Shu-Min Shen

Evaluation of five antibiotic resistance genes in wastewater treatment systems of swine farms by real-time PCR

Antibiotics are widely used in livestock for infection treatment and growth promotion. Wastes from animal husbandry are a potential environmental source of antibiotic-insensitive pathogens, and the removal efficiency of the resistance genotypes in current wastewater treatment plants (WWTPs) is unknown. In this study, quantitative PCR was used for evaluating antibiotic resistance genes in wastewater treatment processes. Six wastewater treatment plants in different swine farms were included in this study, and five antibiotic resistance genes (ARGs) were tested for each treatment procedure. All of the tested ARGs including tetA, tetW, sulI, sulII, and blaTEM genes were detected in six swine farms with considerable amounts. The results showed that antibiotic resistance is prevalent in livestock farming. The ARG levels were varied by wastewater treatment procedure, frequently with the highest level at anaerobic treatment tank and lowest in the activated sludge unit and the effluents. After normalizing the ARG levels to 16S rRNA gene copies, the results showed that ARGs in WWTP units fluctuated partly with the quantity of bacteria. Regardless of its importance in biodegradation, the anaerobic procedure may facilitate bacterial growth thus increasing the sustainability of the antibiotic resistance genotypes. After comparing the copy numbers in influx and efflux samples, the mean removal efficiency of ARGs ranged between 33.30 and 97.56%. The results suggested that treatments in the WWTP could partially reduce the spread of antibiotic-resistant bacteria, and additional procedures such as sedimentation may not critically affect the removal efficiency.

Using magnetic seeds to improve the aggregation and precipitation of nanoparticles from backside grinding wastewater

Backside grinding (BG) wastewater treatment typically requires large quantities of chemicals, i.e. polyaluminum chloride (PAC) coagulant and produces considerable amounts of sludge, increasing the loading and cost of subsequent sludge treatment and disposal processes. This study investigated the effects of the addition of magnetic seeds (FeO*Fe(2)O(3)) of selected particle sizes and of optimized combinations of magnetic seeds and PAC on the aggregation of silica nanoparticles from BG wastewater and on the sedimentation time at various pH values. The results show that the turbidity of BG wastewater was significantly reduced by the magnetic aggregation treatment. The dosage of PAC combined with 2.49gL(-1) or 1.24gL(-1) of magnetic seeds was reduced by 83% (from 60 to 10mgL(-1)) compared to the conventional process of using only PAC as a coagulant. The turbidity of the BG wastewater, initially 1900-2500NTU, could also be successfully decreased about to 23NTU by the addition of 3.74gL(-1) magnetite (FeO*Fe(2)O(3)) only at pH 5 with an applied magnetic field of 1000G. Different coagulation conditions using magnetic seeds combined with coagulant resulted in different aggregation performances. The treatment performance was more effective by using two-stage dosing, in which magnetic seeds and PAC were added separately, than that with one-stage dosing, where the magnetic seeds and PAC were added simultaneously during rapid mixing. The two-stage dosing allowed for a reduction in the optimum dosage of magnetic seeds from 3.74gL(-1) to 2.49gL(-1) or 1.24gL(-1) without affecting performance when coupled with 0.01gL(-1) of PAC coagulant. The developed method effectively reduced the production of waste sludge.

Diversity and Seasonal Impact of Acanthamoeba Species in a Subtropical Rivershed

This study evaluated the presence of Acanthamoeba species in the Puzih River watershed, which features typical subtropical monsoon climate and is located just above the Tropic of Cancer in Taiwan. The relationship between the seasonal and geographical distributions of Acanthamoeba species in this rivershed was also investigated. Acanthamoeba species were detected in water samples using the amoebal enrichment culture method and confirmed by PCR. A total of 136 water samples were included in this study, 16 (11.7%) of which contained Acanthamoeba species. Samples with the highest percentage of Acanthamoeba (32.4%) were obtained during the summer season, mainly from upstream areas. The identified species in the four seasons included Acanthamoeba palestinensis (T2), Acanthamoeba sp. IS2/T4 (T4), Acanthamoeba lenticulata (T5), Acanthamoeba hatchetti (T11), Acanthamoeba healyi (T12), and Acanthamoeba jacobsi (T15). The most frequently identified Acanthamoeba genotype was T4 (68.7%). Acanthamoeba genotype T4 is responsible for Acanthamoeba keratitis and should be considered for associated human health risk potential in the rivershed.

Occurrence and distribution of Naegleria species from thermal spring environments in Taiwan.

Naegleria spp. is a free‐living amoeba that can be found in the natural environment. A number of Naegleria spp. can cause fatal infections in the central nervous system in humans and animals, and the most important source of infection is through direct water contact. In this study, water samples from various thermal springs were taken from four thermal spring areas. Naegleria spp. was detected via culture confirmation and molecular taxonomic identification. Among the 60 samples obtained, Naegleria spp. was identified in 26 (43·3%) samples. The identified species included Naegleria australiensis, Naegleria gruberi, Naegleria lovaniensis and Naegleria mexicana. The presence of living Naegleria spp. was significantly associated with elevated pH value in the water sample.

