Bacillus are known to produce a wide variety of antimicrobial substances and bacteriocins. The main objective of this study was to investigate the effect of these bacteriocins on eukaryotic cells such as fungi, yeast and plant seeds. Several strains were screened for antifungal activities and identified by the means of polymerase chain reaction (PCR) of the 16s rRNA gene and sequencing. Our experiments showed that the Bacillus pumilus ZED17 and DFAR8 strains, had antifungal activities against Rhizoctonia solani and selected for further investigations. Extracellular peptides produced by these strains were purified by ammonium sulfate precipitation and dialysis. Addition of these peptides to Potato Dextrose Agar (PDA) medium inoculated with R. solani indicated significant inhibition of the fungal growth. The antifungal peptides were thermo-stable and remained active after boiling at 100˚C for 15 min. The molecular weight of the peptide with antifungal activity was estimated by electrophoresis on the Sodium Dodecyl Sulfate Poly Acrylamide Gel (SDS-PAGE) as about 5 KDa. Structural nature of this peptide was determined after gel extraction by Fourier-transform infrared spectroscopy (FTIR). Moreover, this peptide showed inhibiting effect on seeds germination of some herbs. This peptide could be applied to control herbal fungal disease induced by R. solani which is a broad host range plant pathogen fungus and its inhibition is very important. The peptide also prevents seed germination. Hence, it can be appropriate for inhibiting weeds growth. No significant effect against Saccharomyces cerevisiae and Candida albicans was observed.The bacteria which are members of the genus  Bacteriocins are natural peptides secreted by many varieties of bacteria for the purpose of destroying other organisms  and are Bacteriocins were first recognized in 1925 by Andre Gratia when he observed that the growth of various bacteria strains was inhibited by a substance with antibacterial properties which was called Colicin V . For sevBacillus are found in diverse environments, which are Gram-positive, aerobic, rod-shaped and endospore-forming bacteria [Bacillus is due to secondary metabolite production like antibiotics and antimicrobial peptides [Bacillus strains are able to survive in various conditions [Bacillus strains are suitable organisms for the production of these antimicrobial compounds. In addition, protein secretion may create better folding conditions in comparison with an intracellular reducing environment by preventing the formation of inclusion bodies. Moreover the separation of protein from the other components will be easier [Members of genus bacteria . They cabacteria . The antpeptides . The compeptides . Beacausnditions . Due to e easier . Fusarium, Nigrospora and Rhizoctonia are involved in plant diseases and cause reduction in yields of important agricultural products such as rice, wheat and potato [Today, resistance to all clinically used antibiotics has reached a point where the World Health Organization (WHO) has expressed concern about the possibility of resistant pathogens becoming incurable . Fungal d potato , 17.Streptomyces, such as amphotericin and nystatin have restrictions such as narrow range spectrum, low permeability to specific tissues, and toxicity for the human body [Sclerotinia, Fusarium, Gaeummanomyces, Nectria, Pythium and Phytophthora, Aspergillus flavus, Aspergillus niger by some strains of Bacillus such as B. cereus, B. subtilis, B. mycoides are reported [Bacillus include mycobacillin, iturin, bacillomycin, surfactin, mycosubtilin, fungistatin, subsporin [Moreover, a serious economic issue for the pulp and paper industry is decaying of the stored wood contaminated with various fungal infections . Many plman body . Thus, mreported , 21. Somubsporin , subtiliubsporin . Many stBacillus strains on eukaryotic cells like the yeasts, fungus and plant seeds. Rhizoctonia is a plant pathogen fungus with a broad host ranges including monocot and dicot plants with economic importance such as rice, wheat, alfalfa, beans, soy, corn, potato and tomato; therefore the inhibition of these fungus is very important.The objective of this study is to examine the effects of the peptides derived from the Bacterial strains and culture conditions: Nine bacteria were isolated from soil and rhizosphere of different plant based on a method used by Shokri and Emtiazi, and identified as Bacillus through biochemical characteristics using standard methods [Bacillus and Saccharomyces cerevisiae strains were cultured in nutrient agar medium at 37˚C. The R. solani, Mucoraceae and Candida albicans strains were grown in PDA (Potato Dextrose Agar) medium at 30˚C, for three days. methods . The obtIsolation of bacteria with antifungal activity: Antimicrobial activity is determined by the spot on lawn method against different fungi and yeasts. The Bacillus strains were tested for their ability to inhibit the growth of various fungi. These strains were grown in 100 ml nutrient broth at 37˚C for 24 h subject to 80 rpm shake. The cells were removed through centrifugation  and the supernatant is refined through a 0.45-mm filter. The strains of fungi including R. solani, Mucor rouxii  DSM1194  and the strains of yeasts including Candida albicans and Saccharomyces cerevisiae were cultured by sterile swabs on PDA medium and a 50 µl of the filtered supernatant was added followed by incubation at 30˚C for 24 h simultaneously. In another experiment, 1×1 mm pieces of each fungi strain were inoculated into 100 ml of nutrient broth medium and 200 µl of the bacterial filtered supernatant (obtained as above) was added to nutrient broth medium inoculated with the fungus, followed by incubation for 7 days at 30˚C and subjected to 80 rpm shake. The fungal dry weight was then calculated. This experiment was performed in triplicate and one specimen including the inoculated medium with fungus without adding the bacterial filtered extract, considered as the control.Identification of bacteriocin producing isolated bacteria: In order to identify the selected Bacillus strains with the best antifungal activity, the strains were grown on nutrient agar medium and incubated at 37˚C for 24 h. The DNA extraction was performed through boiling the bacterial suspension in sterile distilled water at 100˚C for 10 min and centrifuged . The supernatant was used as a DNA template [Identification of Bacteriocin producing gene in Bacillus strains: To determine the types of produced bacteriocins, the PCR is performed on 3 Bacillus strains using bacilysin primers produced by Takapozist Company , under the following conditions: 95˚C for 10 min, 30 cycles of . In addition, the PCR is performed on these strains under temperature gradient. The primers (BACAB-F) 5′-CTT CTC CAA GGG GTG AAC AG-3′ and (BACAB-R) 5′-TGT AGG TTT CAC CGG CTT TC-3′) were used [ere used .Production and extraction of antifungal compounds: Bacillus strains are cultured in 200 ml nutrient agar and incubated at 37˚C under aeration for 24 h. The cells were removed by centrifugation  and the supernatant is filtered through 0.45 µm membranes, and then precipitated with ammonium sulfate to 60% saturation. Then, the saturated solution incubated at 4°C for 24 hours and centrifuged (14500 ×g for 30 min) .The resulting pellets were dissolved in phosphate buffer with pH 7.0 and dialyzed against the same buffer through dialysis tube of 1000 Da. The partially purified compound was tested for antifungal activity by adding 100 µl of this compound simultaneously to an indicator strain of fungi (R.solani) cultured on PDA medium and incubated at 30˚C for 24 h.Molecular weight determination and detection of antifungal activity of purified peptide: To determine the molecular weight of this partially purified antifungal compound, the SDS-PAGE was carried out followed by Zymography techniques Which is an electrophoretic technique to estimate the relative location of the studied peptide . Electrophoresis is conducted at 100V for 2 h. The gel was then sectioned into two parts: one contained a pre-stained protein marker (Chromatein Pre-stained Protein Ladder-vivantis Technologies PR0602) which was stained with Coomassie Brilliant Blue, and the other was studied for direct antifungal activity. To maintain the natural conformation of the proteins, the gels were placed in Triton X-100 for 30 min, washed with sterile distilled water at every 30 minutes for 2 hours and then overlaid on PDA medium inoculated with indicator strain R. solani and incubated at 30˚C [ at 30˚C .Extraction of antifungal peptide from gel and analyzing the petide by FTIR: Antifungal compounds of the two Bacillus strains were subjected to electrophoresis as mentioned above. Sections of the gels that contain the anti-fungal activity were cut horizontally with a sterile scalpel and then 3 ml of elution buffer containing NaCl 150 mM, EDTA 1 mM and Tris-HCL 50 Mm was added on them. The mixture was crushed with a sterile glass rod and incubated overnight at 30˚C under stirring. The mixtures were then centrifuged . Supernatants containing partially purified peptides with antifungal activity were studied again to make sure the activity was maintained. 100µl of the purified peptides was added to PDA medium which contained an indicator strain of fungi (R. solani). The purified peptides from polyacrylamide gel were freeze-dried and analyzed with a (JASCO 6300 JAPAN) Fourier transform infrared (FTIR) spectrophotometer, to determine the natural structure of these peptides.Investigating the effect of antifungal compound on seeds germination: To study the effect of antifungal compounds on seeds germination, the Raphanus sativus and Lepidium sativum seeds were first sterilized as follows: seeds were placed in 70% ethanol for 1 min and washed with distilled water. Sodium hypochlorite was diluted with distilled water with 20 to 80 ratio and the seeds were placed in it for 3-5 minutes and washed 3 times for 2-3 minutes in sterile distilled water. These sterilized seeds were cultured in Murashige and Skoog (MS) medium containing 100 ml macro elements included nitrogen, phosphorus, potassium, calcium, sulfur, magnesium, carbon and 10 ml micro element such as boron, chlorine, manganese , zinc , copper, molybdenum , nickel , 10 ml iron, 10 ml vitamin, 10 gr sugar and 30 gr agar. The purified bacteriocins through ammonium sulfate precipitation and dialysis were added to the seeds in MS medium and then incubated at 30˚C exposed to light for 24 h. Experiments were performed in triplicate and a sample was considered as the control specimen, then the germination rates were measured and compared with the control. Investigating the effect of temperature on activity of antifungal compound: The partially purified bacteriocin was examined to determine the thermal stability. The obtained solution with antifungal activity was apportioned into six vials, and each vial becomes subject to temperatures of -20, 0, 4, 37, 70 and 100˚C for 15 min. The activity was evaluated and 100 µl of each of the portions was added to the culture medium inoculated with the fungi and then incubated for 24 h at 30˚C.R. solani and Mucor rouxii (DSM1194) with a visible growth inhibition zone. However, their effects against Saccharomyces