algal-based water treatment system were investigated (Munasinghe-Arachchige et al., 2019). Low pH and high temperature in the algal system play a crucial role in fecal coliform removal. Moreover, sunlight combined with oxygen supply has been identified as an important factor in the process (Munasinghe-Arachchige et al., 2019). Those factors also influenced the microbial community structure substantially, thereby altering the abundance of ARGs.

3.4. Abundance of ARGs in bacteriophages in conventional system vs. algal system

While the spread of antibiotic resistance through WWTPs has been widely studied, the role of bacteriophages as potential transmission vehicle has not been addressed. A metagenomic analysis of viromes suggests that functional bacterial genes of all types exist in up to 50% to 60% of bacteriophages; these particles can serve as a reservoir for genes in a diversity of environments and as vehicles for their transfer among bacteria (Dinsdale et al., 2008). Virtually any DNA sequence, including antibiotic resistance, found in a bacterial genome can be transferred. In this study, the ARG concentration in phages was investigated to assess their importance in the spread of antibiotic resistance. Out of those 12 ARGs, five (tetW, qnrA, qnrS, sul1, and intI1) were detected in bacteriophages of water samples with considerable abundance. In the conventional system, highest abundance of qnrS, qnrA, sul1 and int I1 from phages was noted (Figure 4). High concentration of bacteriophages in the activated sludge system may promote the ARG transfer through transduction. It has been reported that the concentration of phages in activated sludge systems range from 4.0 × 107 to 3.0 × 109 PFU/mL (Otawa et al., 2007). A study using metagenomic approaches revealed the presence of ARG-like genes in the virome of activated sludge, which could confer resistance to several antibiotics, including tetracycline, ampicillin, and bleomycin (Parsley et al., 2010). Another study of phage DNA from treated effluents of different hospital and urban wastewaters using qPCR assays showed the presence of high levels of genes conferring resistance to β-lactam antibiotics, as well as genes conferring reduced susceptibility to fluoroquinolones (Marti et al., 2014). It has also been demonstrated that bacteriophages could enable their host to acquire ARGs from neighboring cells in the same environment (Haaber et al., 2016). ARGs from phage DNA were transferred to susceptible E. coli strains, which became resistant to ampicillin (Colomer-Lluch et al., 2011). Moreover, it was demonstrated that subclinical concentrations of antibiotics can promote bacteriophage-mediated horizontal transfer of antibiotic resistance genes in agricultural soil microbiomes (Ross and Topp, 2015). The low concentrations of antibiotics in wastewater could also facilitate the phage-mediated HGT, thereby accelerating the spread of ARGs to environment.

Fig 4.

Fig 4.

Open in a new tab

The abundance of ARGs in bacteriophage fraction in primary treatment effluent (Primary), secondary treatment effluent (Secondary), algal reactor (Algal).

Compared to the conventional process, the algal system decreased the abundance of all ARGs in bacteriophages. This high reduction could be attributed to two reasons. Firstly, the unique condition in algal reactor including low pH (4.0) and high temperature (40–46 ℃) suppressed the gene transfer via transduction. Some studies have explored the effect of low pH and high temperature on removal of ARGs or class 1 integrons that help gene transfer were effective (Gao et al., 2012a; Munir et al., 2011; Sun et al., 2016; Xu et al., 2015). On the other hand, simple bacterial community diversity reduced the possibility of transduction among different bacterial species. Most of phages show a specific host range, such as Phikmvlikevirus genus and Twortlikevirus genus, whereas other phages show a broader host range such as Lambdalikevirus genus. Limited diversity of bacteria hosts likely resulted in few varieties of phage and likely narrow host range in algal reactor, thereby reducing the transduction frequency.

