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.

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.

Seasonal distribution and prevalence of diarrheagenic Escherichia coli in different aquatic environments in Taiwan.

Diarrheagenic Escherichia coli (DEC) are the most common agents of diarrhea. Waterborne DEC could pose a potential health risk to human through agricultural, household, recreational, and industrial use. There are few published reports on the detection of DEC and its seasonal distribution in aquatic environments. The presence of DEC in different types of aquatic environments was investigated in this study. Water samples were collected from major rivers, water reservoirs, and recreational hot springs throughout Taiwan. Moreover, an intensive water sampling plan was carried out along Puzih River. The detection of DEC target genes was used to determine the presence of enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and Shiga toxin-producing E. coli (STEC). Among the 383 water samples analyzed, DEC was found in 122 (31.8%) samples. The detection rate varied by genotype, raging from 3.6% for STEC to 17.2% for EPEC. The DEC detection rate was higher from river waters than reservoirs and hot springs. In addition, DEC was detected at a higher rate in spring and summer. The presence of EPEC was significantly associated with total coliform levels among hot spring samples. Moreover, the presence of ETEC in river water samples was associated with heterotrophic plate counts. Water with EPEC differed significantly in pH from Puzih River samples. These results suggest that seasonal characteristics may affect the presence of DEC in different aquatic environments, and water quality indicators may be indicative of the presence of DEC.

Seasonal difference of human adenoviruses in a subtropical river basin based on 1-year monthly survey.

In this study, the seasonal difference and the observable presence/absence of human adenovirus (HAdV) in the Puzih River basin in Taiwan was investigated. A total of 288 water samples were collected from 24 sites from March 2014 to February 2015. Human AdV analysis of sample was subjected to viral concentration using a GN-6 Metricel® filter, followed by DNA extraction, nested-PCR, and qPCR. Human AdV was detected in 34.3 % (99/288) of the entire river water sample. A higher percentage of HAdV (76.4 %) was obtained during the winter. The HAdV median concentration was relatively high in fall (1.4 × 10(3) copies/L) and winter (2.8 × 10(3) copies/L). Significant difference and correlation were found between the seasonal variation of HAdV and water quality parameters, including heterotrophic plate count, total coliform, water temperature, and turbidity. The most frequently identified HAdV (subgenus F) serotype was 41. Human AdV-41 is the main cause of gastroenteritis and should be considered for associated human health risk potential in the Puzih River basin.

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.

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 × 10(2) and 3.3 × 10(5) cells/l in river water and 72.1-5.7 × 10(6) 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.

Antibiotic resistance pattern and gene expression of non-typhoid Salmonella in riversheds.

In this study, antibiotic resistance and major phenol and genotypes of non-typhoid Salmonella spp. from riversheds in Taiwan were examined. In 236 water samples tested, 54 (22.9%) contained Salmonella spp. Fifteen Salmonella serovars were identified from the Salmonella isolates, and some common serovars are associated with infections of human and livestock, including Albany (27.8%), Newport (14.8%), Bareilly (13.0%), Derby (11.1%), and Typhimurium (7.4%). Various environmental factors may also affect the presence and proportion of different serovars in the receiving waters. In contrast, serovars with narrower range of hosts, e.g., Dublin, were rarely detected. The Salmonella isolates were subjected to eight antibiotics for drug resistance, and 51.9% of the samples were resistant to at least one tested antibiotics. Tetracycline and sulfadiazine were the two most ineffective antibiotics against the Salmonella isolates, and the results were indicative of long-term antibiotics abuse as fodder supplements in animal husbandry. The more commonly detected serovars such as Albany, Derby, and Typhimurium were also more likely to be resistant to multiple antibiotics. Finally, a significant correlation was observed between resistance to chloramphenicol and the resistance gene cmlA, suggesting that the resistance genotypes could persist in the environment even long after prohibition of the drug use. The high prevalence of antibiotic-resistant Salmonella spp. infers elevated infection risks that must be further examined.

Seasonal distribution of potentially pathogenic Acanthamoeba species from drinking water reservoirs in Taiwan.