cerevisiae and Candida albicans were insignificant. Moreover, the degree of growth inhibition of yeasts and fungi were tested through direct antagonism on plates by adding the filtered supernatant to the liquid medium inoculated with the fungus followed by fungal dry weight calculation. In this study, bacterial strains showed inhibition effects against Bacillus strains, B. Pumilus ZED17 (0.13±0.06) and DFAR8 (0.12±0.06) showed the best antifungal effect and the best performance was against R. solani (0.91±0.15). Therefore, in this study the focus was on these strains. Identification of isolated bacteria producing bacteriosins by sequencing the PCR product, a new strain of the genus Bacillus pumilus was identified. The result of sequence analysis was compared to the data from the (NCBI) Gene Bank with nucleotide BLAST. The obtained results indicated significant inhibition of the fungal growth compared to the control sample. The results of triplicate experiments were analyzed by applying the Duncan test at the confidence level of 0.05% by SPPSS 6.0 software. This can determine whether differences were statistically significant. Among the 9 Bacillus pumilus DFAR8 with accession number of KC577596 which is recorded in the NCBI database.The phylogenetic tree that was drawn by CLC sequence viewer 6.1.A phylogeny software represents the evolutionary relationships of this strain with 99% homology . This stBacillus, PCR performed on B. pumilus DFAR8 and B. pumilus ZED17 strains using bacAB gene primers that encode the bacilysin. The PCR analysis showed that this gene is not found in Bacillus strains. Therefore compound produced by these strains can be a bacteriocin other than bacilysin.Since bacilysin is found in many strains of R.solani cultured on PDA medium simultaneously. After incubation at 30˚C for 24 h, a growth inhibition zone became visible.Antifungal activity of the partially purified compounds was tested after precipitation by ammonium sulfate and dialysis. 100 µl of the purified compounds was added to the indicator strain of fungi, B. pamilusZED17 and B. pamilus DFAR8 were determined as<10 KDa  and (3197 cm-1) indicate the presence of aliphatic and aromatic chains respectively. In addition, the absorption valley at 3999 cm-1 represents strong H-bonding of acid, amine and amid groups, while the valley at 3350 cm-1 is related to NH stretching of terminal amine.Samples extracted from the gel were freeze dried and analyzed with FTIR. The infrared spectrum of antifungal compound exhibited the characteristic absorption valleys at 1671,1616,1630, 1553, 1296 cmructures . ValleysAdding partially purified bacteriocin to sterilized seeds cultured on MS medium showed inhibition effect on seed germination compared to that of the control. Results of triplicate experiments are analyzed by applying the Duncan test at the confidence level of 0.05% by SPPSS 6.0 software. Different letters indicate significant difference with control sample . The resThe antifungal peptide remained active after exposure to different temperatures -20, 0, 4, 37, 70 and 100˚C. It was also significantly thermo stable, even after boiling at 100˚C for 15 min, as no changes were observed in the antifungal activity.Bacillus strains produce secondary metabolites such as antibiotics, antifungals and siderophores. This ability makes them to be considered as an appropriate biological control agents [B.pumilus ZED17 and B.pumilus DFAR8 are purified and characterized and their effect on eukaryotic cells was evaluated. Antifungal peptides are extracted by precipitation and dialysis process respectively. The molecular weight of compounds is determined to be <10 KDa by SDS-PAGE. Unstained gel in the same area showed antifungal activity. Peptides derived from this gel regions are relatively pure and are analyzed with FTIR. The FTIR provides additional information about the nature of the peptides. Analysis of the FTIR spectrum showed absorption bands related to the presence of the secondary structure of the peptide, CH stretching of aromatic ring, CH group aliphatic, strong H-bonding of acid, amine and amid groups and NH stretching of terminal amine in antifungal peptide. Perhaps since the large number of double bonds are found in the peptide of B.pumilus ZED17 strain, this peptide displays more complex folding condition. The l agents . In thisB.pumilus ZED17 and B.pumilus DFAR8 strains. Therefore, to identify produced bacteriocin in these strains, the PCR can be conducted for the other bacteriocins genes with low molecular weight such as subtilin with molecular weight of 3.3 KDa or mersacidin with molecular weight of 1.8 KDa [ The PCR analysis of bacAB genes related to bacilysin revealed this gene is not observed in Bacillus strains on the eukaryotic cells such as fungi and seeds of plants. The consumed antifungal compounds do not serve the purpose at all times, and might create recipient resistance while being toxic for human. As the members of Genus Bacillus can produce a variety of compounds with biological activity running more investigations on them can be considered as further tasks for researchers.Moreover, the antifungal peptides are thermostable and remain active after boiling at 100˚C for 15 min. This compound prevents seed germination, meaning that it can also be effective in inhibiting weed growth. The obtained result confirmed the effect of peptides derived from


Background: Hydrogen peroxide (H2O2) is a common antiseptic that is available without a prescription in the United States, and it is indicated for minor dermal abrasion; mouth, gum, or dental irritations; and removal of oral secretion. Several other medical uses have also been described, including clot dissolution during endoscopic gastrointestinal evaluation, cleansing of orthopedic surgical sites, and bladder irrigation. However, these uses of H2O2, as well as high-dose ingestion, have been associated with a wide variety of medical complications, including but not limited to air pulmonary embolism and stroke.Case Presentation: Our patient is a 51-year-old female with a medical history of hypertension, familial, hypercholesterolemia, gallstones, depression, coronary artery disease , nephrolithiasis, and recurrent cystitis. She required percutaneous nephrolithotomy and had H2O2 administered for clot dissolution. The clinical and temporal evidence would suggest a transient pulmonary air embolus after the intrarenal administration of or irrigation with H2O2, large amounts under high pressure.Conclusion: This represents the first reported incidence of air embolus as a result of intrarenal administration of H2O2. The complications of these uses are minor and mostly include pain on administration and irritation to areas of administration.1Hydrogen peroxide , nephrolithiasis, and recurrent cystitis. Her first stone event was in 2008 that subsequently required ureteroscopy (URS) and ureteral stent placement. She presented to our outpatient clinic initially in consultation for left-sided abdominal pain, and subsequent CT scan revealed nephrolithiasis with three separate left ureteral stones , requiriAfter urgent decompression with a stent, she was scheduled for bilateral URS with laser lithotripsy (LL) and possible bilateral percutaneous nephrolithotomy (PCNL) for definitive stone management. She ultimately underwent bilateral URS with LL and right-sided PCNL. Postoperative CT scan revealed residual right-sided stone burden, so she was scheduled for a second stage right-sided PCNL. In the interim, her postoperative course was complicated by gross hematuria and anemia, requiring transfusion of 2 U of packed red blood cells.2O2 standard preparation was diluted three times and ∼100 mL was instilled using a 60 mL Toomey syringe through the current indwelling 22 Councill nephrostomy tube to dissolve the extensive clot burden and improve observation. Approximately 45 seconds after instillation, the anesthesiologist noted that the patient's end tidal carbon dioxide (CO2) had suddenly plummeted (0–9) and she had become hypotensive. ST segment elevation was also noted on the telemetry tracings that were a change from her preoperative electrocardiogram. Owing to immediate concern for pulmonary embolus, the procedure was terminated, and the patient was taken out of dorsal lithotomy position and placed in the supine position. The patient's blood pressure increased over the next several minutes, and she became hemodynamically stable; however, her end tidal CO2 remained well below normal. She was immediately transferred from the operating room to radiology for emergent CT angiography (CTA). At the time of CTA, the patient's end tidal CO2 had risen to 30 to 40. Ultimately the CTA revealed no evidence of pulmonary thrombus; however, abdominal follow-through was notable for extensive air in the right renal collecting system  with or without head down to avoid further embolization. Next, attention should be turned to determining hemodynamic stability.2 in our case likely resulted from transient intraoperative usage of H2O2 with moderate irrigating force in a closed cavity. In addition, given the patient's preoperative evidence of hemorrhage, she may have had an increased risk of venous backflow. We consider our experience in addition to the literature to date as adequate evidence for advising caution when considering its use during urologic procedures.We believe that the intraoperative instability and clinical decompensation along with drop in end tidal CO2O2, large amounts under high pressure. This represents the first reported incidence of air embolus as a result of intrarenal administration of H2O2.The clinical and temporal evidence would suggest a transient pulmonary air embolus after the intrarenal administration of or irrigation with H


