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?×?10⁴ and 3.47?×?10⁵?cells/L in river water, 6.92?×?10⁴ and 4.29?×?10⁵?cells/L in raw drinking water, and 5.71?×?10⁴ and 2.12?×?10⁶?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.     

Molecular detection and comparison of Acanthamoeba genotypes in different functions of watersheds in Taiwan

The protistan genus Acanthamoeba is a free-living amoeba existing in various environments. Within this protistan genus, there are some species recognized as potential human pathogens, which may cause granulomatous amoebic encephalitis, Acanthamoeba keratitis and chronic granulomatous lesions of the skin. In this study, 211 water samples were collected from two watersheds in southern Taiwan. We detected Acanthamoeba based on the PCR amplification with a genus-specific primer pair and investigation of Acanthamoeba in Puzih River and Kaoping River in southern Taiwan. Acanthamoeba species were detected in 34 (16.1%) samples. The presence or absence of Acanthamoeba within the water samples showed significant difference with the levels of water temperature and total coliforms. The most frequently identified Acanthamoeba genotype was T4 (n = 19), followed by T5 (n = 8), and then T15 (n = 3). Genotype T6, T7/T8, T11 and T12 were all detected once. Genotype T4, T5, T6, T11 and T15 of Acanthamoeba are responsible for Acanthamoeba keratitis and should be considered a potential health threat associated with human activities in environmental surface water watersheds.     

Occurrence of diarrheagenic Escherichia coli genes in raw water of water treatment plants

Purpose

The high incidences of waterborne diseases are frequently associated with diarrheagenic Escherichia coli (DEC). DEC may pose a health risk to people who contact surface water for recreation or domestic use. However, there is no published report on the monitoring of DEC in drinking water sources in Taiwan. In this study, the occurrence of DEC genes in raw water for water treatment plants in Taiwan was investigated.

Method

Raw water samples were taken from water treatment plants adjacent to the Kaoping River in southern Taiwan. Each water sample was treated with membrane filtration followed by DNA extraction from the concentrate and concentrate enrichment, respectively. The target genes for various DEC strains of genes were identified, including enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC).

Results

Among 55 water samples analyzed, DEC genes were detected in 16 (29.1%) samples. Strain-specific genes for EAEC, EHEC, EIEC, and EPEC were found in the percentages of 3.6%, 10.9%, 9.1%, and 9.1%, respectively. The specific gene for ETEC is not detected in the study. By looking at the presence/absence of specific genes and water sample characteristics, water temperature was found to differ significantly between samples with and without EHEC gene. In addition, pH levels differed significantly for EHEC and EPEC presence/absence genes, and turbidity was significantly different for water with and without EPEC genes.

Conclusion

DEC genes were detected in 29.1% of the raw water samples in the study location. The potential health threat may be increased if the treatment efficiencies are not properly maintained. Routine monitoring of DEC in drinking water sources should be considered.     

IMS Method Performance Analyses for Giardia in Water Under Differing Conditions

Immunomagnetic separation (IMS) has been specified as a standard method for the measurement of Giardia under USEPA Method 1623. In this study, IMS was evaluated on the basis of recovery efficiencies for Giardia cysts under various IMS operation conditions. Significant change in recovery was observed by altering the debris ratio of water samples. Notably, cyst recovery efficiencies utilizing IMS dropped with increased turbidity, and results for varying dosages of magnetic beads and cysts indicate that 1 / 100 immunomagnetic beads is sufficient to conjugate large numbers of cysts. Changing vessel volume and replacing the sample buffer had no significant effect on cyst recovery efficiencies.     

Differential Legionella spp. survival between intracellular and extracellular forms in thermal spring environments

Legionella are commonly found in natural and man-made aquatic environments and are able to inhabit various species of protozoa. The relationship between the occurrence of Legionella spp. within protozoa and human legionellosis has been demonstrated; however, the proportions of intracellular and extracellular Legionella spp. in the aquatic environment were rarely reported. In this study, we developed a new method to differentiate intracellular and extracellular Legionella spp. in the aquatic environment. Water samples from three thermal spring recreational areas in southeastern Taiwan were collected and analyzed. For each water sample, concurrent measurements were performed for Legionella spp. and their free-living amoebae hosts. The overall detection rate was 32 % (16/50) for intracellular Legionella spp. and 12 % (6/50) for extracellular Legionella spp. The most prevalent host of Legionella spp. was Hartmannella vermiformis. The identified Legionella spp. differed substantially between intracellular and extracellular forms. The results showed that it may be necessary to differentiate intracellular and extracellular forms of Legionella spp.     