Linear regression analysis was conducted to describe the relationships between in ARG abundance in bacteriophages and relative abundance of ARG in bacteria (Figure 3 and 4). The dotted lines in Figure 5 indicate the 95% confidence interval of the linear regression. Interestingly, the abundance of most resistance genes in bacteriophage (qnrA, qnrS and tetW) showed positive correlation with their relative ratio in bacteria fraction (Figure 5). Especially for qnrS gene, strong positive liner correlations (R2 = 0.93 and 0.94 for batch 1 and 2, respectively) were observed. This phenomenon can imply that transduction contributed to the spread of quinolone resistance genes substantially. For int I1 gene, a linear curve (R2 = 1.00 and 0.84 for batch 1 and 2, respectively) suggests the critical role of phages in HGT of ARGs. IntⅠ1 gene is able to form the gene cassettes with other genes at recombination site, which is important to transduction. Previous study (Anand et al., 2016) has also found int I1 gene in bacteriophage with a considerable amount. The similar trends of intI1, qnrA, qnrS and tetW (Figure 5) gene between bacterial fraction and phage fraction confirmed that bacteriophage could play a significant role in the horizontal transfer of resistance genes in WWTPs. It was noticed that stronger correlation usually occurred when ARGs concentration in bacteriophages was high (Batch 1) (Figure 5). Especially for IntI1 gene (Batch 1), the p-value of intI1 is 0.04 which less than 0.05 indicates strong evidence against the null hypothesis when performing a hypothesis test. Despite the positive correlation observed in this study, further studies with more data are needed to establish the extent to which phages contribute to the spread of ARGs in WWTPs (Calero-Cáceres et al., 2019). For instance, shotgun metagenomics could be a useful tool to identify bacterial origin of ARG detected in bacteriophages. Additionally, lab study by using pure strains could be designed to quantify the spread of ARG through phage transduction.

Fig 5.

Fig 5.

Open in a new tab

The correlations of relative abundance of ARGs in bacteria fraction and abundance of ARGs in bacteriophage fraction.

4. Conclusion

This study demonstrated that the algal-based wastewater treatment system utilizing Galdieria sulphuraria could reduce not only the abundance of ARB but also the relative abundance of ARGs (qnrA, qnrS, tetW) in surviving bacteria. These reductions in the algal system were superior to those in a conventional secondary treatment system. The role of bacteriophages in ARG transfer was also investigated in this study through detection of ARGs in bacteriophages and its correlation with relative ARGs abundance in surviving bacteria. Four of the five detectable genes were significantly reduced in the algal system in bacteriophage, suggesting the low frequency of phage-mediated ARG transfer in algal treatment system. Overall, the results of this study confirmed that the algal wastewater treatment system has merits over the conventional secondary wastewater treatment process in controlling ARGs and ARB.

Supplementary Material

1

Highlights.

  • Algal system reduced ARB and the relative abundance of ARGs in bacteria more

  • Algal system could also reduce detectable ARG abundance in bacteriophages

  • Bacteriophages played a role on horizontal gene transfer of ARGs

  • The phage-mediated ARG transfer decreased in algal treatment system

  • Algal system could minimize the spread of antibiotic resistance to the environment

Acknowledgements

This work was supported in part by the National Institutes of Health Support of Competitive Research (SCORE) Pilot Project Award program (grant number 1SC2GM130432), the National Science Foundation Engineering Research Center for Reinventing the Nation’s Urban Water Infrastructure (ReNUWIt) (award EEC 1028968), College of Engineering at New Mexico State University, Interdisciplinary IMPACT Mini-Grants Program at New Mexico State University, and New Mexico Water Resources Research Institute student grant.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reference