Nitrogen concentration and flux-related watershed response metrics exhibited significantly strong negative correlations across case study watersheds, with absolute correlations (|r|) ranging from 0.48 to 0.97 for IWI values, and 0.31 to 0.96 for ICI values. Nitrogen-stable isotope ratios measured in chironomids and periphyton from streams and benthic organic matter from lake sediments also demonstrated strong negative correlations with IWI values, with |r| ranging from 0.47 to 0.92, and 0.35 to 0.89 for correlations with ICI values. This evaluation of the performance of national watershed and catchment integrity metrics and their strong relationship with site level responses provides weight-of-evidence support for their use in state, local and regionally focused applications.Watershed integrity, the capacity of a watershed to support and maintain ecological processes essential to the sustainability of services provided to society, can be influenced by a range of landscape and in-stream factors. Ecological response data from four intensively monitored case study watersheds exhibiting a range of environmental conditions and landscape characteristics across the United States were used to evaluate the performance of a national level Index of Watershed Integrity (IWI) at regional and local watershed scales. Using Pearson’s correlation coefficient ( Watersheds provide a functional context for assessing and managing aquatic ecosystems, as the water and materials from the surrounding landscapes drain to rivers, lakes, wetlands, groundwater, and downstream estuaries ,2, whichTo effectively manage aquatic resources using a holistic watershed perspective, managers, stakeholders, and decision-makers need assessment approaches that integrate and synthesize information related to the functional attributes affecting watershed condition. There have been several recent developments in assessing and evaluating ‘watershed health’ that unite holistic ecosystem approaches with fundamental concepts from the field of landscape ecology. These examine functional ecohydrological processes and aquatic and landscape connectivity while accounting for the hierarchical nature of these processes occurring at multiple spatiotemporal scales ,8–13. WoIn a recent effort to integrate the assessment and management of aquatic ecosystems within watersheds, Flotemersch et al.  construcFocusing on the key functional elements and processes necessary to maintain watershed provision of services and the risk factors that degrade these functions, Flotemersch et al.  utilizedThe six key watershed functions described in detail in Flotemersch et al.  and ThorUsing the operational definition of watershed integrity provided by Flotemersch et al.  and landSince ‘watershed integrity’ is a theoretical concept, and not physical property that can be measured, it is not possible to directly validate the performance of the IWI and ICI. However, Thornbrugh et al.  hypothesThe objectives of this study were twofold: (1) to evaluate the performance of the IWI, ICI and their respective six associated functional components using local scale  metrics and response variables; and (2) to identify specific aquatic characteristics and indicator responses that are correlated with the national watershed integrity metrics, as well as data for specific local site-level characteristics that may need refinement to improve the validity and robustness of these national metrics. The IWI and ICI values derived by Thornbrugh et al. , along wWe selected four intensively monitored watersheds from across the United States having data from multiple sites throughout the watershed. The four case study watersheds range in size, elevation, landscape, and demographic characteristics .2 with elevation ranging from 1562 m at the headwaters and summit of Tidbits Mountain to less than 57 m at the confluence with the Willamette River. Land cover in the higher elevations is largely evergreen forest, with lower elevations more dominated by agricultural practices  is part of theWillamette drainage and Columbia River basin within the Western Cascades mountain range in Oregon. The CRW encompasses an area of 945 kmractices . Developractices . While t2 in the headwaters of the Upper Choptank and Tuckahoe Creek sub-basins within the northern portion of the Choptank River watershed (The Choptank River (CHOP) is a major tributary of the Chesapeake Bay that originates in Choptank Mills, Delaware and flows southwest through Maryland’s Eastern Shore. The entire watershed is located within the coastal plain of the Delmarva Peninsula in the Mid-Atlantic region. The Choptank was a U.S. Department of Agriculture Conservation Effects Assessment Project (CEAP) Benchmark watershed . The Choatershed . The majatershed . The Chohttp://www.epa.state.oh.us/dsw/tmdl/LittleMiamiRiver.aspx. The case study area, the East Fork Little Miami River (EFLMR) watershed, is a major tributary of the Little Miami River watershed and encompasses an area of 1293 km2. Along its course the river drops from an elevation of 365 m to 149 m with an average gradient of 1.4 m/km. In 1978, the U.S. Army Corps of Engineers impounded a section of the East Fork Little Miami River by constructing an earthen dam at river km 33, creating an 874-hectare reservoir (Harsha Lake) stretching approximately 16 km upstream from the dam. Draining agricultural lands, Harsha Lake functions as both a significant sink and source of nitrogen [The Little Miami River, located in southwestern Ohio in the Ohio River drainage basin, contains some of Ohio’s most scenic and diverse riverine habitat and is designated a State and National Scenic River by the Ohio Environmental Protection Agency: nitrogen . The resnitrogen . The preThe EFLMR watershed has a temperate climate characterized by well-defined winter and summer seasons. The average annual total precipitation ranges from 104–109 cm, with about 40% falling during the growing season between May and August. The months with the least amount of precipitation are January, February, and October, all with average monthly totals of less than 7 cm .2 and is one of the most densely populated watersheds in the United States, with 442 persons/km2 [Narragansett Bay is the largest estuary in New England and is located primarily in Rhode Island; however, 60% of the watershed contributing to the Bay is in Massachusetts. The Narragansett Bay Watershed (NBW) area covers 4421 kmsons/km2  and rougsons/km2 . Three msons/km2 . RoughlyThe four case studies discussed herein were independently conceived and executed from this study, and represent an array of aquatic ecosystem conditions, as well as temporal and spatial scales. Consequently, each study relied on a unique set of response variables and metrics. These site-level response metrics were derived independently to characterize and represent stressors and impacts within the focal watershed. Taken collectively, these differences present an opportunity to comprehensively evaluate how the watershed and catchment integrity indices perform across a range of conditions and settings typical of those encountered in the United States and abroad.Eighteen watershed attribute response variables or metrics were developed in the CRW: nine nitrogen-related response variables, five response variables related to the physical habitat of streams, three stream temperature response variables, and a multimetric fish index . Descrip15N/14N expressed as δ15N) measured from chironomids collected at 31 stream site locations within the CRW from 2013–2015 [Nitrogen-related response variables included the monthly average and range in monthly average nitrate concentrations at 53 stream sampling sites ,37, tota013–2015 ; see detPhysical habitat data were collected from 2013–2015 for 20 streams within the CRW during the summer low-flow season. The following two stream physical habitat metrics were extracted for comparison and analysis to capture functional processes of interest that had been previously identified by Kaufmann  and KaufStream water temperature was measured using temperature loggers  placed in a well-mixed portion of the stream channel following methods of Dunham et al. . The maxFish sampling data used to derive multimetric indices (MMIs) in the Calapooia basin were collected between 2010 and 2014 using methodologies consistent with those used by US EPA’s Environmental Monitoring and Assessment Program (EMAP)  and the There have been numerous studies evaluating the various methods for characterizing and measuring the physical, biological, and chemical interactions and connectedness of wetlands and streams, principally for the determination of wetland jurisdiction and protection ,46–50. Thttps://www.fws.gov/wetlands/data/Mapper.html.For this study several hydrologic connectivity metrics (described below) were developed using GIS data layers to measure the interconnectivity of streams and wetlands within the Choptank Study area . By measSince riverine wetlands are connected to waterbodies by definition, these wetland polygons were removed (there were no estuarine or marine wetlands in the study area) from the NWI V2 GIS data layer and analysis, leaving only palustrine wetland polygons. Therefore, our analyses focus on quantifying ‘non-riverine’ wetland and stream connectivity at the catchment scale. Two different stream network representations were used to characterize the Choptank study area to evaluate if this affected connectivity estimates and subsequent correlations with the IWI/ICI values: the 1:100,000-scale NHDPlusV2; and the semi-automated high resolution Light Detection and Ranging (LiDAR) derived stream network developed by Lang et al. .Based on numerous wetland connectivity studies and a body of literature ,51–54 weFive nitrogen metrics  were calculated using two different summary metrics , for a total of 10 nitrogen-related response variables . The dat43−, NO3-N (as NO3+NO2−N), NH4+, dissolved organic carbon (DOC), SO42−, Cl−, Ca2+, Mg2+, Na+, and K+, and unfiltered samples were used to measure total P and total N using US EPA approved protocols [3-N were measured using the cadmium reduction method and NH4+ was measured using the phenolate method. Of the 77 sampled stream sites, 71 sites were associated with IWI/ICI values; Cl−, TN, NO3-N and NH4+ were chosen as response variables reflecting watershed development impacts and anthropogenic sources of nitrogen in streams to relate to IWI/ICI and six associated functional component indices  [15N/14N expressed as δ15N; see Periphyton was simultaneously collected at the same 77 stream sites within NBW where single grab sample surface water chemistry was collected (described in section above) between July and October 2012 . Stable nd δ13C) . Of the 15N and δ13C of benthic organic matter (BOM) collected from surficial sediments in lakes are associated with increases in impervious surface and population density and decreases in forested land in watershed and buffer zones surrounding lakes [15N and δ13C in periphyton  for each stream segment that was sampled. To reduce spatial autocorrelation with nested sampling sites, in cases where there was more than one sampling site within a catchment the sampling site that was the furthest downstream within the catchment was used for the response variable value. For lake samples (sampled only in the Narragansett Bay watershed) we associated the IWI/ICI values for the NHDPlusV2 stream segment that runs through each lake waterbody with the corresponding sampled lake site.Response variables from each study site were also examined for correlation with select individual landscape explanatory variables from StreamCat  using the unique COMID for each stream segment sampled. This was done because any effect observed with the IWI or ICI could be due to one of these dominant land use variables. In such a case, development of the IWI or ICI, which is complex and requires significant effort, would be unnecessary. Comparing correlations between response variables and both the integrity indices and the separate land use indicators allowed us to determine whether there was value added from the IWI and ICI. For each response variable and scale (watershed or catchment), we calculated the following: %Max, equal to the IWI or ICI absolute correlation value divided by the maximum absolute correlation value; Rank, which is the rank of the IWI or ICI absolute correlation value; and #Exceed, or the number of individual landscape variable absolute correlations that the IWI or ICI absolute correlation exceeded.r) to measure the strength of the linear relationship and association between the IWI/ICI values and the response variables within each watershed. The six functional components or indices associated with each of the IWI and