Using the Flow Cytometry to Quantify the Giardia Cysts and Cryptosporidium Oocysts in Water Samples

The flow cytometry (FC) has been used to detect Giardia cysts and Cryptosporidium oocysts quantitatively and instantaneously in this study. The experimental results showed that FC is potential to become a more precise method for the detection of Giardia and Cryptosporidium in water. This study also evaluated the staining efficiencies for three commercial antibodies. After staining Cryptosporidium oocysts with direct immunofluorescent antibodies in water samples, two populations were detected in the scatter-plots (FL1 versus SSC) of the FC. The Cryptosporidium oocysts and Giardia cysts are significantly separated from other particles while stained with direct immunofluorescent antibodies produced by Meridian Diagnostics and Waterborne^TM Inc.     

First detection of enteric adenoviruses genotype 41 in recreation spring areas of Taiwan

Environmental Science and Pollution Research - Human adenoviruses (HAdVs) are DNA viruses found in recreational water, such as water parks and swimming pools. Human adenovirus 41 (HAdV-41) is the...     

Utilization of polymerase chain reaction and selective media cultivation to identify Legionella in Taiwan spring water samples

In this study, we investigated the distribution of Legionella in sources of spring water and wastewater throughout Taiwan. Legionella were detected with culture method and PCR method from water samples, combined with molecular methods that allow the taxonomic identification of Legionella species. Factors associated with the distribution of Legionella including the physical?Cchemical and microbiological water quality characteristics were also measured. Legionella were detected in 18 of the 68 samples. Additionally, 16 of the 68 samples analyzed by polymerase chain reaction (PCR) and eight of the 68 samples evaluated using the culture method were found to be positive for Legionella. The Legionella species identified included Legionella pneumophila, unnamed Legionella genotypes, uncultured Legionella spp., and Legionella lytica. Legionella species were found in water with temperatures ranging from 10.0 to 50.6??C and with pH values ranging from 5.59 to 9.32. Significant differences (Mann?CWhitney U test, P?<?0.05) were observed in the presence/absence of Legionella in samples with different pH values when Legionella were detected by the culture method. The results of this survey confirmed that Legionella is ubiquitous in the water of spring recreational areas in Taiwan. Long-term investigations of the occurrence of Legionella in hot spring recreational areas throughout Taiwan are needed.     

Intensive water quality monitoring in a Taiwan bathing beach

A total of 357 water samples were collected from a public beach in northern Taiwan during beach season, and the densities of enterococci were analyzed by Enterolert methods. The mean enterococci level was 356 MPN/100 ml and ranged from <10 to 2,005 MPN/100 ml, which was classified as high contamination level according to the WHO water quality guideline (95 percentile >1,000 MPN/100 ml). Most of the deteriorated water quality conditions occurred during rainfall. By excluding data from the rain days, the overall beach water quality would be considered in the moderate contamination level (95 percentile 200-1,000 MPN/100 ml). Among the selected microbiological parameters tested, the densities of total coliforms and enterococci exhibited the highest correlation (r = 0.449, p = 0.009), followed by the concentrations of total coliforms and fecal coliforms (r = 0.403, p = 0.02). Nonetheless, no significant correlation was found between enterococci and fecal coliform levels (r = 0.197, p = 0.271).     

Evaluation of enterovirus recovery in surface water by different adsorption and elution procedures

The performance of six concentration method combinations and two quantitative analysis techniques were evaluated in terms of enterovirus recovery efficiencies by adjusting the pH and salt concentration of water samples. Of the six concentration method combinations, adsorption on nitrocellulose membranes followed by an acid rinse elution consistently gave the highest recovery efficiencies. In theory, an electropositive membrane should be the most appropriate technique for adsorption of electronegative viruses in pure water. However, it displayed the greatest loss in natural waters. For adsorption and elution procedures, both the electronegative membrane, accompanied by an acid rinse step, and the electropositive membrane, accompanied by a glycine rinse step, provided higher recovery efficiencies. MPN-RT-PCR, a statistically quantitative analysis method, performed more efficiently, in economic terms, but had a similar enterovirus recovery trend to real-time RT-PCR, which is the authoritative quantitative analysis method for nucleic acid.