In order to detect the presence/absence of Acanthamoeba along with geographical variations, water quality variations and seasonal change of Acanthamoeba in Taiwan was investigated by 18S ribosomal RNA (rRNA) gene TaqMan quantitative real-time PCR. Samples were collected quarterly at 19 drinking water reservoir sites from November 2012 to August 2013. Acanthamoeba was detected in 39.5 % (30/76) of the water sample, and the detection rate was 63.2 % (12/19) from samples collected in autumn. The average concentration of Acanthamoeba was 3.59 × 10(4) copies/L. For geographic distribution, the detection rate for Acanthamoeba at the northern region was higher than the central and southern regions in all seasons. Results of Spearman rank test revealed that heterotrophic plate count (HPC) had a negative correlation (R = -0.502), while dissolved oxygen (DO) had a positive correlation (R = 0.463) in summer. Significant differences were found only between the presence/absence of Acanthamoeba and HPC in summer (Mann-Whitney U test, P < 0.05). T2 and T4 genotypes of Acanthamoeba were identified, and T4 was the most commonly identified Acanthamoeba genotypes. The presence of Acanthamoeba in reservoirs presented a potential public health threat and should be further examined.

Seasonal variation of Legionella in Taiwan's reservoir and its relationships with environmental factors.

In this study, the presence of Legionella in major water reservoirs of Taiwan was examined with respect to seasonal variation, geographical variation, and water quality parameters using TaqMan real-time qPCR. Water samples were collected quarterly at 19 reservoirs in Taiwan between November 2012 and August 2013. The detection rate for Legionella was 35.5% (27/76), and Legionella was detected in all seasons. The Legionella concentration was relatively high in spring and summer, reaching 3.86 × 10(8) and 7.35 × 10(8) cells/L, respectively. By sampling the area, Legionella was detected at a higher proportion in reservoirs in the northern and southern areas, and the difference was consistent in all seasons. Significant association was found between detection of Legionella and various water quality parameters, including conductivity, chlorophyll a, and dissolved oxygen (Mann-Whitney U test, P < 0.05). Results of Spearman rank test showed negative correlation for Legionella detection with pH (P = 0.030, R = -0.497) and dissolved oxygen (P = 0.007, R = -0.596) in fall and positive correlation with Carlson's trophic state index (P = 0.049, R = 0.457) in spring. The identified species included Legionella pneumophila and Legionella drancourtii. The detection of Legionella in reservoirs was indicative of a potential public health risk and should be further evaluated.

Prevalence, quantification, and typing of human adenoviruses detected in river water in Taiwan.

The prevalence of human adenoviruses (HAdV) in river waters was investigated in this study. Water samples were collected from 13 rivers in Taiwan, concentrated, and assessed for the presence of HAdVs using nested polymerase chain reaction (PCR). Human AdV positive samples were then subjected to real-time PCR (qPCR) to quantify the viral genomes and further subjected to primer-based genotyping to identify the various serotypes present. For each water sample, several water quality parameters were evaluated, including heterotrophic plate count, total coliform, Escherichia coli, water temperature, pH, conductivity, and dissolved oxygen. Among the 13 rivers examined, four rivers (30.8 %) were found to contain HAdVs. The major genotype was F species HAdV serotype 41. The mean HAdVs concentrations ranged from 6.10 × 10(2) to 8.51 × 10(2) copies/L. No significant differences were observed between the presence of HAdVs, and all of the water quality parameters evaluated (heterotrophic plate count, total coliform, E. coli, water temperature, pH, conductivity, and dissolved oxygen). Given the potential health risks posed by the presence of enteric viruses in environmental waters, further assessment is desirable with respect to possible sources, virus transport, and survival of viruses in the aquatic environment.

Evaluation of diarrheagenic E. coli in riversheds by quantitative PCR in combination with enrichment.

Diarrheagenic Escherichia coli (DEC) is a group of the most common agents of diarrhea. Highly virulent DEC strains could cause illness with dozens of organisms. Waterborne DEC may be detected using polymerase chain reaction (PCR); however, environmental contaminants can interfere with PCR reaction, thus causing the prevalence of DEC to be underestimated. In this study, we propose an approach to efficiently quantify trace amounts of DEC. An enrichment procedure was performed to amplify total E. coli including DEC in the water samples. By normalizing the number of pathotype-specific genes to the amplification rate of a housekeeping gene in all E. coli, the quantity of DEC in original samples could be assessed. This method allows detection of trace amounts of DEC in receiving waters. The results showed that the presence of DEC in water samples was partially associated with riverside settlement. The DEC concentration was substantially higher at a few sampling sites, suggesting that evaluation of DEC along the river may help identify previously unknown pollution sources. Although the sustainability of DEC in the receiving waters may be low, the risk of DEC infection from the watershed warrants further examination.

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.

