Bing-Mu Hsu

Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area.

Acanthamoeba, Hartmannella, and Naegleria are free-living amoebae, ubiquitous in aquatic environments. Several species within these genera are recognized as potential human pathogens. These free-living amoebae may facilitate the proliferation of their parasitical bacteria, such as Legionella. In this study, we identified Acanthamoeba, Hartmannella, Naegleria, and Legionella using various analytical procedures and investigated their occurrence at a mud spring recreation area in Taiwan. We investigated factors potentially associated with the prevalence of the pathogens, including various water types, and physical and microbiological water quality parameters. Spring water was collected from 34 sites and Acanthamoeba, Hartmannella, Naegleria, and Legionella were detected in 8.8%, 35.3%, 14.7%, and 47.1%, respectively. The identified species of Acanthamoeba included Acanthamoeba castellanii and Acanthamoeba polyphaga. Nearly all the Hartmannella isolates are identified as Hartmannella vermiformis. The Naegleria species included Naegleria australiensis and its sister groups, and two other isolates referred to a new clade of Naegleria genotypes. The Legionella species identified included unnamed Legionella genotypes, Legionella pneumophila serotype 6, uncultured Legionella spp., Legionella lytica, Legionella drancourtii, and Legionella waltersii. Significant differences (Mann-Whitney U test, P<0.05) were observed between the presence/absence of Hartmannella and total coliforms, between the presence/absence of Naegleria and heterotrophic plate counts, and between the presence/absence of Legionella and heterotrophic plate counts. This survey confirms that pathogenic free-living amoebae and Legionella are prevalent in this Taiwanese mud spring recreation area. The presence of pathogens should be considered a potential health threat when associated with human activities in spring water.

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 Acanthamoeba from Taiwan spring recreation areas using culture enrichment combined with PCR

In the study, 52 spring water samples were collected from three hot spring recreation areas in northern Taiwan and Acanthamoebae were isolated from 11 samples (21.2%) on two hot spring recreation areas and mainly present in the hot spring water, hot tubs and wastewater. The most frequently identified Acanthamoeba genotype was T15, followed by T6, and then T5. Genotype T1, T2, T3 and T4 were detected once, respectively. The presence or absence of Acanthamoeba within the spring water samples showed significant difference with the levels of heterotrophic plate counts (HPC). Genotype T2-T6 and genotype T15, the organism responsible for Acanthamoeba keratitis, and the Acanthamoeba species organism, retained pathogenic Legionella, and should be considered a potential health threat associated with human activities in spring recreation areas.

Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments

This study compares five genera of free-living amoebae (FLA) hosts by Legionella spp. in the fixed and floating biofilm samples from spring environments. Detection rate of Legionella spp. was 26.9% for the floating biofilms and 3.1% for the fixed biofilms. Acanthamoeba spp., Hartmanella vermiformis, and Naegleria spp. were more frequently detected in floating biofilm than in fixed biofilm samples. The percentage of pathogenic Acanthamoeba spp. among all the genus Acanthamoeba detected positive samples was 19.6%. The potential pathogenic Naegleria spp. (for example, Naegleria australiensis, Naegleria philippinensis, and Naegleria italica) was 54.2% to all the Naegleria detected positive samples. In the study, 12 serotypes of possible pneumonia causing Legionella spp. were detected, and their percentage in all the Legionella containing samples was 42.4%. The FLA parasitized by Legionella included unnamed Acanthamoeba genotype, Acanthamoeba griffini, Acanthamoeba jacobsi, H. vermiformis, and N. australiensis. Significant differences were also observed between the presence/absence of H. vermiformis and Legionella parasitism in FLA. Comparisons between the culture-confirmed method and the PCR-based detection method for detecting FLA and Legionella in biofilms showed great variation. Therefore, using these analysis methods together to detect FLA and Legionella is recommended.

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.