  1. Anand T, Bera BC, Vaid RK, Barua S, Riyesh T, Virmani N, et al. 2016. Abundance of antibiotic resistance genes in environmental bacteriophages. J. Gen. Virol 97, 3458–3466. [DOI] [PubMed] [Google Scholar]
  2. Andersson DI, Hughes D 2010. Antibiotic resistance and its cost: Is it possible to reverse resistance? Nat. Rev. Microbiol 8, 260. [DOI] [PubMed] [Google Scholar]
  3. Andersson DI, Hughes D 2012. Evolution of antibiotic resistance at non-lethal drug concentrations. Drug Resist. Updat 15, 162–172. [DOI] [PubMed] [Google Scholar]
  4. Antunes P, Machado J, Sousa JC, Peixe L 2005. Dissemination of sulfonamide resistance genes (sul1, sul2, and sul3) in portuguese salmonella enterica strains and relation with integrons. Antimicrob. Agents Chemother 49, 836–839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Auerbach EA, Seyfried EE, McMahon KD 2007. Tetracycline resistance genes in activated sludge wastewater treatment plants. Water Res 41, 1143–1151. [DOI] [PubMed] [Google Scholar]
  6. Aw TG, Howe A, Rose JB 2014. Metagenomic approaches for direct and cell culture evaluation of the virological quality of wastewater. Journal of virological methods 210, 15–21. [DOI] [PubMed] [Google Scholar]
  7. Baharoglu Z, Mazel D 2014. Sos, the formidable strategy of bacteria against aggressions. FEMS Microbiol. Rev 38, 1126–1145. [DOI] [PubMed] [Google Scholar]
  8. Balcazar JL 2014. Bacteriophages as vehicles for antibiotic resistance genes in the environment. PLoS Path 10, e1004219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bengtsson-Palme J, Kristiansson E, Larsson DJ 2017. Environmental factors influencing the development and spread of antibiotic resistance. FEMS Microbiol. Rev 42, fux053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bouhajja E, Efthymiopoulos T, George IF, Moreels D, Van Houdt R, Mergeay M, et al. 2016. Conjugative transfer of broad host range plasmids to an acidobacterial strain, edaphobacter aggregans. J. Biotechnol 221, 107–113. [DOI] [PubMed] [Google Scholar]
  11. Calero-Cáceres W, Ye M, Balcázar JL 2019. Bacteriophages as environmental reservoirs of antibiotic resistance. Trends Microbiol 27, 570–577. [DOI] [PubMed] [Google Scholar]
  12. Canal N, Meneghetti KL, Almeida C.P.d., Bastos M.d.R., Otton LM, Corção G 2016. Characterization of the variable region in the class 1 integron of antimicrobial-resistant escherichia coli isolated from surface water. Braz. J. Microbiol 47, 337–344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cantalupo PG, Calgua B, Zhao G, Hundesa A, Wier AD, Katz JP, et al. 2011. Raw sewage harbors diverse viral populations. MBio 2, e00180–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Challacombe J, Kuske C 2012. Mobile genetic elements in the bacterial phylum acidobacteria. Mob. Genet. Elements 2, 179–183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chee-Sanford JC, Aminov RI, Krapac I, Garrigues-Jeanjean N, Mackie RI 2001. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl. Environ. Microbiol 67, 1494–1502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Colomer-Lluch M, Jofre J, Muniesa M 2011. Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples. PLoS One 6, e17549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Da Silva MF, Tiago I, Veríssimo A, Boaventura RA, Nunes OC, Manaia CM 2006. Antibiotic resistance of enterococci and related bacteria in an urban wastewater treatment plant. FEMS Microbiol. Ecol 55, 322–329. [DOI] [PubMed] [Google Scholar]
  18. Davidson M, Summers AO 1983. Wide-host-range plasmids function in the genus thiobacillus. Appl. Environ. Microbiol 46, 565–572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Davies J 2012. Sanitation: Sewage recycles antibiotic resistance. Nature 487, 302. [DOI] [PubMed] [Google Scholar]
  20. Davies J, Spiegelman GB, Yim G 2006. The world of subinhibitory antibiotic concentrations. Curr. Opin. Microbiol 9, 445–453. [DOI] [PubMed] [Google Scholar]
  21. Delanka-Pedige HM, Munasinghe-Arachchige SP, Cornelius J, Henkanatte-Gedera SM, Tchinda D, Zhang Y, et al. 2019. Pathogen reduction in an algal-based wastewater treatment system employing galdieria sulphuraria. Algal Res 39, 101423. [Google Scholar]
  22. Di Cesare A, Eckert EM, D’Urso S, Bertoni R, Gillan DC, Wattiez R, et al. 2016. Co-occurrence of integrase 1, antibiotic and heavy metal resistance genes in municipal wastewater treatment plants. Water Res 94, 208–214. [DOI] [PubMed] [Google Scholar]
  23. Dinsdale EA, Edwards RA, Hall D, Angly F, Breitbart M, Brulc JM, et al. 2008. Functional metagenomic profiling of nine biomes. Nature 452, 629. [DOI] [PubMed] [Google Scholar]