ICI values were also compared to each of the response variables using Pearson’s correlation coefficient. One exception was for the Choptank Watershed study area; because the response variables developed within this watershed were not normally distributed, Spearman’s rank correlation coefficient  was used as a non-parametric rank statistic to measure the strength of the association between response variables and the IWI/ICI and six functional component indices.We calculated Pearson’s correlation coefficient , for a total of 406 correlations. We characterize the strength of these correlations using five classification categories: ‘very strong’ (0.80–1.0); ‘strong’ (0.60–0.79); ‘moderate’ (0.40–0.59); ‘weak’ (0.20–0.39); ‘very weak’ (0.0–0.19). All correlation analyses were performed in R Software Version 3.4.1 ; all datntegrity .Maps of the four case study watersheds show the entire range and spatial distribution of the indices of watershed integrity and catchment integrity throughout each entire watershed . The comEach of the case study watersheds exhibited a more limited range of IWI and ICI values when considered for the specific sampling sites where response variables were obtained. The lowest IWI/ICI values for sampled sites of 0.40 and 0.39, respectively, were in the Choptank study area watershed and the highest IWI/ICI values of 0.98 and 0.98, respectively, were in the Calapooia watershed . The medOf the 29 response variables from the four case study watersheds, 15% of the response variables demonstrated ‘very strong’ (0.80–1.0) correlations, 26% demonstrated ‘strong’ (0.60–0.79) correlations, 32% exhibited ‘moderate’ (0.40–0.59) correlations, 15% revealed ‘weak’ (0.20–0.39) correlations and 12% of the response variables demonstrated ‘very weak’ (0.0–0.19) correlations with IWI/ICI values and their associated six functional component indices –10. Resur), of −0.97 and −0.96, respectively, indicating that as TN input increases IWI and ICI decreases. All six of the associated functional component indices for IWI and ICI were also significantly correlated with TN input with |r| values ranging from 0.92 for CCONN  to 0.99 for both WCHEM  and WHYD  (r = −0.93) and ICI (r = −0.82) values is the metric log10Ndif, which measures the log of the fluctuation in annual stream nitrate concentration from 54 streams within the Calapooia watershed (r = −0.92) and ICI (r = −0.77) (15N) in chironomids collected from 22 streams within the Calapooia watershed also demonstrated a highly significant negative correlation with IWI (r = −0.92) and ICI (r = −0.89) values . Four additional stream response metrics including percent riparian vegetation cover (vegcovrip), sediment embeddedness (sedembed), maximum summer stream temperature (max_tempC_summer) and index of thermal regime timing (phase), all exhibited strong correlations with IWI values with |r| values ranging from 0.65 (vegcovrip) to 0.77 (phase and sedembed) and ICI values ranging from 0.52 (vegcovrip) to 0.91 (max tempC_summer). All the six functional components associated with the IWI and ICI values demonstrated similarly strong correlations for each corresponding response variable metric  , Table 7atershed . The mon) values , Table 7s = 0.49) while the area of wetland intersecting stream channels in the catchment metric (wetareasqm) demonstrated the strongest positive correlation with IWI (rs = 0.36)  , Table 8 = 0.36) , Table 8Eight of the ten nitrogen metrics measured in the EFLMR demonstrated significantly negative correlations with IWI, while nine of the ten were significant for ICI. The strongest negative correlations were associated with annual average of total nitrogen (log10Tavg) and IWI (r = −0.78), and the annual range in total nitrate/nitrite (log10TNoxdif) and ICI (r = −0.61). All the IWI and ICI associated six functional component indices demonstrated similar strengths and correlations with each of the nitrogen metrics , Table 93-N and NH4+) and watershed development metrics (Cl−) measured in streams within NBW all demonstrated significant negative correlations with IWI, ICI and their six associated functional component indices. Log10 chloride concentrations had the strongest negative correlations with IWI and ICI with r = −0.68 and r = −0.57, respectively. Of the nitrogen metrics, log10 NO3-N concentrations exhibited the strongest negative correlation with IWI (r = 0.60) and ICI (r = −0.52) values. The measured nitrogen-stable isotope ratios (δ15N) of periphyton (pN15) also demonstrated a significant negative correlation with IWI and ICI values with r = 0.47 and r = 0.35, respectively  collected from lakes showed a significantly negative correlation with IWI and ICI values with r = −0.58 and r = −0.64, respectively. All the six functional components associated with IWI and ICI showed similar relationships as with the IWI and ICI correlations for the δ15N BOM  associated with response variables at the watershed and catchment scales varied widely across locations . HoweverIn this study, we examine how a range of response variables measured in four watersheds in four different ecoregions across the CONUS that have different geophysical and ecological conditions compare with national indices of integrity. These response variables varied across case study watersheds thematically  and were measured over varying temporal scales , spatial scales, and ecosystems . Using these independently collected response variables across characteristically different watersheds allows us to evaluate the robustness of this national index of watershed integrity.very strong (0.80–1.0) correlations with 57% of the response variables significantly correlated with IWI/ICI values. The CRW nitrogen-related response metrics were all significantly and negatively correlated with the IWI/ICI values, with |r| for IWI ranging from 0.92 to 0.97, and 0.82 to 0.96 for ICI. Comparing nitrogen-related response metrics across watersheds  with varying sources of nutrients , and varying temporal scales , it is noteworthy that the majority (74%) of nitrogen-related metrics exhibited significantly moderate to very strong (|r| = 0.40–1.0), negative linear correlations with IWI and ICI values. Comparing site level response metrics across watersheds, for example, NO3 concentration, which was measured at three of the four watersheds, reveals a pattern of lower correlation strength with the IWI as the size of the watershed increases . The IWI exhibiting higher correlations with stressors in systems where there are fewer risk factors could be related to the complex and highly variable nature of antagonistic interactions where multiple stressors (risk factors) exist. As systems get bigger  their degree of disturbance increases , and as disturbance increases, the correlations with IWI decrease.Comparing response variable correlations with IWI/ICI values within each case study watershed, the CRW had the highest percentage of r = 0.40–1.0) with IWI and ICI values. Stable isotope ratios of periphyton and stream macroinvertebrates  have been shown to be effective indicators of watershed development effects on stream ecosystems [Another nitrogen-related response metric that demonstrated strong negative correlations with IWI and ICI values that can be compared across watersheds is the measured nitrogen-stable isotope ratios of chironomids (in CRW streams), periphyton (in NBW streams), and benthic organic matter (in NBW lakes). Again, the nitrogen sources varied , as well as the aquatic ecosystems , and temporal scales , yet 83% of the nitrogen-stable isotope ratio response measures revealed moderate to very strong negative correlations . Relative to issues of data source resolution, it is reaffirming that the correlation relationships with IWI and ICI values were very similar regardless of the resolution of stream characterization method. The wetpercentage response variable developed for the Choptank watershed study area is the variable that most pragmatically represents aquatic connectivity as it quantifies the connection between streams and wetlands within each catchment. Considering that the Choptank watershed study area is essentially at sea level with very little elevation, it makes intuitive sense that the catchment values  would be more closely correlated as the catchment would be more influential absent any effects from upstream watersheds.2 values for the 18 nitrogen response metrics , IWI accounts for 39% and ICI accounts for 32% of the variation in nitrogen response across the Calapooia River, East Fork Little Miami River and Narragansett Bay watersheds. In the Calapooia River watershed, IWI accounts for 88% and ICI accounts for 80% of the variation in the nitrogen response metrics.In a previous study, Thornbrugh et al.  used sit2 = 0.27) with the water quality indicator, followed by the % agriculture at the watershed scale (adjusted R2 = 0.25), while the % urban at the watershed scale exhibited a much weaker linear relationship (adjusted R2 = 0.01). These analyses demonstrated that there was an added benefit or value in applying the watershed integrity index to explain the variance in water quality, versus using individual landscape indicators to characterize aquatic condition . In thiRecently Aho et al.  conductehttps://www.epa.gov/hwp/download-2017-preliminary-healthy-watersheds-assessments). The IWI and ICI can also be applied in conjunction with the US EPA’s Recovery Potential Screening (RPS) (https://www.epa.gov/rps) which provides technical methods and tools for comparing large numbers  of hydrologic unit codes (HUC) based on the USGS HUC framework, a dataset based on drainage subdivisions of land surface areas at several hierarchical levels [Within the US there are opportunities to apply this watershed integrity approach in coordination with the US EPA’ s Healthy Watershed Program which uses integrated assessments ranging from screening-level assessments using GIS data layers to statistical and geospatial modeling of ecological attributes .One of the main objectives of the US Clean Water Act (CWA), is “to restore and maintain the chemical, physical, and biological integrity of the nation’s waters”. The intent of the term “integrity” in the CWA was to recognize the importance of preserving natural ecosystems that support key watershed processes, water quality, and the condition of aquatic ecosystems . Much ofThis study demonstrates that the two integrity indices are related to site-scale response variables for streams, lakes, and wetlands and across four study areas that vary in geophysical and ecological conditions. In contrast to three other landscape variables, the integrity indices are robust since they incorporate multiple stressors and so perform well across all four locations. An empirically based, national-scale mapping and assessment of watershed integrity provides EPA and state programs with additional information for fulfilling objectives of the CWA and supporting tenets of the Healthy Watersheds Program by providing a consistent and systematic geospatial framework for evaluating watershed integrity based on risks to key watershed functions. A nationally consistent map of watershed integrity facilitates comparisons among regions and can assist states and other agencies in identifying and prioritizing protection for healthy watersheds, as well as targeting critical functional elements of watersheds for restoration efforts.Supplement1


