The results of water quality tests indicated different pollution levels between the upstream and the downstream. It showed different variation patterns of physicochemical and bacteriological parameters along the watercourse. According to the PCA results, we found different degrees of pollution among the sampling sites.
Physicochemical parameters
Overall there was an increasing trend of temperature, conductivity, TDS, salinity and BOD values in the downstream. While pH and DO values went in an opposite trend. Temperature values gradually increased at downstream, it ranged from 20°C to 25°C in the upstream and from 25°C to 30°C in the downstream. For conductivity, TDS, salinity and BOD, the mean values slightly varied in the upstream from site 1 to site 6 (conduct. = 244.00 ─ 362.00 μS cm-1, TDS = 120.20 ─ 174.00 mg L-1, Sal. = 128.67 ─ 210.33 mg L-1, BOD = 0.20 ─ 0.33 mg L-1). For each of these values, there was a substantial increase in the downstream from site 7 to site 9 (conduct. =
557.33 ─ 758.33 μS cm-1, TDS = 303.33 ─ 419.33 mg L-1, Sal. = 381.67 ─ 519.00 mg L-1, BOD = 0.97 ─ 1.27 mg L-1). pH values were > 8.00 in the upstream and < 8.00 in the downstream. The DO were > 8.00 mg L-1 in the upstream and < 8.00 mg L-1 in the downstream (Table 1.1).
Bacteriological parameters
A total of 5 bacterial species were predominantly isolated and from samples inoculated on nutrient agar: Bacillus subtilis (B. subtilis), Bacillus megaterium (B. megaterium), Bacillus cereus (B. cereus), Acinetobacter sp. and Serratia marcescens (S. marcescens); Enterobacteriaceae including Escherichia coli (E. coli), Klebsiella pneumonia (K. pneumonia), Enterobacter sp. and S. marcescens were isolated on MacConkey agar; Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) were isolated on Mannitol salt agar. Bacillus species, Acinetobacter sp. and S. marcescens began to occur at site 1, E. coli and K. pneumonia and S. aureus at site 3, S. epidermidis at site 4 and Enterobacter sp. at site 5. For abundance analysis, we calculated total viable counts (TVC), total Enterobacteriaceae (TE), total coliforms (TC), E. coli (EC), total Staphylococcus (TS), Acinetobacter sp. (AB) and total Bacillus (TB). As for representation analysis, we calculated the proportion of total Enterobacteriaceae (TEP), total coliforms (TCP), E. coli (ECP), total Staphylococcus (TSP), Acinetobacter sp. (ABP) and total Bacillus (TBP) (Table 1.2).
We present here the abundances of different bacteria or bacterial groups at different sampling sites. There was an increasing trend of CFU numbers for TVC, TE, TC and each of these bacteria in the downstream. Compared with the site where the bacteria or bacterial groups began to occur in the upstream (TVC = 4.35 × 104 CFU mL-1, TE = 4.48 × 103 CFU mL-1, TC = 3.46 × 103 CFU mL-1, EC = 2.45 × 103 CFU mL-1, TS = 2.45 × 103 CFU mL-1), the mean values slightly increased at site 6 (TVC = 7.82 × 104 CFU mL-1, TE = 1.37 × 104 CFU mL-1, TC = 7.11 × 103 CFU mL-1, EC = 3.11 × 103 CFU mL-1, TS = 5.65 × 103 CFU mL-1). For TVC, TE, TC, EC and TS, there were substantial increases of the values from site 6 to site 7 (TVC = 1.66 × 105 CFU mL-1, TE = 3.06 × 104 CFU mL-1, TC = 1.73 × 104 CFU mL-1, EC = 8.76 × 103 CFU mL-1, TS = 9.38 × 103 CFU mL-1), and from site 7 to site 8 (TVC = 3.27 × 105 CFU mL-1, TE = 6.46 × 104 CFU mL-1, TC = 3.89 × 104 CFU mL-1, EC = 1.75 × 104 CFU mL-1, TS = 2.53 × 104 CFU mL-1). At site 9, the values were close to site 8 (TVC = 3.27 × 105 CFU mL-1, TE = 6.32 × 104 CFU mL-1, TC = 3.87 × 104 CFU mL-1, EC = 1.82 × 104 CFU mL-1, TS = 2.65 × 104 CFU mL-1). The AB and TB increase gradually. AB ranged from 8.08 × 103 CFU mL-1 to 1.12 × 104 CFU mL-1 in the upstream and from 1.67 × 104 CFU mL-1 to 2.63 × 104 CFU mL-1 in the downstream. TB ranged from 1.88 × 104 CFU mL-1 to 3.05 × 104 CFU mL-1 in the upstream and from 4.61 × 104 CFU mL-1 to 7.04 × 104 CFU mL-1 in the downstream. For representations of different bacteria or bacterial groups at different
sampling sites, TBP maintained the highest value of the community along the river. Compared with site 1 (ABP = 18.59 %, TBP = 43.25 %), the mean values of ABP and TBP slightly decreased at site 6 (ABP = 14.25 %, TBP = 38.91 %). There was a substantial decrease of ABP and TBP from site 6 to site 7 (ABP = 10.06 %, TBP = 27.76 %) and from site 7 to site 8 (ABP = 7.28 %, TBP = 21.12 %). At site 9, the value was close to site 8 (ABP = 8.04 %, TBP = 21.54 %). Compared with the site where the bacteria or bacterial groups began to occur in the upstream (TEP = 10.31 % ─ 17.52 %, TCP = 5.68 % ─ 9.09 %, ECP = 3.97 % ─ 4.43 %, TSP = 4.43 % ─ 7.22 %), TEP, TCP, ECP, and TSP overall maintained the values in the downstream (TEP = 18.46 % ─ 19.73 %, TCP = 10.42 % ─ 11.88 %, ECP = 5.28 % ─ 5.55 %, TSP = 5.65 % ─ 8.11 %).
Principal components analysis
River water was characterized by 7 physicochemical parameters and 13 bacteriological parameters. The PCA analysis showed that of the 20 components, the first principal components accounted for 99.21%, while the second, third, fourth and fifth principal components accounted for 0.69%, 0.05%, and 3.83%, respectively (Table S4). Here we present a scatter plot consisting of PC1 and PC2 (Fig. 2). It demonstrates two clusters, one accommodates the samples which are from the upstream (Upstream cluster), while the other comprises those from the downstream (Downstream cluster). The two clusters differ in dispersion. The distribution of the 3 samples in the Downstream cluster are more dispersed than those in the Upstream cluster. This finding might imply that the pollution scenarios are similar among the sampling sites in the upstream but varied among the 3 sites downstream. Thus, for the following antibacterial resistance tests, we decided to make a comparison among 4 sites: a site where the isolate began to occur at the upstream of Qishan river, compared to the following downstream sites: Qishan town (site 7), WWTP (site 8) and Kaoping river (site 9).
Antibacterial resistance tests
Resistance level could be indicated by zone diameter in disk diffusion and MIC in micro-dilution. For each antibacterial activity on bacteria, resistance level that significantly increased and reached the breakpoints of above intermediate resistance were considered as increased resistance (Table 3). The results showed variable patterns of antibacterial resistance levels among sampling sites along the watercourse. Most increased resistance occurred at site 8 and site 9. Overall, the two methods showed that there were mainly 5-6 types of antibacterials showing increased resistance with bacteria in the downstream.
