Tsui-Kang Hsu

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.

Surveillance and evaluation of the infection risk of free-living amoebae and Legionella in different aquatic environments

Free-living amoebae (FLA) are ubiquitous in various aquatic environments. Several amoebae species are pathogenic and host other pathogens such as Legionella, but the presence of FLA and its parasites as well as the related infection risk are not well known. In this study, the presence of pathogenic FLA and Legionella in various water bodies was investigated. Water samples were collected from a river, intake areas of drinking water treatment plants, and recreational hot spring complexes in central and southern Taiwan. A total of 140 water samples were tested for the presence of Acanthamoeba spp., Naegleria spp., Vermamoeba vermiformis, and Legionella. In addition, phylogenetic characteristics and water quality parameters were also assessed. The pathogenic genotypes of FLA included Acanthamoeba T4 and Naegleria australiensis, and both were abundant in the hot spring water. In contrast, Legionella pneumophila was detected in different aquatic environments. Among the FLA assessed, V. vermiformis was most likely to coexist with Legionella spp. The total bacteria level was associated with the presence of FLA and Legionella especially in hot spring water. Taken together, FLA contamination in recreational hot springs and drinking water source warrants more attention on potential legionellosis and amoebae infections.

Leber's hereditary optic neuropathy masquerading as optic neuritis with spontaneous visual recovery

We report a case of Lebers hereditary optic neuropathy (LHON) masquerading as optic neuritis with late visual recovery. A 28‐year‐old man had gradual visual loss in both eyes for two weeks. Visual acuity was 0.4 in the right eye and 0.7 in the left. Fundus examination revealed hyperaemic discs in each eye. Fluorescein angiography revealed dye leakage at both optic discs in the late phase. Static perimetry (Humphrey 30‐2) revealed bilateral relative central scotomata. Magnetic resonance imaging of the optic nerves was normal and his lumbar puncture showed normal opening pressure.

Application of TaqMan fluorescent probe-based quantitative real-time PCR assay for the environmental survey of Legionella spp. and Legionella pneumophila in drinking water reservoirs in Taiwan.

In this study, TaqMan fluorescent quantitative real-time PCR was performed to quantify Legionella species in reservoirs. Water samples were collected from 19 main reservoirs in Taiwan, and 12 (63.2%) were found to contain Legionella spp. The identified species included uncultured Legionella spp., L. pneumophila, L. jordanis, and L. drancourtii. The concentrations of Legionella spp. and L. pneumophila in the water samples were in the range of 1.8×10(2)-2.6×10(3) and 1.6×10(2)-2.4×10(2) cells/L, respectively. The presence and absence of Legionella spp. in the reservoir differed significantly in pH values. These results highlight the importance that L. pneumophila, L. jordanis, and L. drancourtii are potential pathogens in the reservoirs. The presence of L. pneumophila in reservoirs may be a potential public health concern that must be further examined.

Control strategies for biohydrogen production by immobilized co-culture of Clostridium butyricum and Rhodopseudomonas palustris

ABSTRACT In this study, we evaluated biohydrogen production of Clostridium butyricum and Rhodopseudomonas palustris by immobilized co-culture. Effects of free cells and immobilized cells, immobilized biomass ratio, sucrose concentration, and initial pH on hydrogen production were investigated. The immobilized co-culture can achieve high cumulative hydrogen volume yield (604 mL) as compared to free co-culture cumulative hydrogen volume (513 mL) while the initial sucrose concentration was 17.8 g/L. The optimum C. butyricum/R. palustris ratio was 1:10, yielding the highest cumulative hydrogen (728 mL). High sucrose concentration (above 35.6 g/L) would inhibit hydrogen production. The optimal initial pH value for hydrogen production of immobilized co-culture was 7.0 (cumulative hydrogen volume 830 mL).

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.

Nested-PCR and TaqMan real-time quantitative PCR assays for human adenoviruses in environmental waters

Human adenovirus (HAdV) infections can occur throughout the year. Cases of HAdV-associated respiratory disease have been more common in the late winter, spring, and early summer. In this study, to provide viral pollution data for further epidemiological studies and governmental actions, the presence of HAdV in the aquatic environment was quantitatively surveyed in the summer. This study was conducted to compare the efficiencies of nested-PCR (polymerase chain reaction) and qPCR (quantitative PCR) for detecting HAdV in environmental waters. A total of 73 water samples were collected from Puzi River in Taiwan and subjected to virus concentration methods. In the results, qPCR had much better efficiency for specifying the pathogen in river sample. HAdV41 was detected most frequently in the river water sample (10.9%). The estimated HAdV concentrations ranged between 6.75 × 10(2) and 2.04 × 10(9) genome copies/L. Significant difference was also found in heterotrophic plate counts, conductivity, water temperature, and water turbidity between presence/absence of HAdV. HAdV in the Puzi River may pose a significant health risk.

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.

Application of modified JDP-DGGE-based molecular genotyping method to predict Acanthamoeba genotype and to analyse community diversity in aquatic environments

AbstractAcanthamoeba spp. are ubiquitous, opportunistic potential human pathogens, causing granulomatous amoebic encephalitis and keratitis. They are classified as protozoa, and they include at least 20 different genotypes (T1–T20) based on variation in the 18S rRNA gene. Acanthamoeba spp. are diverse in their production of toxins and in their ability to resist environmental factors. Therefore, it is necessary to develop a rapid genotyping method for Acanthamoeba spp. in aquatic environments. Although the denaturing gradient gel electrophoresis (DGGE) method for analysing microbial genotypes is potentially useful for rapid identification of aquatic environmental species, the technique has been compromised by artificial DGGE profiles in which many DNA fragments of identical sequences are segregated and displayed as different bands. The results indicate that PCR-DGGE genotyping with a GC clamp results in many segregated weaker bands of identical DNA sequences. In contrast, PCR-DGGE genotyping without a GC clamp displays genotype-dependent patterns in the major bands. Thus, DGGE without a GC clamp was performed to compare genotyping efficiency for Acanthamoeba in 21 water samples from rivers and reservoirs in Taiwan. Among them, four samples were found to demonstrate a banding pattern with more than one major band, and these band profiles of major bands were identical to those of positive controls. DNA cloning further confirmed that the sequences of the major bands were identical. In conclusion, more than two genotypes of Acanthamoeba in the four samples were identified by this method, suggesting that PCR-DGGE genotyping without a GC clamp is a useful approach for studying the diversity of Acanthamoeba communities. Graphical abstract