Klebsiella pneumoniae meningitis, bacteremia, and hepatic abscess. Despite intravenous antibiotic therapy and percutaneous drainage of the hepatic abscess, the patient developed worsening meningitis with ventriculitis, which led to her death. This invasive Klebsiella syndrome is characterized by metastatic infections with a hypervirulent strain and a poor clinical prognosis. Important elements of this patient’s case—including the widespread and highly aggressive course—and of those reported in the literature are reviewed. Although the invasive Klebsiella syndrome is an increasingly recognized clinical entity, most often in the Eastern hemisphere, it remains rare in the United States with approximately 20 cases reported in the literature, and given its high morbidity, it is vitally important for clinicians to recognize.In this article, we describe the case of a 61-year-old woman who presented with altered mental status and was found to have  Klebsiella pneumoniae causes nosocomial and community-acquired infections, including pneumonia, bacteremia, urinary tract infection, surgical wound infections, liver abscess, cholangitis, and peritonitis. K pneumoniae has also been increasingly recognized as a cause of community-acquired pyogenic liver abscess, in the absence of underlying hepatobiliary disease, especially in patients from Eastern countries  and in those with underlying risk factors such as diabetes mellitus.1 Metastatic infection has also been associated with K pneumoniae liver abscess.2 However, the biology, and especially the natural history of K pneumoniae liver abscess and associated invasive syndrome is substantial, with mortality rates ranging from 3% to 42%.3 Here, we report on a patient with invasive K pneumoniae syndrome with pyogenic liver abscess associated with meningitis and ventriculitis that was fatal.Pyogenic liver abscesses are typically treated with management of the underlying condition , drainage, and antibiotics and typically have an excellent prognosis.A 61-year-old Chinese female with no known past medical history presented to the hospital with altered mental status for 1 week with associated fever, neck pain, nausea, and vomiting. It was not possible to obtain history from her due to her altered mental status; her family denied complaints of abdominal pain, headache, visual changes, focal weakness, chest pain, or dyspnea. She had been waking up in the middle of the night to cook meals and clean her house, and was intermittently somnolent. The patient did not smoke, drink alcohol, or use illicit drugs. She was born in China and immigrated to the United States, where she resided for the past 30 years with no foreign travel during that time. She had not seen a physician in her adult life and took no medications. Given her altered mental status and concern about the stability of her airway, she was intubated in the emergency department.2 (fraction of inspired oxygen) of 40%. Physical examination demonstrated a positive Brudzinski sign and neck stiffness, even while sedated. Pupils were equal round and reactive to light, and she responded to painful stimuli. Her lungs were clear to auscultation bilaterally, and cardiac examination was unremarkable without a murmur. Her abdominal examination was normal with no hepatomegaly or ascites.On admission to the intensive care unit, the patient’s vital signs were as follows: temperature 36.5°C, heart rate 90 beats/min, respiratory rate 22 breaths/min, blood pressure 108/61 mm Hg, and oxygen saturation of 100% on an FiO3 (81% neutrophils), hemoglobin 11.8 g/dL, platelet count 170 000/µL, creatinine 0.5 mg/dL, bilirubin 0.6 mg/dL, aspartate transaminase 49 IU/L, alanine transaminase 81 IU/L, alkaline phosphatase 142 IU/L, and albumin 1.9 g/dL. Electrolytes were within normal limits. C-reactive protein was 4.19 mg/dL, and erythrocyte sedimentation rate was >100 mm/h. She had mild hyperglycemia on admission, with glucose 132 mg/dL and hemoglobin A1c of 6.4%.Admission laboratory data demonstrated white blood cell count of 19 900/mm2O, a white blood cell count of 5051/mm3 (neutrophils 87%), protein 803 mg/dL, and a glucose 4 mg/dL. CSF culture was positive for K pneumoniae.Magnetic resonance imaging of the brain demonstrated diffuse leptomeningeal enhancement most conspicuous along the bilateral temporal lobes and insula, subarachnoid space of the basal cisterns, and the ventricular system, and proteinaceous material and pus were visualized within the subarachnoid space and bilateral lateral ventricles . NeurosuK pneumoniae; a catheter was placed into the hepatic abscess. Blood cultures were positive for K pneumoniae.Magnetic resonance imaging of the abdomen demonstrated a complex multiloculated cystic space occupying lesion 6.3 × 4.6 cm with a small amount of surrounding edema and no intrahepatic or extrahepatic biliary dilatation . PercutaShe was initially treated with meropenem and intrathecal gentamicin. She subsequently developed seizures. With concern of lowered seizure threshold from meropenem, this was changed to cefepime. Ciprofloxacin was added as she developed worsened mental status, and repeat central nervous system (CNS) imaging revealed increasing leptomeningeal enhancement and fluid within the occipital horns, consistent with worsening meningitis and ventriculitis. Her external ventricular devices repeatedly clogged and a total of 6 external ventricular drains were placed. Thirty days after admission, she developed diabetes insipidus, uncal herniation, and progressed to brain death.Klebsiella pneumoniae is recognized as a common pathogen in both nosocomial and community-acquired infections. It is a classic cause of pneumonia, especially in alcoholic and diabetic patients. It is also well known to cause bacteremia and pyogenic liver abscess. To date, some 800 cases of K pneumoniae liver abscess have been reported from Eastern countries and Southeast Asia, but a substantially lower number have been reported in Europe, North America, and South America.1 Several case series in the United States have evaluated pyogenic liver abscess, with those involving K pneumoniae being reported to occur in 7% to 35%.5 A very small proportion  of those reported in the United States appear to have been caused by the hypervirulent strain.6K pneumoniae infection has also become a major cause of community-acquired meningitis in Asia, and remarkably has been linked to pyogenic liver abscess.3 Particularly noteworthy are the points that invasive K pneumoniae syndrome is associated with primary hepatic abscess and systemic infection is associated with a high morbidity and mortality.K pneumoniae was first reported in Taiwan starting in the mid-1980s, where patients without hepatobiliary disease presented with community-acquired hepatic abscess with a propensity for disseminated spread.6 This particular strain appears to be different from classic K pneumoniae strains, because it has a propensity for metastatic spread to unusual sites and because it often occurs in previously healthy hosts3; the metastatic spread and aggressive nature of the disease has led to the term “Invasive Klebsiella Syndrome.” It has been proposed that the syndrome is “definitive” in the presence of a K pneumoniae liver abscess with extrahepatic infection, including in the CNS, endophthalmitis, or necrotizing fasciitis.1 Our patient met the clinical criteria for the definitive invasive syndrome as evidenced by liver abscess and concomitant meningitis and ventriculitis.This hypervirulent strain of K pneumoniae liver abscess have been reported to develop metastatic infection at a rate of 8% to 24%,2 with the most common metastatic foci being meningitis or endophthalmitis1 in the invasive syndrome. In addition to meningitis, other CNS infections including brain abscess and subdural empyema have been described in the metastatic syndrome.3 Other sites of systemic infection include the lungs (septic pulmonary emboli or empyema), musculoskeletal system ,1 or the spleen (splenic abscess) or the peritoneum.7 High mortality has been noted in patients with meningitis, septic pulmonary emboli, or empyema.2Patients with primary 2 Interestingly, abdominal pain and gastrointestinal symptoms are not always present in patients with the invasive form of the disease.1 Importantly, several risk factors have been put forward for the invasive syndrome, including Asian descent and diabetes mellitus.1 The invasive syndrome has been reported to be prominently associated with diabetes mellitus.5 Additionally, typical laboratory findings include leukocytosis, thrombocytopenia, hyperglycemia, abnormal liver function tests, and elevated C-reactive protein. Our patient was of Asian descent and had impaired glucose tolerance without meeting diagnostic criteria for diabetes mellitus.The most common clinical manifestations in patients with liver abscesses are fever, chills, abdominal pain, nausea, and vomiting.K pneumoniae strain and also serotypes K1 or K2.1 This hypermucoviscous variant phenotype is identified by a positive “string test” in the laboratory. The positive “string test” is defined as an inoculation loop generating a viscous string greater than 5 mm in length when manipulating bacterial colonies on an agar plate.3 Our patient’s colonies were reported to exhibit a positive string test. It has been hypothesized that the hypermucoviscosity phenotype of the hypervirulent K pneumoniae strain may make catheter drainage difficult.3 In our patient, we hypothesize that the high viscosity of this organism likely explains the difficulty draining the hepatic abscess and the recurrent clogging of the external ventricular drains with need for repeated drain replacement.Patients with the invasive syndrome have been found to have a hypermucoviscous K pneumoniae abscess includes the cephalosporins.1 Extended spectrum β-lactamase producing K pneumoniae are rare but can be treated with carbapenem therapy.2 The most appropriate duration of therapy ranges from a minimum of 2 to 4 weeks for solitary liver abscess to over 6 weeks for complicated (multiple) abscesses. In patients with diabetes mellitus, strict glycemic control appears to be important as this can prevent development of extrahepatic complications.1 Treatment of metastatic infection is typically guided by the site of foci involved.The most effective antibiotic for treatment of K pneumoniae syndrome are substantial, with mortality rates ranging from 3% to 42%.3K pneumoniae meningitis has a particularly high mortality, with rates from 38% to 91%.8 Thrombocytopenia has been associated with a 3-fold increased mortality rate over patients with normal platelet counts (37% vs 11%).5Morbidity and mortality from the invasive K pneumoniae syndrome is unclear. Given the predilection for those of Asian descent, possibilities include genetically predisposed susceptibility or perhaps pathogen exposure based on geography. Studies have demonstrated that a substantial proportion of individuals of Asian descent are colonized with hypervirulent K pneumoniae strains, with the dominant site of colonization being the gastrointestinal tract.3 For example, studies of