Survey of Naegleria and its resisting bacteria-Legionella in hot spring water of Taiwan using molecular method

Naegleria is a free-living amoebae existing in soil and aquatic environments. Within the genus Naegleria, N. fowleri is most recognized as potential human pathogen causing primary amoebic meningoencephalitis (PAM). Furthermore, the Naegleria spp. can serve as vehicles for facultative pathogens, such as Legionella. In this study, we identified Naegleria and Legionella based on the PCR amplification with a genus-specific primer pair and investigated the distribution of Naegleria and Legionella at five spring recreation areas in Taiwan. In this study of hot spring and other water sources in Taiwan, five Naegleria spp. were detected in 15 (14.2%) of the water samples. The most frequently detected was N. lovaniensis (n = 6), followed by N. australiensis (n = 5), and then N. clarki (n = 2). N. americana and N. pagei were detected once, respectively. The pathogenic species N. fowleri was not detected; however, N. australiensis considered to be a potential pathogen species in humans was found. Legionella spp., an endosymbiont of Naegleria, was detected in 19 (17.9%) of the water samples in this study. Overall, 5.7% of the water samples contained both Naegleria and Legionella. The Legionella spp. identified were L. pneumophila and L. erythra. Results of this survey confirm the existence of Naegleria and Legionella in Taiwan spring recreation areas. It should be considered a potential threat for health associated with human activities in spring recreation areas of Taiwan.

Prevalence of enteroviruses in hot spring recreation areas of Taiwan.

Enteroviruses can be introduced into the water environment as a result of human activity. Contaminations within hot tubs, spas and public baths are also possible. We investigated the distribution of enteroviruses at six hot spring recreation areas throughout Taiwan. Spring water was collected from 34 sites and enteroviruses were detected in 13 (38.2%). The most frequently detected was coxsackievirus A2, followed by echovirus 11. Enterovirus 71 (EV 71) and porcine enterovirus 9 were detected once. Water quality indicators were not statistically associated with the occurrence of enteroviruses, although the enterovirus-positive samples were positive for a greater number of microbiological indicators and showed a link to pH and water temperature. The results confirm the ubiquity of enteroviruses in Taiwan spring recreation areas. Coxsackievirus A2, echovirus 11 and EV 71, the enteroviruses responsible for disease outbreaks identified at these sites, should be considered a potential public health threat in spring recreation areas of Taiwan.

Water quality parameters associated with prevalence of Legionella in hot spring facility water bodies.

Some species of Legionella are recognized as opportunistic potential human pathogens, such as Legionella pneumophila, which causes legionnaires disease. Indeed, outbreaks of legionellosis are frequently reported in areas in which the organism has been spread via aerosols from contaminated institutional water systems. Contamination in hot tubs, spas and public baths are also possible. As a result, in this study, we investigated the distribution of Legionella at six hot spring recreation areas throughout Taiwan. Legionella were detected in all six hot spring recreation areas, as well as in 20 of the 72 samples that were collected (27.8%). Seven species of Legionella identified from samples by the direct DNA extraction method were unidentified Legionella spp., Legionella anisa, L. pneumophila, Legionella erythra, Legionella lytica, Legionella gresilensis and Legionella rubrilucen. Three species of Legionella identified in the samples using the culture method were L. pneumophila, unidentified Legionella spp. and L. erythra. Legionella species were found in water with temperatures ranging from 22.7 °C to 48.6 °C. The optimal pH appeared to range from 5.0 to 8.0. Taken together, the results of this survey confirmed the ubiquity of Legionella in Taiwan spring recreational areas. Therefore, a long-term investigation of the health of workers at hot spring recreational areas and the occurrence of Legionella in hot spring recreational areas throughout Taiwan are needed.

Differentiation and identification of Shigella spp. and enteroinvasive Escherichia coli in environmental waters by a molecular method and biochemical test.

Both Shigella spp. and enteroinvasive Escherichia coli (EIEC) are important human pathogens that are responsible for the majority of cases of endemic bacillary dysentery. However, they are difficult to identify and differentiate by biochemical tests or molecular methods alone. In this study, we developed a procedure to detect Shigella spp. and EIEC from environmental water samples using membrane filtration followed by nutrient broth enrichment, isolation using selective culture plates, and identification of the invasion plasmid antigen H (ipaH) gene by PCR amplification and DNA sequencing. Finally, we used a biochemical test and a serological assay to differentiate between Shigella and EIEC. Among the 93 water samples from nine reservoirs and one watershed, 76 (81.7%) water samples of culture plates had candidate colonies of Shigella and EIEC and 5 water samples were positive (5.4%) for a Shigella- and EIEC-specific polymerase chain reaction targeting the ipaH gene. Guided by the molecular method, the biochemical test, and the serological assay, 11 ipaH gene-positive isolates from 5 water samples were all identified as EIEC.

Molecular detection and prevalence of enterovirus within environmental water in Taiwan

Aims:  To investigate the presence of enterovirus RNA in various samples of environmental water collected in Taiwan during 2004–2005, and to characterize the genotypes and distribution of the viruses identified in Taiwan.