Disk diffusion
Fig. 3 shows the results of disk diffusion. All comparisons were made between a site where the isolate began to occur in upstream and sites in downstream. For E. coli, by comparison with site 3, increased resistances were found with ampicillin (p < 0.0001), chloramphenicol (p < 0.0001), ciprofloxacin (p < 0.0001) and tetracycline (p < 0.0001). Site 7 showed smaller inhibition zones with chloramphenicol. Both sites 8 and 9 showed smaller inhibition zones with ampicillin, chloramphenicol, ciprofloxacin and tetracycline (Fig. 3A). For K. pneumonia, by comparison with site 3, increased resistances were found with ampicillin (p < 0.0001), chloramphenicol (p < 0.0001), ciprofloxacin (p < 0.0001), and tetracycline (p < 0.0001). Site 7 showed smaller inhibition zones with chloramphenicol. Site 8 showed smaller inhibition zones with chloramphenicol, ciprofloxacin and tetracycline. Site 9 showed smaller inhibition zones with ampicillin, chloramphenicol, ciprofloxacin, and tetracycline (Fig. 3B). For Enterobacter sp., by comparison with site 5, increased resistances were found with ampicillin (p < 0.0001), chloramphenicol (p < 0.0001), ciprofloxacin (p < 0.0001) and tetracycline (p < 0.0001). Both sites 7 and 8 showed smaller inhibition zones with chloramphenicol and tetracycline. Site 9 showed smaller inhibition zones with ampicillin, chloramphenicol, ciprofloxacin and tetracycline (Fig. 3C). For S. marcescens, by comparison with site 1, increased resistances were found with ampicillin (p < 0.0001), ciprofloxacin (p < 0.0001), trimethoprim/ sulfamethoxazole (p < 0.0001), and tetracycline (p = 0.0002). Site 7 showed smaller inhibition zones with tetracycline. Site 8 showed smaller inhibition zones with tetracycline. Site 9 showed smaller inhibition zones with ampicillin, ciprofloxacin, trimethoprim/ sulfamethoxazole and tetracycline (Fig. 3D). The zone diameter breakpoints of Enterobacteriaceae were recommended by CLSI (CLSI 2018) (Table S7.1).
For Acinetobacter sp., by comparison with site 1, increased resistance was found with ciprofloxacin (p < 0.0001) and tetracycline (p < 0.0001). Both sites 7 and 8 showed smaller inhibition zones with tetracycline. Site 9 showed smaller inhibition zones with ciprofloxacin and tetracycline (Fig. 3E). The zone diameter breakpoints of Acinetobacter spp. were recommended by CLSI (CLSI 2018) (Table S7.2).
For S. aureus, by comparison with site 3, increased resistances were found with chloramphenicol (p < 0.0001), erythromycin (p < 0.0001) and tetracycline (p < 0.0001). Site 7 showed smaller inhibition zones with chloramphenicol. Both sites 8 and 9 showed smaller inhibition zones with chloramphenicol, erythromycin and tetracycline (Fig. 3F). For S. epidermidis, by comparison with site 4, the increased resistances were found with chloramphenicol (p < 0.0001), erythromycin (p < 0.0001) and tetracycline (p < 0.0001). Site 7 showed smaller inhibition zones with chloramphenicol. Both sites 8 and 9 showed smaller inhibition zones with chloramphenicol, erythromycin and tetracycline (Fig. 3G). The zone diameter breakpoints of Staphylococcus spp. were recommended by CLSI (CLSI 2018) (Table S7.3).
For B. megatium, by comparison with site 1, the increased resistances were found with ciprofloxacin (p = 0.0001), erythromycin (p = 0.0008) and tetracycline (p = 0.0056). Both sites 7 and 8 showed smaller inhibition zones with erythromycin. Site 9 showed smaller inhibition zones with ciprofloxacin, erythromycin and tetracycline (Fig. 3H). For B. cereus, by comparison with site 1, the increased resistances were found with ciprofloxacin (p = 0.0001), erythromycin (p = 0.0147) and tetracycline (p < 0.0001). Site 8 showed smaller inhibition zones with erythromycin and tetracycline, site 9 showed smaller inhibition zones with ciprofloxacin, erythromycin and tetracycline (Fig. 3I). For B. subtilis, by comparison with site 1, the increased resistances were found with erythromycin (p < 0.0001) and tetracycline (p < 0.0001). Both sites 8 and 9 showed smaller inhibition zones with erythromycin and tetracycline (Fig. 3J). The zone diameter breakpoints of Bacillus spp. were recommended by CLSI (CLSI 2018) (Table S7.4).
