Antibiotic Resistance Genes Dynamics at the Different Stages of the Biological Process in a Full-Scale Wastewater Treatment Plant

Wastewater treatment plants (WWTPs) highly contribute to the transmission of antibiotic resistance genes (ARGs) in the environment. In this work, the diversity of ermF, ermB, sul1 and int1-enconding genes was examined in the influent, the mixed liquor and the effluent of a full-scale WWTP. Based on the clones analyzed, similar genotypes were recorded at all process stages. However, distinct genotypes of int1 were responsible for the expression of sul1 and ermF genes in Gammaproteobacteria and Bacteroidetes, respectively. Due to the detection of similar ARGs profiles throughout the biological process, it is concluded that additional treatment is needed for their retention.


Introduction
The main source of antibiotics in the environment is the excretion of incompletely metabolized antibiotics by humans and animals. Thus, WWTPs are important reservoirs of antibiotic resistant bacteria, transmitting them into the aquatic ecosystems [1].
There are several studies examining the effectiveness of the different stages of the treating processes at reducing the concentrations of various antibiotics. Lin et al. [2] determined the effectiveness of primary and secondary treatment process, which was followed by a disinfection step, on the reduction of sulfonamides, cephalosporins, quinolones and macrolides. Batt et al. [3] studied the fate of four different antibiotics belonging to quinolones, sulfonamides and tetracyclines, during their treatment in four WWTPs, which differed in design and operating conditions. Moreover, Li and Zhang [4] and Watkinson et al. [5] recorded concentration removals within 25 and 80% during treatment of antibiotics in conventional treatment plants.
Despite that WWTPs are suitable for reducing the concentrations of pollutants in municipal wastewater, they appear to be insufficient in reducing antibiotic gene content [6]. By contrast, the activated sludge of municipal wastewater treatment plants is an ideal environment for the proliferation of antibiotic resistance genes since ARGs transmission can occur within the diverse microbial species of the flocs [7,8]. For instance, tetracycline, sulfonamide, macrolide and quinolone resistance genes (tet, sul, erm, and qnr, respectively) have been detected in the treated effluent of WWTPs [9]. Moreover, Chen and Zhang [10] detected low number of copies of the sulfonamide antibiotic resistance genes sul1 and sul2, and the integrase 1 gene (int1) in the effluent of certain WWTPs. The same authors reported a strong correlation within the amount of sul1 and int1 genes, indicating the involvement of integrase in sul1 gene transmission [10].
Thus, the current study was conducted to investigate the distribution of antibiotic resistance genes (ARGs) in a sewage treatment plant, based on the molecular identification of three antibiotic resistance genes, i.e., sul1, ermB and ermF, and the integrase 1 gene (int1), which favors the spread of multiple genes to the microbial constituents of the activated sludge.

Materials and Methods
Samples from the influent, the mixed liquor and the effluent of WWTP were obtained by using sterile glass bottles. Genomic DNA extraction was conducted based on commercially available kit (Vivantis, Selangor Darul Ehsan, Malaysia). The macrolide (i.e., ermB and ermF) and the sulfonamide (i.e., sul1) resistance genes as well as the class 1 integron integrase (int1) genes were amplified by the use of the primer pairs erm(B)-454rc/erm(B)-91fc, erm(F)-189f/erm(F)-497r, sul1-F/sul1-B and int1-F/int1-R, respectively [11,12]. The amplification reaction was carried out in a TaKaRa Dice TP600 PCR thermocycler (Japan). The amplicons obtained were ligated into the bacterial vector pGEM-T Easy (Promega, Madison, WI, USA), and the recombinant DNA were transformed into DH5a competent cells. The plasmid DNA was extracted by the Vivantis plasmid kit (Malaysia) from the recombinant Escherichia coli cultures, and sequencing of the PCR inserts was performed at Macrogen using primers SP6 and T7 (Promega). The closest relatives of the amplicons obtained were subjected to BLAST searches. The amplified gene sequences and their closest relatives were translated to amino-acids by Emboss Transeq [13] and aligned by the Clustal Omega bioinformative tool [14].