fecal carriers of K pneumoniae in healthy Chinese people have found a prevalence of K pneumoniae of 75% in healthy adults with high prevalence of the K1 or K2 serotype isolates (23%), which is considerably higher than prevalence in European fecal samples (10% to 19%).1 Thus, it is possible that colonization with the virulent K1 or K2 serotype may predispose to translocation of K pneumoniae in the setting of dysregulated gut barrier function from the gastrointestinal tract to the hepatic circulation.9The reason for the aggressive pathogenesis of the invasive Klebsiella syndrome, particularly with its increasing spread outside of Asia. Additionally, microbiologists should be aware that K pneumoniae with hypermucoviscous phenotype suggests an invasive K pneumoniae strain,1 which should prompt notification of clinicians to ensure appropriate screening for extrahepatic manifestations. Additionally, in any patient with K pneumoniae meningitis, endophthalmitis, or other extrahepatic infection (particularly in those of Asian descent or with uncontrolled diabetes mellitus), patients should be investigated for possible liver abscess. By recognizing the features of the invasive Klebsiella syndrome, rapid diagnosis and treatment may improve outcomes.It is important for clinicians to be aware of the invasive K pneumoniae syndrome is associated with significant morbidity and mortality. Atypical Klebsiella infections associated with this invasive syndrome include meningitis, endophthalmitis, and necrotizing fasciitis. Additionally, in patients with atypical Klebsiella infection, a high index of suspicion for the presence of pyogenic liver abscess is important. Although the disseminated K pneumoniae has been previously reported in the literature, our case demonstrates the importance of recognizing disseminated K pneumoniae infection as part of an invasive syndrome with unique extrahepatic manifestations. Finally, this disease causes significant morbidity and mortality and appears to be underappreciated by clinicians in Western nations. Indeed, early recognition of the disorder is vitally important.The invasive


Vibrio cholerae hazards were poorly associated with plate count faecal coliforms, but flagged up by the MinION screening method, and confirmed by a qPCR assay. Plate counting methods remain important to assess viability of faecal coliforms in disinfected water sources. We outline a systematic approach for data collection and interpretation of such complementary results. In the Kathmandu Valley, there is high variability of water quality from different sources, including for treated water samples, illustrating the importance of disinfection at the point of use.Nucleic acid based techniques, such as quantitative PCR (qPCR) and next generation sequencing (NGS), provide new insights into microbial water quality, but considerable uncertainty remains around their correct interpretation. We demonstrate, for different water sources in informal settlements in the Kathmandu Valley, Nepal, significant Spearman rank correlations between conventional and molecular microbiology methods that indicate faecal contamination. At family and genera level, 16S rRNA amplicon sequencing results obtained with the low-cost, portable next generation sequencer MinION from Oxford Nanopore Technologies had significant Spearman rank correlations with Illumina MiSeq sequencing results. However, method validation by amplicon sequencing of a MOCK microbial community revealed the need to ascertain MinION sequencing results for putative pathogens at species level with complementary qPCR assays.  In Nepal waterborne diseases account for 15% of all illness and 8% of total deaths2. With waterborne diseases accounting for 38% of deaths in children under the age of five3, water quality in Nepal is a major public health concern6. According to the Central Bureau of Statistics, Government of Nepal, one out of five families in the Kathmandu Valley does not have access to municipal drinking water, and in most areas availability is <4–7 h/week7. Many people depend on alternative sources such as groundwater from boreholes and wells, stone spouts, bottled drinking water or water supplied by delivery truck8. The quality of these water sources is not routinely monitored.An estimated 1.8 billion people are still exposed to drinking water sources contaminated with faecal matter9. Escherichia coli are World Health Organization (WHO) recommended faecal indicator bacteria for drinking water10, while Enterococci are indicator bacteria for bathing water11. FIO are used because there is good epidemiological evidence that they correlate with disease outcomes13. However, FIO are a poor proxy for organisms with quite different physiologies (e.g. viruses and protozoa) and it is difficult to distinguish FIO from human or animal sources14. The routine isolation of all pathogens is impractical15, as each requires a unique microbiological isolation technique17. In addition, culture dependent approaches may require long incubation periods, and there is a demand for more rapid and comprehensive screening methods to detect FIO and/or their markers14 and putative pathogens in water samples21. Culture independent methods such as quantitative real-time PCR (qPCR)14, next generation sequencing (NGS)22, DNA hybridisation platforms and immunoassays23 allow direct measurement of cellular properties that may identify pathogens  without incubation. These methods may reduce the detection and quantification time to a few hours24. NGS methods potentially allow simultaneous detection of gene fragments from different types of faecal indicators and putative pathogens, such as thermo-tolerant coliforms, faecal coliforms, Vibrio cholerae, Streptococci, Bacteroides, etc., especially through 16S rRNA gene amplicon sequencing (from here on referred to as amplicon sequencing). However, NGS techniques are typically more expensive and require more sophisticated equipment and reagents than culturing methods26. Also, NGS techniques may have limited taxonomic resolution; due to an inability to capture the near-complete genomes of rare taxa in shot-gun sequencing or an inability to reliably classify taxa down to the species level with amplicon sequencing due to the relatively short fragment sizes that can be sequenced on some NGS platforms, or the presence of highly conserved 16S rRNA genes in some families and genera28. This compromises their ability to reliably detect waterborne hazards. To circumvent some of these limitations, different approaches can be combined. For instance, Cui et al. combined Illumina-based amplicon sequencing and qPCR to evaluate the pathogen diversity in urban recreational water29. Ahmed et al. used host associated molecular markers with qPCR and Illumina-based amplicon sequencing to identify faecal pollution sources in environmental waters in Brisbane, Australia30. An exciting development for NGS is the MinION, a low cost, memory-stick sized real time sequencer from Oxford Nanopore Technologies Ltd. Its portability opens up the possibilities for sequencing to be done in the field. Recently, Hu et al. compared culturing, MinION shotgun sequencing and Illumina MiSeq amplicon sequencing methods to trace faecal contamination from wastewater in urban stormwater systems24. They demonstrated high correlations between E. coli culturing counts, the relative abundance of human gut microbiome related amplicon sequences, and the frequencies of human gut microbiome genes from shotgun sequencing data. However, they did not assess their method validity using samples of known composition. When applied to drinking water quality monitoring, qPCR and NGS data were not always found to be well-correlated with culture based methods31. Reliable identification of putative pathogens at species level remains a challenge, especially for water samples containing low amounts of DNA.The current standard method for microbial examination of both drinking and bathing water requires the isolation and enumeration of organisms that indicate the presence of faecal contamination To address these uncertainties, we first evaluated various molecular microbiology methods  using samples of known composition (i.e. MOCK communities containing DNA of FIO and putative pathogens). Since our samples of interest would include groundwater and treated drinking water samples with low amounts of DNA, we focused on the evaluation of qPCR and NGS 16S rRNA amplicon sequencing methods. We combined these molecular with conventional methods to assess water quality for different types of household water sources in the Kathmandu Valley, Nepal. To our knowledge, this is the first study using amplicon sequencing with the MinION for microbial water quality analysis, and comparing its results with those obtained with other methods and known sample compositions. Based on the results, we propose a tool-box approach to make the most of complementary technologies for water quality monitoring in areas with a significant waterborne disease burden.Escherichia coli, and Enterococcus faecalis, respectively) and other putative pathogens   and E. coli (i.e. rodA), but not Vibrio cholerae and human E. coli genes. These results are consistent with the true or actual composition of the MOCK community, since the E. coli DNA in the MOCK community was not from a strain associated with the human gut microbiome.For method validation, we used a MOCK community consisting of genomic DNA from eight bacterial and two fungal species. The MOCK community included Gram-negative and Gram-positive faecal indicator bacteria (a) Table . The pera) Table . Figure ty Table . In party Tables  and S2. % Tables  and S2. % Tables  and S2. Bacteroides, Prevotella, Enterobacteriaceae), and other groups containing putative pathogens , distinguished at family (Enterobacteriaceae) or genus level . A significant rank correlation was observed  between the relative abundances of Bacteroides, Prevotella, Enterobacteriaceae and all other putative pathogenic genera determined by MinION and Illumina NGS , but not with human E. coli qPCR  gene copy numbers. Likewise, Enterobacteriaceae MinION data , but not with human E. coli qPCR  gene copy numbers. As expected from the MOCK community results, only the NGS results from the MinION contained several reads aligned with putative pathogens at species level , which were also the only samples for which NGS with the MinION matched some reads to Vibrio cholerae at species level. With regards to the agreement between molecular and plate count methods, all MinION NGS data had a significant and positive correlation with the total coliform plate count data . Positive correlations were also observed between faecal E. coli plate count and total coliform qPCR, total E. coli qPCR, and human E. coli qPCR data, respectively . The correlation between faecal E. coli plate count and total E. coli qPCR data was particularly strong . It is noteworthy that Vibrio cholerae MinION and Vibrio cholerae qPCR data were negatively correlated with the faecal E. coli plate count  and molecular microbial water quality assessment methods was thus generally good, there were noteworthy discrepancies for disinfected water samples. Water sampled from location 2, 3 and 13 were disinfected waters , and