Micro-dilution
Fig. 4 shows the results of micro-dilution. All comparisons were made between a site where the isolate began to occur in upstream and sites in downstream. For E. coli, by comparison with site 3, increased resistances were found with ampicillin (p < 0.0001), chloramphenicol (p = 0.0001) and tetracycline (p < 0.0001). Site 7 showed higher MICs with chloramphenicol and tetracycline. Both sites 8 and 9 showed higher MICs with ampicillin, chloramphenicol and tetracycline (Fig. 4A). For K. pneumoniae, by comparison with site 3, increased resistances were found with ampicillin (p = 0.0001), chloramphenicol (p = 0.0012), ciprofloxacin (p = 0.0118) and tetracycline (p = 0.0001). Site 8 showed higher MICs with chloramphenicol and tetracycline. Site 9 showed higher MICs with ampicillin, chloramphenicol, ciprofloxacin and tetracycline (Fig. 4B). For Enterobacter sp., by comparison with site 5, increased resistances were found with ampicillin (p < 0.0001), chloramphenicol (p < 0.0001), ciprofloxacin (p < 0.0001) and tetracycline (p < 0.0001). Site 8 showed higher MICs with ampicillin, chloramphenicol and tetracycline. Site 9 showed higher MICs with ampicillin, chloramphenicol, ciprofloxacin and tetracycline (Fig. 4C). For S. marcescens, by comparison with site 1, increased resistances were found with chloramphenicol, ciprofloxacin and tetracycline. Site 7 showed higher MICs with tetracycline. Site 8 showed higher MICs with chloramphenicol and tetracycline. Site 9 showed higher MICs with chloramphenicol (p < 0.0001), ciprofloxacin (p < 0.0001) and tetracycline (p = 0.0054) (Fig. 4D). The MIC breakpoints of Enterobacteriaceae were recommended by CLSI (CLSI 2018) (Table S7.1).
For Acinetobacter sp., by comparison with site 1, the increased resistance was found with tetracycline. Both sites 8 and 9 showed higher MICs with tetracycline (p < 0.0001) (Fig. 4E). The MIC breakpoints of Acinetobacter spp. were recommended by CLSI (CLSI 2018) (Table S7.2).
For S. aureus, by comparison with site 3, the increased resistances were found with chloramphenicol (p < 0.0001), erythromycin (p < 0.0001), tetracycline (p < 0.0001) and vancomycin (p < 0.0001). Site 7 showed higher MICs with chloramphenicol, erythromycin and tetracycline. Both sites 8 and 9 showed higher MICs with chloramphenicol, erythromycin, tetracycline and vancomycin (Fig. 4F). For S. epidermidis, by comparison with site 4, increased resistances were found with chloramphenicol (p < 0.0001), erythromycin (p = 0.0006), tetracycline (p < 0.0001) and vancomycin (p < 0.0001). Site 7 showed higher MICs with chloramphenicol and erythromycin. Both site 8 and site 9 showed higher MICs with chloramphenicol, erythromycin, tetracycline and vancomycin (Fig. 4G). The MIC breakpoints of Staphylococcus spp. were recommended by CLSI (CLSI 2018) (Table S7.3).
For B. megatium, by comparison with site 1, the increased resistances were found with erythromycin and tetracycline. Both site 8 and site 9 showed higher MICs with erythromycin (p = 0.0012) and tetracycline (p < 0.0001) (Fig. 4H). For B. cereus, by comparison with site 1, the increased resistances were found with erythromycin (p = 0.0013). Sites 7, 8 and 9 showed higher MICs with erythromycin (Fig. 4I). For B. subtilis, by comparison with site 1, the increased resistances were found with erythromycin (p < 0.0001). Both sites 8 and 9 showed higher MICs with erythromycin (Fig. 4J). The MIC breakpoints of Bacillus spp. were recommended by CLSI (CLSI 2018) (Table S7.4).