Results and Discussion
A total of twelve clone libraries were constructed, each one for any antibiotic resistance gene and sampling point examined, i.e., the influent, the mixed liquor and the effluent of the full-scale WWTP. In the case of macrolides, the screening of ermB gene clone libraries resulted in the identification of three distinct genotypes. The major genotype consisting of 19 clones, which were identical to known sequences deposited in the International Sequencing database, while the second genotype comprised of 3 clones. Interestingly, the last genotype consisted of a single clone, which was only detected in the effluent of WWTP. On the other hand, the two major genotypes were detected throughout the whole biological treatment, a fact that indicates dispersion of this gene throughout the processing. The major ermB gene genotype showed high genetic relationship with respective genes carried out by strains of the genera Streptococcus, Nocardia, Staphylococcus, Clostridium, Lactococcus and Listeria (Table 1). Based on protein level analysis, the ermB genes detected were responsible for encoding a protein consisting of 107 amino acids, where similar gene translation patterns were identified. In comparison to other studies, Lee et al. [15] detected ermB genes in the effluent of two WWTPs, even after UV application as the disinfection method. Moreover, Wang et al. [6] showed the ineffectiveness of WWTPs in the removal of antimicrobial resistant genes, which were prevailing in all samples analyzed.
Regarding the detection of ermF genes in the WWTP examined, three distinct clone groups were identified, with the major clone cluster being consisted of 17 members. The ermF genes of the three genotypes were differentiated up to three nucleotide bases. It was also observed that the second group of ermF gene clones showed a close genetic relationship with the members of the first group and therefore, it could be considered as a subgroup of the dominant genotype. This led us to the conclusion that the dominant genotype of the ermF gene was detected throughout the biological treatment process. Although the third genotype consisted only of 3 clones, such genetic pattern was detected throughout the whole biological process. At protein level, only two distinct amino-acid patterns were identified as the result of the close relationship within the members of the first and the second genotype. Almost all microorganisms carrying the ermF gene showed high genetic relation with respective genes detected only in members of the phylum Bacteroidetes, except of the gammaproteobacterium Bibersteinia trehalosi (Table 2). In comparison to other studies, Fahrenfeld et al. [16] detected ermF genes in the reclaimed water derived from a WWTP. Based on metagenomic analysis, Szczepanowski et al. [17] reported the prevalence of ermF gene in both activated sludge and the treated effluent of a WWTP. Regarding sulfonamides resistance genes, there was a dominant cluster consisting of 23 over 24 clones analyzed (carrying the sul1 gene). The single representative of the second genotype possessed a distinct genetic position as compared to the dominant genotype, where genetic distance was greater than 4 base pairs. However, at protein level, its amino-acid sequence was similar with the major sul1 cluster. Most of the microorganisms, which carried the sul1 gene, exhibited high genetic similarity with sul1 genes detected in strains of the class Gammaproteobacteria (Table 3). In the studies of Ben et al. [7] and Du et al. [18], sul1 genes were the most abundant gene among the ARGs examined in various WWTPs. Moreover, Lupan et al. [19] confirmed the spread of sul1 genes in a river 10 km downstream of a WWTP.  Considering the genetic analysis of integrase class 1 gene, a prevalent clone cluster was detected, which included 25 out of 26 clones analyzed. Nevertheless, at protein level, these integrase class 1 clusters differed by a single amino acid. Interestingly, the microbial species of the major genotype carrying the int1 gene were related to those carrying sul1 genes. On the other hand, the bacterial strain possessing the int1 gene of the minor cluster was associated with microbiota carrying ermF genes (Table 4). A strong correlation between the concentrations of sul1 and int1 genes have been recently reported [6,7,18], which demonstrates the importance of integrons in the spread of these antibiotic resistance genes in the environment, indicating that sul1 gene is frequently located on class 1 integron.

Conclusions
Based on the molecular determination of sul1, ermB, ermF and int1 genes, it is concluded that antibiotic resistant genes spread occurred throughout the biologic process in WWTPs. The similar genotyping profiles detected in the influent and the effluent point out the necessity for the implementation of effective tertiary treatment methods, regarding gene removal from the treated effluent. Further experiments on the use of advanced oxidation systems and membrane technologies will elucidate issues regarding the removal of antibiotic resistance genes. Moreover, different class 1 integrons appeared to be responsible for the transmission of sul1 and ermF genes among members of distinct phyla, a fact that indicates a microbial specificity in antibiotic resistant gene transmission.