  24. Gao P, Mao D, Luo Y, Wang L, Xu B, Xu L 2012a. Occurrence of sulfonamide and tetracycline-resistant bacteria and resistance genes in aquaculture environment. Water Res 46, 2355–2364. [DOI] [PubMed] [Google Scholar]
  25. Gao P, Munir M, Xagoraraki I 2012b. Correlation of tetracycline and sulfonamide antibiotics with corresponding resistance genes and resistant bacteria in a conventional municipal wastewater treatment plant. Sci. Total Environ 421, 173–183. [DOI] [PubMed] [Google Scholar]
  26. Gross W 2000. Ecophysiology of algae living in highly acidic environments. Hydrobiologia 433, 31–37. [Google Scholar]
  27. Gross W, Schnarrenberger C 1995. Heterotrophic growth of two strains of the acido-thermophilic red alga galdieria sulphuraria. Plant and Cell Physiology 36, 633–638. [Google Scholar]
  28. Haaber J, Leisner JJ, Cohn MT, Catalan-Moreno A, Nielsen JB, Westh H, et al. 2016. Bacterial viruses enable their host to acquire antibiotic resistance genes from neighbouring cells. Nat. Commun 7, 13333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hall RM, Collis CM 1995. Mobile gene cassettes and integrons: Capture and spread of genes by site-specific recombination. Mol. Microbiol 15, 593–600. [DOI] [PubMed] [Google Scholar]
  30. Henkanatte-Gedera S, Selvaratnam T, Caskan N, Nirmalakhandan N, Van Voorhies W, Lammers PJ 2015. Algal-based, single-step treatment of urban wastewaters. Bioresour. Technol 189, 273–278. [DOI] [PubMed] [Google Scholar]
  31. Jacoby GA 2017. Plasmid-mediated quinolone resistance. Antimicrobial drug resistance Springer, pp. 265–268. [Google Scholar]
  32. Ju F, Beck K, Yin X, Maccagnan A, McArdell CS, Singer HP, et al. 2019. Wastewater treatment plant resistomes are shaped by bacterial composition, genetic exchange, and upregulated expression in the effluent microbiomes. ISME J 13, 346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kim S, Yun Z, Ha U-H, Lee S, Park H, Kwon EE, et al. 2014. Transfer of antibiotic resistance plasmids in pure and activated sludge cultures in the presence of environmentally representative micro-contaminant concentrations. Sci. Total Environ 468, 813–820. [DOI] [PubMed] [Google Scholar]
  34. Kim Y, Van Bonn W, Aw TG, Rose JB 2017. Aquarium viromes: Viromes of human-managed aquatic systems. Frontiers in microbiology 8, 1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Koczura R, Mokracka J, Barczak A, Krysiak N, Kaznowski A 2013. Association between the presence of class 1 integrons, virulence genes, and phylogenetic groups of escherichia coli isolates from river water. Microb. Ecol 65, 84–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kruse H, Sørum H 1994. Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments. Appl. Environ. Microbiol 60, 4015–4021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mach P, Grimes D 1982. R-plasmid transfer in a wastewater treatment plant. Appl. Environ. Microbiol 44, 1395–1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Marano RB, Cytryn E 2017. The mobile resistome in wastewater treatment facilities and downstream environments. Antimicrobial resistance in wastewater treatment processes John Wiley & Sons Inc, pp. 129. [Google Scholar]
  39. Marti E, Variatza E, Balcázar J 2014. Bacteriophages as a reservoir of extended-spectrum β-lactamase and fluoroquinolone resistance genes in the environment. Clin. Microbiol. Infect 20. [DOI] [PubMed] [Google Scholar]
  40. McCann CM, Christgen B, Roberts JA, Su J-Q, Arnold KE, Gray ND, et al. 2019. Understanding drivers of antibiotic resistance genes in high arctic soil ecosystems. Environ. Int 125, 497–504. [DOI] [PubMed] [Google Scholar]
  41. McMinn BR, Ashbolt NJ, Korajkic A 2017. Bacteriophages as indicators of faecal pollution and enteric virus removal. Lett. Appl. Microbiol 65, 11–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Munasinghe-Arachchige SP, Delanka-Pedige HMK, Henkanatte-Gedera SM, Tchinda D, Zhang Y, Nirmalakhandan N 2019. Factors contributing to bacteria inactivation in the galdieria sulphuraria-based wastewater treatment system. Algal Res 38, 101392. [Google Scholar]
  43. Munir M, Wong K, Xagoraraki I 2011. Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in michigan. Water research 45, 681–693. [DOI] [PubMed] [Google Scholar]
  44. Nwosu VC 2001. Antibiotic resistance with particular reference to soil microorganisms. Res. Microbiol 152, 421–430. [DOI] [PubMed] [Google Scholar]
  45. O’Brien E, Munir M, Marsh T, Heran M, Lesage G, Tarabara VV, et al. 2017. Diversity of DNA viruses in effluents of membrane bioreactors in traverse city, mi (USA) and la grande motte (france). Water research 111, 338–345. [DOI] [PubMed] [Google Scholar]