showed no evidence of thermo-tolerant coliform bacteria by the plate count method (Table NGS (Illumina and MinION), qPCR and plate count methods were combined to investigate microbial water quality for thirteen water sources in the Kathmandu Valley, Nepal Fig. . The datGS Table 21. When ata Fig.  had a siata Fig.  had a siata Fig.  were welata Fig. , which wunt Fig. . Consequod Table .Figure 3Exceedances of the Nepalese guidance values were observed for the following chemical water quality parameters and locations: Nitrate in water from the stone spout (location 11) and a tube well (location 8), ammonia in one tube well (location 7), iron in location 5 (deep borehole) and location 7 (tube well), manganese in location 5 (deep borehole) and location 7 (tube well), and aluminum in location 4 (tube well). More details are provided in Tables 27. In this study, the presence of the genera Escherichia and Salmonella in the MOCK community was not identified from the Illumina data, and none of the sequencing reads from Illumina were classified to species level. In the context of water quality monitoring, this may then result in false negative outcomes at genus and species level for these putative pathogens. MinION sequencing has the advantage of a longer amplicon size, but the disadvantage of a reported higher error rate/lower accuracy than Illumina sequencing27. These MinION sequencing errors caused significant underreporting of the relative abundance of actual MOCK community members such as Escherichia coli. At the same time, assignment of partially erroneous reads to species by the MinION 16S bioinformatics workflow resulted in mismatched identities and taxonomic classification at species level, i.e. false positive results. While MinION sequencing detected all the species present in the MOCK community, these species were not always those that were most frequently detected within a certain genus. In particular, reads falsely classified as Escherichia fergusonii had a higher relative abundance than Escherichia coli. Phylogenetic analyses revealed that the 16S rRNA sequences for different strains of E. coil, E. fergusonii, and other strains from the genera Shigella, Citrobacter, and Salmonella, are closely related  would make it difficult to reliably distinguish related species, such as those within the Enterobacteriaceae, that share a high sequence similarity. Recently developed methods such as Metagenomic phylogenetic analysis (MetaPhlAn) for species-level profiling in large scale microbial community studies36 or Electronic probe Diagnostic for Nucleic acid Analysis (EDNA) to detect pathogens in metagenomics database37 might overcome these challenges and contribute in reliably detecting putative pathogens at species level. In addition, both of these techniques use species-specific markers, and can decrease the probability of both false positive and false negative profiling of microorganisms. When considering the implications of these findings for water quality analysis, Illumina sequencing with its shorter read length may result in false negative results, i.e. E. coli is present in the sample, but not detected at genus and species level. MinION sequencing with its longer read lengths, but lower accuracy, may result in underreported abundances (i.e. E. coli), and also false positives (i.e. E. fergusonii and other closely related species). Although both NGS methods for 16S rRNA amplicon sequencing, with their associated bioinformatics platforms, have short-comings when it comes to reliably establishing identities at species level, they can provide initial insight into the likely microbial community composition, which is not readily available from other analytical methods. While species level identities need to be interpreted with great caution, more reliable information is obtained at genus and family level, and can guide further investigations of microbial water quality. A combination of NGS for screening, and qPCR methods for validation, can thus help identify false negative or false positive results. When using total 16S rRNA, total coliforms, total E. coli, human E. coli and Vibrio cholerae marker gene primers to analyse the MOCK community, the results were consistent with the true composition of the MOCK community. For example, detection of E. coli by qPCR would help identify one of the Enterobacteriaceae species in the MOCK community, which Illumina NGS had classified only to family level. The absence of the cholera toxin excretion protein gene by qPCR would flag up a few MinION NGS reads mis-classified as Vibrio cholerae as potential false positives. For the water samples from the Kathmandu Valley, the Spearman rank correlations between various methods to target FIO were generally positive and significant in this study, in line with previous findings38. However, there were discrepancies between thermo-tolerant coliform bacteria by the plate count method, and those analysed with qPCR for several of the disinfected water samples. These discrepancies suggest that coliform bacteria were inactivated by the disinfection, but inactivated cells and/or their DNA were still present in the water. For example, DNA based methods such as qPCR and NGS (both Illumina and MinION) will not differentiate between viable and dead bacterial cells, or extracellular DNA, and therefore may have poor comparability with culture based techniques31, especially in disinfected waters. In such instances, the combined information of DNA based and culture based methods adds meaningful value to the water quality survey, because it suggests that some water samples were fit for consumption due to treatment (i.e. disinfection by the bottling company), but that the water sources were influenced by faecal contamination. Nucleic acid extraction methods incorporating PMA (propidium monoazide) can distinguish between active/live and dead bacteria, and may provide an avenue for the reliable detection of viable pathogens in future work39. Other minor discrepancies between various methods may also be due to method limitations , which were also the only samples for which NGS with the MinION matched some reads to Vibrio cholerae at species level. The combined data gives a much higher level of confidence in the presence of potentially pathogenic Vibrio cholerae bacteria in these water samples, than NGS data on its own. The Vibrio cholerae hazard was not detected with Illumina NGS, and Vibrio cholerae MinION and Vibrio cholerae qPCR data were negatively correlated with the faecal E. coli plate count. Screening with MinION NGS can flag up such hazards and guide the choice of subsequent testing protocols, such as qPCR, to verify the hazard. In addition to the method development and cross-comparison we also wanted to demonstrate their applicability to comprehensive water quality monitoring in informal settlements. A discussion of chemical water quality parameters is provided as supporting information. WHO and Nepalese drinking water quality guidelines state that water intended for drinking or in the distribution system for drinking purposes should not contain faecal coliforms in a 100 mL sample42. The water from four locations in the Kathmandu Valley (one community tube well (location 8), spring water (location 9), stone spout (location 11) and one piped supply (location 12)), out of thirteen locations, was contaminated with culturable faecal coliforms, and therefore was not fit for consumption according to standard methods. The water from a spring (location 9) had the highest faecal E. coli concentration. These observations raise concerns, because springs are commonly used as a water source, also for the production of bottled or jar water and even tap water. Molecular methods also identified DNA from faecal bacteria in treated water samples . 16S rRNA gene copy numbers were high in piped water from location 12, indicating a high amount of bacterial DNA in the tap water. Indicator gene copy numbers for the total coliforms were relatively higher as compared to other locations in the water sampled from location 9 (spring water), 10 (delivery truck) and 12 (piped water). Indicator genes for human E. coli pollution were only detected in water sampled from location 9 (spring water) and 12 (piped water). The molecular data raises strong concerns about the quality of spring water as a drinking water source, while total coliforms detected by plate count in one sample from the piped supply , and delivery truck water (location 10) raises strong concerns about the effectiveness of water treatment, as previously reported by other studies8. Clearly, water treatment needs to be more robust, if water sources such as springs have very poor microbial quality. The MinION NGS and qPCR Vibrio cholerae data also raises serious concerns about the safety of water from shallow tube wells . Knowledge of common waterborne disease-causing agents prevailing in the sampling region , UK43. While the majority of OTUs in Illumina data were not resolved at species level, the pattern for the relative abundance of Pseudomonas and Erysipelothrix across sampling locations was consistent with the MinION NGS data for putative pathogen species within these genera, showing an unusually high number of MinION reads matched to Pseudomonas aeruginosa in location 4, and Erysipelothrix rhusiopathiae in location 6. The relative abundance of MinION reads matched to putative pathogens on the HSE list was on average higher in water from tube wells compared to other water types. As mentioned earlier, due to the risk of false positive identifications at species level with the MinION NGS method, additional tests using qPCR and culturing techniques (to check for viability) would be required to confirm the presence of these putative pathogens in the water samples. MinION NGS data is helpful in providing direction for such future work. With careful interpretation and cross-validation of results, NGS data adds significant value to water quality assessments, as demonstrated in this survey. As a low-cost, portable NGS tool generating long sequencing reads, the MinION of ONT is especially promising. The significant rank correlations between the relative abundances of Bacteroides, Prevotella, Enterobacteriaceae and all other putative pathogenic genera determined by MinION and Illumina NGS suggest that the portable, memory-stick sized MinION provides a valid alternative to the Illumina platform, which is that currently most widely used for NGS community-based microbial water quality monitoring and environmental surveillance25. However, further improvements of protocols, nanopore technology, and bioinformatics workflows are needed to improve read accuracy and avoid false assignment of reads to species. For instance, Calus et al. have recently developed a workflow for MinION sequencing to estimate the diversity of MOCK communities with average sequence accuracy of 99.5%. While their library preparation protocol using rolling circle amplification and enhanced bioinformatics dramatically improved the accuracy of MinION NGS, the total number of accepted reads generated per sequencing run was reduced accordingly44. The MinION NGS has already been applied in the field of public and animal health, and water security to identify specific