Application of TaqMan qPCR for the detection and monitoring of Naegleria species in reservoirs used as a source for drinking water.

Naegleria spp. can be found in the natural aquatic environments. Naegleria fowleri 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, PCR of 5.8S ribosomal RNA (rRNA) gene and internal transcribed spacer (ITS) region was performed in order to identify Naegleria isolates and quantify the Naegleria spp. by TaqMan real-time quantitative PCR in reservoir water samples. The occurrence of Naegleria spp. was investigated in 57 water samples from reservoirs with culture and PCR positive in 2 of them (3.5%), respectively. The total detection rate was 7.0% (4/ 57) for Naegleria spp. The identified species included Naegleria spp., Naegleria canariensis, and Naegleria clarki. N. fowleri was not found in Taiwan's reservoirs used for drinking purposes. The concentrations of Naegleria spp. in detected positive reservoir water samples were in the range of 599 and 3.1 × 10(3) cells/L. The presence or absence of Naegleria spp. within the reservoir water samples showed significant difference with the levels of water temperature. The presence of Naegleria spp. in reservoirs considered a potential public health threat if pathogenic species exist in reservoirs.

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.

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.

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.

Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay.

In this study, a SYBR green quantitative real-time PCR was developed to quantify and detect the Legionella spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and thermal spring area in Taiwan. Legionella was detected in 13.6 % (24/176), and the detection rate for river water, raw drinking water, and thermal spring water was 10, 21.4, and 16.6 %, respectively. Using real-time PCR, concentration of Legionella spp. in detected samples ranged between 9.75 × 10(4) and 3.47 × 10(5) cells/L in river water, 6.92 × 10(4) and 4.29 × 10(5) cells/L in raw drinking water, and 5.71 × 10(4) and 2.12 × 10(6) cells/L for thermal spring water samples. The identified species included Legionella pneumophila (20.8 %), Legionella jordanis (4.2 %), Legionella nautarum (4.2 %), Legionella sp. (4.2 %), and uncultured Legionella sp. (66.6 %). The presence of L. pneumophila in aquatic environments suggested a potential public health threat that must be further examined.

Quantitative detection and identification of Naegleria spp. in various environmental water samples using real-time quantitative PCR assay.

Naegleria spp. is a free-living amoeba that can be found in various aquatic environments. There are some Naegleria spp. that can cause fatal infections in animals and humans, and the most important source of infection is through direct water contact. In this study, a real-time quantitative PCR was developed to detect and quantify the Naegleria spp. in various environmental water samples. The water samples were taken from rivershed, water treatment plants, and thermal spring recreation areas. The total detection rate was 4.0% (7/176) for Naegleria spp. The percentages of samples containing Naegleria spp. from river water, raw drinking water, and thermal spring water were 0% (0/100), 10.7% (3/28) and 8.3% (4/48), respectively. The concentration of Naegleria spp. in detected positive raw drinking water and thermal spring water samples was in the range of 3.9-12.6 and 1.1-24.2 cells/L, respectively. The identified species included Naegleria australiensis, Naegleria lovaniensis, and Naegleria spitzbergeniensis. The presence of Naegleria spp. in various aquatic environments is considered a potential public health threat.

Real-time PCR method for the detection and quantification of Acanthamoeba species in various types of water samples.

In this study, a quantitative real-time PCR was developed to detect and quantify Acanthamoeba spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and three thermal spring recreation areas. The overall detection rate was 14.2 % (25/176) for Acanthamoeba spp. The percentages of samples containing Acanthamoeba spp. from river water, raw drinking water, and thermal spring water were 13 % (13/100), 25 % (7/28), and 10.4 % (5/48), respectively. Acanthamoeba spp. concentrations were determined according to SYBR Green quantitative real-time PCR. A plasmid-based standard curve was constructed to determine the Acanthamoeba concentration using dilution factors for achieving 1.36 × 10(9) gene copies per PCR for 18S rRNA gene in Acanthamoeba spp. The resulting concentrations varied by the type of water, in the range of 46-2.6 × 10(2) cells/l in positive raw drinking water, 2.7 × 10(2)-1.5 × 10(4) cells/l in river water, and 54-1.7 × 10(3) cells/l in thermal spring water. The presence of Acanthamoeba spp. in the raw drinking water samples was also found to have a significant difference with heterotrophic plate count. The presence of Acanthamoeba spp. in various aquatic environments may be a potential health hazard and must be further evaluated.