Studies on the biosystematics of Trichodinid ciliates parasitic on British freshwater fish.

Of the numerous reports of trichodinids from British freshwater fish only four have been to species level, representing three Trichodina species and one Trichodinella species. Twenty freshwater fish species were sampled from approximately sixty sites in Scotland, England and Wales. Skin and gill smears were prepared using Klein’s silver staining technique, and morphometric measurements made from features of the adhesive di.sc taken from enlarged photomicrographs according to Lorn’s (1958) taxonomic criteria. The adoral cilia, used as a feature of generic classification were described for each trichodinid population from live preparations. Species discrimination and investigation of mtraspecific morphological variation was aided by the u.se of Principal Components and Cluster analysis. Thirteen trichodinid species were identified from British freshwater fish during this study including: Trichodina acuta Lorn, 1961 from Cyprinus carpio, Carassius auratus, Oncorhynchus mykiss, and Phoxinus phoxinus; Trichodina domerguei Wallengren, 1897 from Gasterosteus aculeatus; Trichodina tenuidens Faure-Fremiet, 1944 from Gasterosteus aculeatus; Trichodina pediculus Ehrenberg, 1838 from Gasterosteus aculeatus; Trichodina modesta Lorn, 1970 from Ahramis hrama; Irichodina nif>ra Lorn, I960 from Cyprinus carpio, Salmo trutta and Oncorhynchus mykiss; Trichodina mutahilis Kazubski & Migala, 1968 from Phoxinus phoxinus; Trichodina rostrata Kulemina, 1968 from Phoxinus phoxinus; Trichodina intermedia Lorn, I960 from Phoxinus phoxinus; Trichodinella epizootica Raabe, 1950 from Perea fluviatilis and Oncorhynchus mykiss; Tripartiella lata Lorn, 1963 from Phoxinus phoxinus; Tripartiella copiosa Lorn, 1959 from Phoxinus phoxinus and Paratrichodina incisa Lorn, 1959 from Phoxinus phoxinus. Ahramis brama and Rutilas rutilas. Many of the previous species demonstrated considerable inter and intrapopulational morphological variation. Immature specimens of Trichodina acuta, Trichodina domerguei, Trichodina tenuidens and Trichodina intermedia were examined, elucidating developmental morphology and its taxonomic relevance. A .study of intraspecific morphological variation was undertaken by sampling trichodinids from a population of Gasterosteus aculeatus over a twelve month period. Regression analysis indicated statistically significant negative correlations between water temperature and measurements of the adhesive disc in Trichodina domerfpiei and Trichodina tenuidens. Principal Components Analysis was utilised to differentiate extreme morphological variants of the two previous species. A sonication technique modified from Shinn et al. (1993) was used to i.solate skeletal structures of the adhesive disc from Trichodina domerf^uei, Trichodina intermedia, Paratrichodina incisa and Trichodinella epizoótica for .scanning electron micro.scopy. New structures which were previously undescribed are discussed, including denticle apophyses, denticular pins and a small peg-like structure on the central parts ot the denticles. Actual denticle structure is compared to that visualised by silver staining using light microscopy, and its implications for generic classification of the trichodinids is commented upon

Predicting selection for antimicrobial resistance in UK wastewater and aquatic environments: ciprofloxacin poses a significant risk

Antimicrobial resistance (AMR) is a threat to human and animal health, with the environment increasingly recognised as playing an important role in AMR evolution, dissemination, and transmission. Antibiotics can select for AMR at very low concentrations, similar to those in the environment, yet their release into the environment, e.g., from wastewater treatment plants, is not currently regulated. Understanding the selection risk antibiotics pose in wastewater and receiving waters is key to understanding if environmental regulation of antibiotics is required. We investigated the risk of selection occurring in UK wastewater and receiving waters by determining where measured environmental concentration data (n = 8187) for four antibiotics (ciprofloxacin, azithromycin, clarithromycin, and erythromycin) collected in England and Wales 2015–2018 (sites n = 67) exceeded selective concentration thresholds derived from complex microbial community evolution experiments undertaken previously. We show that selection for AMR by ciprofloxacin is likely to have occurred routinely in England and Wales wastewater during the 2015–2018 period, with some seasonal and regional trends. Wastewater treatment reduces the selection risk posed by ciprofloxacin significantly, but not completely, and predicted risk in surface waters remains high in several cases. Conversely, the potential risks posed by the macrolides (azithromycin, clarithromycin, and erythromycin) were lower than those posed by ciprofloxacin. Our data demonstrate further action is needed to prevent selection for AMR in wastewater, with environmental quality standards for some antibiotics required in the future, and that selection risk is not solely a concern in low/middle income countries

Cultivation-independent screening revealed hot spots of IncP-1, IncP-7 and IncP-9 plasmid occurrence in different environmental habitats

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Cultivation-independent screening revealed hot spots of IncP-1, IncP-7 and IncP-9 plasmid occurrence in different environmental habitats

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Cultivation-independent screening revealed hot spots of IncP-1, IncP-7 and IncP-9 plasmid occurrence in different environmental habitats

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Pathogen quantitation in complex matrices : a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition

Background: Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. Methodology/Principal Findings: We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pretreatment step, the FastDNA® Spin kit, the UltraCleanTM and PowerSoilTM soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. Conclusions: M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25 x 105 cells g-1 using Griffiths and 4.25 x 106 cells g-1 using FastDNA® Spin kit

Cultivation-Independent Screening Revealed Hot Spots of IncP-1, IncP-7 and IncP-9 Plasmid Occurrence in Different Environmental Habitats

Dealtry S, Ding G-C, Weichelt V, et al. Cultivation-Independent Screening Revealed Hot Spots of IncP-1, IncP-7 and IncP-9 Plasmid Occurrence in Different Environmental Habitats. PLoS ONE. 2014;9(2): e89922.IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC- DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots'' of plasmids potentially carrying catabolic genes

Generation of probes for Southern blot hybridization.

<p>Generation of probes for Southern blot hybridization.</p