pathogenic strains48. Theuns et al. successfully used MinION NGS as a diagnostic tool for porcine viral enteric disease and revealed porcine kobuvirus as the main enteric disease causing virus in swine45. Rames and Macdonald were able to detect Enteroviruses (EV) in wastewater (WW) samples with MinION sequencing, after spiking EV into the WW prior to sequencing46.Analysis of a MOCK community revealed that Illumina sequencing data showed better taxonomic resolution as compared to MinION data at the family level, while MinION sequencing data showed better taxonomic resolution at genus and species level. The amplicon size of the targeted 16S rRNA gene sequences was shorter with Illumina than those targeted using the MinION, and short amplicon size is known to compromise the achieved taxonomic resolution when classifying sequences to taxaWater samples in this study were collected from the most common water sources used by residents in the Kathmandu Valley. These comprised shallow tube wells, deep boreholes, stone spouts , piped water, water supplied by delivery truck, bottled drinking water, and jar water . Water samples were collected from 13 sites across the Kathmandu Valley. The locations of these water sources are presented in Fig. From each of the sources, 3 litres of water were collected in sterile 1 litre bottles. During sampling, the lid of the sterile bottle was opened and closed aseptically, and bottles were rinsed thoroughly with water from the same source before sample collection. From the piped water supply and stone spout, water was allowed to flow directly into the sterile bottles. For tube wells and boreholes, the water was pumped using a vacuum pump and polyethylene pipe. Sufficient purging was performed to prevent the collection of standing water already present in the pipe. Prior to collecting the water, the outlet of the tube was sterilized using 70% ethanol and flamed with a burning cotton swab, and water was then allowed to flow directly into the sterile bottle. The bottled drinking water and jar water were bought from local vendors. The sample bottles were stored in an insulated cold-box with ice packs inside and transported to the laboratory to be processed within two hours.49. Different volumes of water (250 mL up to 2 L) were filtered through 0.22 µm membranes  depending on the turbidity of the water from each sampling site and were immediately stored at −20 °C to preserve DNA for subsequent molecular microbiology. The molecular work was then conducted at Newcastle University (NU), Newcastle upon Tyne, UK. The total DNA from prokaryotic biomass retained by the membrane was extracted using a PowerWater® DNA Isolation Kit as per the manufacturer’s instructions . DNA purity and concentration were determined using a DS-11 FX + Spectrophotometer/Fluorometer . 30 ng of DNA was used to build a 16S rRNA prokaryote gene sequencing library for nanopore sequencing using a 16S Barcoding kit (SQK-RAB204 from Oxford Nanopore Technologies (ONT), Oxford, UK) as per the manufacturer’s instructions and loaded onto a MinION sequencing apparatus flow cell . Table Total coliform and faecal E. coli bacteria were determined by membrane filtration in duplicate at Nobel College (NC), Kathmandu, Nepal, following Standard Methods for the Examination of Water and Wastewater50. The amplicon data from Illumina were processed using an open source software package: Quantitative Insights Into Microbial Ecology, QIIME 2 (https://qiime2.org/). Denoising and dereplication of pair end sequencing, including chimera removal and trimming of reads based on positional quality scores, were performed using the Divisive Amplicon Denoising Algorithm 2 (DADA2)51. The VSEARCH clustering method was used to cluster the quality-filtered sequences into ASVs (amplicon sequencing variants) that were converted into OTUs (operation taxonomic units), with a threshold of 97% identity52. Finally, taxonomy of each OTU was assigned by matching to the GreenGenes database (v13_8), based on a naïve Bayesian classifier with default parameters.Further, the extracted DNA were also sequenced  in duplicate with an Illumina Miseq platform  using the primer set targeting the V4 region of the bacterial 16S rRNA Table  as descrE. coli, E. fergusonii, closely related other strains from the genera Shigella, Citrobacter, and Salmonella, and a more distant strain  recorded in the NCBI reference database  to a final volume of 10 μL. Reaction conditions for quantification of each target gene were 98 °C for 3 min (1x), then 98 °C for 15 s, and the Primer Annealing Temperature (Ta) for 60 s  were performed to quantify the number of target genes on a BioRad CFX C1000 system  using the primers shown in Table  s Table   approaches was performed in R-studio.Supplementary information


Eucalyptus. FPCs are widely investigated due to their herbivore deterrence properties and various bioactivities of pharmaceutical relevance. Despite the increasing number of studies elucidating new FPCs structures and bioactivity, little is known about the role of those compounds in planta, and the effects of environmental stresses on FPC concentration. Ozone (O3) and wounding are key stress factors regularly confronted by plants. In this study, we investigated how O3, wounding, and their combination affected individual and total FPC foliar concentration of the economically important species Eucalyptus globulus. Six individual FPCs, including five macrocarpals and one sideroxylonal, showed different response patterns to the single and combined stresses. Total macrocarpals only increased under single O3 treatment, whereas total sideroxylonals only increased in response to wounding treatment, suggesting different physiological roles played by the two groups of FPCs predominantly existing in E. globulus foliage. Total FPCs increased significantly under individual wounding and O3 treatments but not under the combined treatment. A principal component analysis indicated that all different treatments had unique FPC fingerprints. Total phenolic contents increased in all O3 and wounding treatments, and a marginally positive correlation was found between total FPCs and total phenolic contents. We suggest that, depending on the concentration and composition, FPCs play multiple physiological roles in planta, including serving as antioxidants to scavenge the reactive oxygen species brought about by O3 and wounding stresses. Formylated phloroglucinol compounds (FPCs) are a class of plant specialized metabolite present in the Myrtaceae family, especially in the genus  Calibration curves covered the range 0.5–75 µM and were used for absolute quantification of the six compounds, macrocarpal A, macrocarpal D, macrocarpal J, macrocarpal L, macrocarpal N, and sideroxylonal A corresponding to the analytical standards. The other FPCs detected were labeled as FPC1, FPC3, etc., and further divided into two major groups, macrocarpals and sideroxylonals due to their predominance in E. globulus foliage  foliage . Total m2O, v/v) was added to 50 mg frozen, homogenized leaf powder on ice and vortexed. After incubating the extraction mixture at room temperature with shaking for 15 min, the mixture was vortexed again and centrifuged at 10000× g for 5 min. The supernatant was transferred to new tubes, 1 mL 50% acetone was added to the pellet, and the extraction procedure was repeated twice. The supernatant of the three extractions was combined and stored on ice in darkness. For the Folin-Ciocalteu assay, gallic acid  was used as the standard. The reaction mixture containing 40 µL standard solution/sample extracts, 1560 µL deionized H2O, and 100 µL Folin-Ciocalteu reagent  was incubated for 8 min at room temperature and 300 µL 20% Na2CO3 (w/v in deionized H2O) was added to each tube and vortexed. After 2 h incubation at room temperature, the absorbance was determined at 765 nm with a Shimadzu UV2550PC spectrophotometer . The total phenolic contents were expressed as gallic acid equivalents (mg of GAE g−1 DM) using the standard curve with gallic acid .The total phenolic contents were determined by a Folin-Ciocalteu assay ,92 with 3, and O3 + wounding combination, and different recovery times on time-dependent changes of the concentration of individual and total FPCs, total macrocarpals and sideroxylonals, the ratio of individual FPCs to the total FPCs, and the total phenolic contents were analyzed separately by using linear mixed models  with treatments  and recovery time as fixed effects. Log10 transformation was used where necessary to satisfy the assumption of the linear mixed model. In all cases, paired comparisons among the levels of the same factor were tested for significance by comparing differences of least squares means, at P < 0.05. Principal component analysis (PCA) was used to evaluate the effects of different treatments on the coordinated changes of individual FPCs concentrations across the recovery times. Loading and score plots were initially derived after mean-centering and cube root transformation by MetaboAnalyst version 3.0 [In this study, three replicates from different plants were used in all the treatments. The statistical impacts of wounding, Osion 3.0 ,94 and t3 and its combination with wounding were shown to influence the abundance of FPCs, resulting in temporal variations of specific compounds in the leaves of the ecologically and economically important tree E. globulus. These novel findings open the debate for different roles of this class of specialized metabolite in abiotic stresses, besides the defense against herbivores. The two predominant FPC classes in E. globulus leaves, macrocarpals and sideroxylonals, exhibited different response patterns to single and combined treatments of O3 and wounding. The single O3 treatments induced increased accumulation in individual and total macrocarpals but not in sideroxylonal A and total sideroxylonals; while the single wounding treatment had no effects on the total macrocarpals but resulted in changes in concentrations of several individual macrocarpals, sideroxylonal A, and total sideroxylonals. Combined O3 and wounding treatments only led to changes in total sideroxylonals and some individual FPCs but did not affect the concentration of total macrocarpals and total FPCs in E. globulus leaves. In particular, among the six quantified individual FPCs, the concentration of macrocarpal J decreased in all O3 and wounding treated leaves. A positive correlation between total FPCs and total phenolic contents and negative correlations between total FPCs and terpenoid emissions from stressed E. globulus leaves were observed. Changes in the concentration and composition of FPCs suggest their multiple roles in plant resistance mechanisms to environmental stresses. We presume that the group of sideroxylonals is of great importance in wounding stress, not only caused by herbivore feeding but also by other abiotically mechanical damages, while macrocarpals might play multiple roles such as antioxidants induced by both wounding and O3 treatments. Given the negative correlations between total FPC concentrations and terpenoid emissions, syntheses of FPC and terpenoid may be closely connected by competing for the same precursors that exist in terpenoid synthesis pathways.In this study, O