  46. Otawa K, Lee SH, Yamazoe A, Onuki M, Satoh H, Mino T 2007. Abundance, diversity, and dynamics of viruses on microorganisms in activated sludge processes. Microb. Ecol 53, 143–152. [DOI] [PubMed] [Google Scholar]
  47. Parsley LC, Consuegra EJ, Kakirde KS, Land AM, Harper WF, Liles MR 2010. Identification of diverse antimicrobial resistance determinants carried on bacterial, plasmid, or viral metagenomes from an activated sludge microbial assemblage. Appl. Environ. Microbiol 76, 3753–3757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Poté J, Ceccherini MT, Rosselli W, Wildi W, Simonet P, Vogel TM 2003. Fate and transport of antibiotic resistance genes in saturated soil columns. Eur. J. Soil Biol 39, 65–71. [Google Scholar]
  49. Quentmeier A, Friedrich CG 1994. Transfer and expression of degradative and antibiotic resistance plasmids in acidophilic bacteria. Appl. Environ. Microbiol 60, 973–978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rizzo L, Manaia C, Merlin C, Schwartz T, Dagot C, Ploy M, et al. 2013. Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: A review. Science of the total environment 447, 345–360. [DOI] [PubMed] [Google Scholar]
  51. Rodriguez-Mozaz S, Chamorro S, Marti E, Huerta B, Gros M, Sànchez-Melsió A, et al. 2015. Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river. Water Res 69, 234–242. [DOI] [PubMed] [Google Scholar]
  52. Ross J, Topp E 2015. Abundance of antibiotic resistance genes in bacteriophage following soil fertilization with dairy manure or municipal biosolids, and evidence for potential transduction. Appl. Environ. Microbiol., AEM 02363-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schmieder R, Edwards R 2012. Insights into antibiotic resistance through metagenomic approaches. Future Microbiol 7, 73–89. [DOI] [PubMed] [Google Scholar]
  54. Sims A, Gajaraj S, Hu Z 2012. Seasonal population changes of ammonia-oxidizing organisms and their relationship to water quality in a constructed wetland. Ecological Engineering 40, 100–107. [Google Scholar]
  55. Sun W, Qian X, Gu J, Wang X-J, Duan M-L 2016. Mechanism and effect of temperature on variations in antibiotic resistance genes during anaerobic digestion of dairy manure. Sci. Rep 6, 30237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tchinda D, Henkanatte-Gedera S, Abeysiriwardana-Arachchige I, Delanka-Pedige H, Munasinghe-Arachchige S, Zhang Y, et al. 2019. Single-step treatment of primary effluent by galdieria sulphuraria: Removal of biochemical oxygen demand, nutrients, and pathogens. Algal Res 42, 101578. [Google Scholar]
  57. Touchon M, de Sousa JAM, Rocha EP 2017. Embracing the enemy: The diversification of microbial gene repertoires by phage-mediated horizontal gene transfer. Curr. Opin. Microbiol 38, 66–73. [DOI] [PubMed] [Google Scholar]
  58. Tran JH, Jacoby GA 2002. Mechanism of plasmid-mediated quinolone resistance. Proc. Natl. Acad. Sci 99, 5638–5642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Xu H, Huo C, Sun Y, Zhou Y, Xiong Y, Zhao Z, et al. 2019. Emergence and molecular characterization of multidrug-resistant klebsiella pneumoniae isolates harboring blactx-m-15 extended-spectrum β-lactamases causing ventilator-associated pneumonia in china. Infection and drug resistance 12, 33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Xu J, Xu Y, Wang H, Guo C, Qiu H, He Y, et al. 2015. Occurrence of antibiotics and antibiotic resistance genes in a sewage treatment plant and its effluent-receiving river. Chemosphere 119, 1379–1385. [DOI] [PubMed] [Google Scholar]
  61. Yahya M, Hmaied F, Jebri S, Jofre J, Hamdi M 2015. Bacteriophages as indicators of human and animal faecal contamination in raw and treated wastewaters from t unisia. J. Appl. Microbiol 118, 1217–1225. [DOI] [PubMed] [Google Scholar]
  62. Yang Y, Shi W, Lu S-Y, Liu J, Liang H, Yang Y, et al. 2018. Prevalence of antibiotic resistance genes in bacteriophage DNA fraction from funan river water in sichuan, china. Sci. Total Environ 626, 835–841. [DOI] [PubMed] [Google Scholar]
  63. Yoshida H, Bogaki M, Nakamura M, Nakamura S 1990. Quinolone resistance-determining region in the DNA gyrase gyra gene of escherichia coli. Antimicrob. Agents Chemother 34, 1271–1272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhang Y, Riley LK, Lin M, Purdy GA, Hu Z 2013. Development of a virus concentration method using lanthanum-based chemical flocculation coupled with modified membrane filtration procedures. J. Virol. Methods 190, 41–48. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1545909-supplement-1.docx (267.6KB, docx)

RESOURCES