Po-Min Kao

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.

Isolation and identification of Legionella and their host amoebae from weak alkaline carbonate spring water using a culture method combined with PCR

Legionella were detected with the direct DNA extraction method, Legionella culture method, and free-living amoebae (FLA) culture method from weak alkaline carbonate spring water in Taiwan. Moreover, we also investigated the existence of Acanthamoeba, Hartmannella, and Naegleria, ubiquitous FLA in aquatic environments, to identify the correlations between existing Legionella. This study reports detecting Legionella in 15 of the 51 weak alkaline carbonate spring water samples (29.4%). This work also found five of the 51 samples (9.8%) analyzed by the direct DNA extraction method, three of the 51 (5.9%) samples analyzed by the Legionella culture method, and 11 of the 51 samples (21.6%) evaluated using the FLA culture method to be positive for Legionella. The most frequently identified Legionella species was the Legionella-like amoebal pathogen (n = 5), followed by unidentified Legionella spp. (n = 4), and Legionella pneumophila (n = 4), Legionella fairfieldensis (n = 3), and then Legionella rubrilucens (n = 2). Legionella waltersii was detected once. The occurrence of Acanthamoeba, Hartmannella, and Naegleria were 5.9% (3/51), 52.9% (27/51), and 5.9% (3/51), respectively. All Hartmannella isolates were identified as Hartmannella vermiformis, and Naegleria isolates were all identified as Naegleria australiensis. The three Acanthamoeba isolates were identified as one Acanthamoeba polyphaga and two Acanthamoeba jacobsi. H. vermiformis (40.7%) were Legionella hosts, including all of the amoebae-resistant Legionella detected in the present study. Therefore, the important correlations between Legionella and H. vermiformis require further clarification. The combined results of this survey confirm that Legionella and FLA are ubiquitous in weak alkaline carbonate spring water in Taiwan.

Isolation and identification of Acanthamoeba species from thermal spring environments in southern Taiwan

Acanthamoeba species are free-living amoebae found in a range of environments. Within this genus, a number of species are recognized as human pathogens, potentially causing Acanthamoeba keratitis, granulomatous amoebic encephalitis, and chronic granulomatous lesions. In this study, 60 water samples were taken from four thermal spring recreation areas in southern Taiwan. We detected living Acanthamoeba spp. based on culture-confirmed detection combined with the molecular taxonomic identification method. Living Acanthamoeba spp. were detected in nine (15%) samples. The presence or absence of Acanthamoeba spp. in the water samples depended significantly on the pH value. The most frequently identified living Acanthamoeba genotype was T15 followed by T4, Acanthamoeba spp., and T2. Genotypes T2, T4, and T15 of Acanthamoeba, are responsible for Acanthamoeba keratitis as well as granulomatous amoebic encephalitis, and should therefore be considered a potential health risk associated with human activities in thermal spring environments.

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.

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.

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.

Application of molecular biological techniques to analyze Salmonella seasonal distribution in stream water

Salmonella is a leading cause of waterborne diseases. Salmonella can survive for a long time in aquatic environments, and its persistence in the environment is of great concern to public health. Nonetheless, the presence and diversity of Salmonella in the aquatic environments in most areas remain relatively unknown. In this study, we examined three analytical processes for an optimum Salmonella detection method, and the optimized method was used to evaluate seasonal variations of Salmonella in aquatic environments. In addition, Salmonella strains were isolated by selective culture medium to identify the serotypes by biochemical testing and serological assay, and to identify the genotypes by pulsed-field gel electrophoresis based on the genetic patterns. A total of 136 water samples were collected in the study area in 9 months. Forty-one (30.1%) samples were found to contain Salmonella-specific invA gene, and most (24/41) of the detections occurred in summer. The serovars of Salmonella enterica were identified, including Bareilly, Isangi, Newport, Paratyphi B var. Java, Potsdam and Typhimurium.

Identification and quantification of the Acanthamoeba species and genotypes from reservoirs in Taiwan by molecular techniques

The occurrence of Acanthamoeba was investigated from 21 main reservoirs of Taiwan with 12 (57.1%) testing positive. Analysis of the 18S rRNA gene PCR product was performed in order to identify the Acanthamoeba isolates. Acanthamoeba spp. concentrations were determined according to TaqMan real-time qPCR. Acanthamoeba genotypes of all isolates were identified T4. The species were categorized to Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba hatchetti. The concentration of Acanthamoeba spp. in detected positive reservoir water samples was in the range of 3.0-1.8 × 10(3) cells/L. These results highlight the importance of Acanthamoeba in reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

Approach to determine the diversity of Legionella species by nested PCR-DGGE in aquatic environments

In this study, we describe a nested PCR-DGGE strategy to detect Legionella communities from river water samples. The nearly full-length 16S rRNA gene was amplified using bacterial primer in the first step. After, the amplicons were employed as DNA templates in the second PCR using Legionella specific primer. The third round of gene amplification was conducted to gain PCR fragments apposite for DGGE analysis. Then the total numbers of amplified genes were observed in DGGE bands of products gained with primers specific for the diversity of Legionella species. The DGGE patterns are thus potential for a high-throughput preliminary determination of aquatic environmental Legionella species before sequencing. Comparative DNA sequence analysis of excised DGGE unique band patterns showed the identity of the Legionella community members, including a reference profile with two pathogenic species of Legionella strains. In addition, only members of Legionella pneumophila and uncultured Legionella sp. were detected. Development of three step nested PCR-DGGE tactic is seen as a useful method for studying the diversity of Legionella community. The method is rapid and provided sequence information for phylogenetic analysis.

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.