## 3 Results and Discussion
### Genetic Location of ARGs  
The genetic location of ARGs in the influent, activated sludge, and effluent of HX WWTP was variable (Figure 4A). The influent contained 42.2% of ACCs from chromosome sequences, and only 22.6 % were plasmid sequences (Figure 4A). This differed from the genetic location of ARGs in the influent of Hong Kong WWTPs, with 30.6% from chromosomes and 55.3% of ACCs derived from plasmids and ICEs [48], and may be related to the diverse antibiotic pressure on ARGs in different regions. Antibiotic selection pressure significantly increased the abundance and proportion of plasmid-related ARGs [18]. The  
\begin{table}
\begin{tabular}{c c c} \hline
**ARP** & **ACCs** & **Carrying ARG Subtypes** \\ \hline _Escherichia coli_ & 39 & aminoglyoxide: _aph_(3\({}^{\prime\prime}\))-I, _aph_(6)-I, _ac_(3)-II; bacitracin: _lacA_; beta-lactam: _TEM_-1; fosmidomycin: _rosA_, _rosB; kasugamycin: ksgA; \\  & & MLS: _macA_; multidrug: _arcA_, _orB_, _ber_, _emx_, _emx_, _emx_, _emx_, _emx_, _emx_, _emx_, _emx_, _emx_, _emx_, _emx_, _emx_, _emx_, _mdf_, \(A\), _mdf_,  
proportion of chromosomal ACCs and plasmid ACCs gradually decreased and increased, respectively, in activated sludge and effluent. The effluent had the highest proportion of plasmid ACCs (58.3%), with 8.3% of chromosomal ACCs (Figure 4A). This indicated that plasmid-related ARGs may be mobile and HGT occurred in the activated sludge. Moreover, plasmid-related ARGs are approximately two-fold more likely to be transcriptionally expressed than chromosome-related ARGs in Taiwan WWTPs [60]. Bacitracin and multidrug resistance genes were mostly carried by chromosomes, while MLS, vancomycin, sulfonamide, beta-lactam, tetracycline, chloramphenicol, and aminoglycoside resistance genes were mainly carried by plasmids (Figure 4B). This agrees with previous work showing that multidrug resistance genes are primarily located on chromosomes [18,19] and the transcript abundance of aminoglycoside, sulfonamide, and tetracycline resistance genes are mainly contributed by plasmid-related ARGs [60]. Bactracin resistance genes tend to be chromosomally coded, probably because bacitracin resistance genes are inherent in bacteria [11]. The ARG types carried by plasmids are closely linked to commonly used antibiotics in humans: aminoglycoside, beta-lactam, MLS, sulfonamide, and tetracycline. The vast majority of ACCs carry only one ARG with only 9% of ACCs carrying at least two ARGs (Figure S6). There was a tendency for multiple ARGs to be located on the same contig in this study, which was previously suggested to be driven by the same resistance mechanism [10]. For example, the multidrug efflux protein combination _adc_-_adel_-_ade_K and multidrug transporter _md_I-_md_C was observed in the influent (Table S2). Similarly, fosindomycin resistance (_ros_A-_ros_B) and MLS resistance (_macA-_mac_B) were observed, amongst others (Table S2). _macB_ and _macA_ gene products form an ATP-binding cassette (ABC) transporter, which is ubiquitous in chicken manure, pig manure, influent, and human intestinal samples [52]. In activated sludge and effluent, most of the ARG combinations were composed of different ARG types, such as the chloramphenicol, sulfonamide, and tetracycline resistance gene combination (_flo_R-_sull_-_tet_G; Table S4). The different ARGs co-located on the same contig enhance the spread of multidrug resistance. The co-occurrence mechanism of ARG subtypes is common in various environments such as _tet_A-_aph_(3_)-VID-_aph_(3_)-Ib-_aph_(6)-Id from a WWTP [61], _sull_-_cat_-_a_(6_)-Ib from pKp368/10  
Figure 4: Genetic location of ARGs in HX WWTP. (**A**) The percentage of ACCs belonged to plasmid, chromosomal and unclassified sequences in the influent, activated sludge, and effluent. (**B**) Genetic location and frequency of ARGs were carried by chromosomes or plasmids summarized by ARG type. plasmid [62], _aad_A5-_dfr_A17 of chicken feces [52], and _aad_A-_aac_A4-_bla_OxA-10 from a gene cassette [63].