Evaluation of Immunomagnetic Separation for the Detection of Salmonella in Surface Waters by Polymerase Chain Reaction

Salmonella spp. is associated with fecal pollution and capable of surviving for long periods in aquatic environments. Instead of the traditional, time-consuming biochemical detection, polymerase chain reaction (PCR) allows rapid identification of Salmonella directly concentrated from water samples. However, prevalence of Salmonella may be underestimated because of the vulnerability of PCR to various environmental chemicals like humic acid, compounded by the fact that various DNA polymerases have different susceptibility to humic acid. Because immunomagnetic separation (IMS) theoretically could isolate Salmonella from other microbes and facilitate removal of aquatic PCR inhibitors of different sizes, this study aims to compare the efficiency of conventional PCR combined with immunomagnetic separation (IMS) for Salmonella detection within a moderately polluted watershed. In our study, the positive rate was increased from 17.6% to 47% with nearly ten-fold improvement in the detection limit. These results suggest the sensitivity of Salmonella detection could be enhanced by IMS, particularly in low quality surface waters. Due to its effects on clearance of aquatic pollutants, IMS may be suitable for most DNA polymerases for Salmonella detection.

Surveillance of parasitic Legionella in surface waters by using immunomagnetic separation and amoebae enrichment.

Free-living amoebae (FLA) are potential reservoirs of Legionella in aquatic environments. However, the parasitic relationship between various Legionella and amoebae remains unclear. In this study, surface water samples were gathered from two rivers for evaluating parasitic Legionella. Warmer water temperature is critical to the existence of Legionella. This result suggests that amoebae may be helpful in maintaining Legionella in natural environments because warmer temperatures could enhance parasitisation of Legionella in amoebae. We next used immunomagnetic separation (IMS) to identify extracellular Legionella and remove most free Legionella before detecting the parasitic ones in selectively enriched amoebae. Legionella pneumophila was detected in all the approaches, confirming that the pathogen is a facultative amoebae parasite. By contrast, two obligate amoebae parasites, Legionella-like amoebal pathogens (LLAPs) 8 and 9, were detected only in enriched amoebae. However, several uncultured Legionella were detected only in the extracellular samples. Because the presence of potential hosts, namely Vermamoeba vermiformis, Acanthamoeba spp. and Naegleria gruberi, was confirmed in the samples that contained intracellular Legionella, uncultured Legionella may survive independently of amoebae. Immunomagnetic separation and amoebae enrichment may have referential value for detecting parasitic Legionella in surface waters.

Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay

Abstract Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 102 and 3.3 × 105 cells/l in river water and 72.1–5.7 × 106 cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

Identification of the Naegleria Species in Natural Watersheds Used for Drinking and Recreational Purposes in Taiwan

AbstractNaegleria species (spp.) is a free-living amoeboflagellate that is widespread in natural environments. Some Naegleria spp. cause fatal human infections, and the most important source of these infections is water. This genus is recognized as a potential human pathogen that causes primary amoebic meningoencephalitis. In this study, 211 water samples were collected from two watersheds in Taiwan. The Naegleria spp. on the basis of polymerase chain reaction (PCR) amplification with a genus-specific primer pair and an investigation of Naegleria spp. in the Puzih Rivers and Kaoping Rivers in Taiwan. The percentage of positive samples obtained from the Kaoping River (21.8%) was higher than that from the Puzih River (0.6%). The presence/absence of Naegleria spp. within the water samples showed a significant difference with levels of water temperature and pH value. The most frequently identified Naegleria spp. was N. philippinensis (n=3), followed by N. clarki (n=2), N. gallica (n=2), and N. americana (n=2)...

Tracking biological pollution sources using PCR-DGGE technology at Ta-An Beach.

The environmental authority of Taiwan has announced that ocean quality standard A, with E. coli less than 1,000 CFU/100 mL is safe for swimming. Ta-An Beach in central Taiwan was found to have exceeded 1,000 CFU/100 mL, which is 51% of the total monitoring records. Sewage, piggery and duck wastewater are discharged directly into this area. The traditional pollution source trace methods did not clearly identify the pollution source. This study used PCR-DGGE technology to establish micro-organisms fingerprints from water samples using comparative analysis with microbiological composition, and then determined the possible sources of biological contamination. The E. coli colonies at all samples were processed using linear regression analysis and compared with each other. The R(2) is 0.4102-0.7387 for the livestock farm and beach. This shows a positively relation. The piggery impact is more obvious. The beach microbial communities exhibited high similarity with piggery wastewater from the upstream region. We confirmed that the major pollution source for Ta-An Beach comes from piggery wastewater. The proposed method has high stability and reliability. It can be used as the basis for rapid identification of pollution sources at other beach water sites in the future.

Wastewater treatment design parameter analysis using the Nan-Men contact bed treatment facility in Northern Taiwan

Contact bed treatment is one of the methods used for constructed wetlands. Taiwan introduced contact bed treatment to treat sewage in 2004. The reference design parameters are based on data obtained from developed countries. These foreign designs ignore the unique hydrological environment and climate of Taiwan. This study tried to analyze the water quality of the contact bed treatment system and to assess the efficiency of design parameter, based on that to propose design parameters to similar facilities on Taiwan. This study shows that the out-site contact bed treatment design should be changed to increase the aeration and disinfection parameters with the DO greater than 5 mg/L, BOD(5) optimal concentration of 10-25 mg/L, COD of 32-60 mg/L, SS of 15-25 mg/L, NH(3)-N concentration can be greater than 16 mg/L, hydraulic retention time of two hours, gravel size of 10-15 cm, porosity of 30-50%, with the water flow rate less than 10 cm/s and sludge accumulation of 10 to 15 days. These studies conducted long term observation in accordance with local conditions. The above data are available to provide local design parameters for future follow-up designs.