DOI: 10.17033/data.00000226
title: Drinking-water under a "One Health" lens: quantifying microbial contamination pathways between livestock and drinking- water

description: Diarrhoeal disease and lack of access to safe water remain significant public health issues in developing countries. There is also growing concern about the potential for disease, including diarrhoeal infections, to be transmitted from livestock to humans. This project addresses the potential drinking-water contamination risks to human health in rural sub-Saharan Africa, where people and livestock often live in close proximity. Preliminary fieldwork will be carried out in rural Kenya, building on an ongoing study that is simultaneously recording human and livestock disease in ten villages. The fieldwork will test different techniques to identify contamination hazards from livestock, alongside water quality testing and recording of diarrhoea in children. These techniques will include the use of GPS collars to track cattle movements, maps of hazardous areas created by the communities themselves, and also checklists for recording signs of livestock hazards at water sources and around water stored in the home. We will look at how feasible it is to record hazards using these techniques. We will also statistically assess whether we find greater water contamination and greater diarrhoea in children where there are more recorded hazards. Since measurement of water contamination used in such areas is based on bacteria found in both livestock and humans, the project will also work on affordable ways of testing for micro-organisms that are specifically found in livestock faeces versus those found in human faeces. If successful, such techniques could be used to investigate the importance of different sources of faecal contamination of drinking-water. This in turn could help manage the safety of rural water sources like wells and rainwater and better protect drinking-water stored in the home from contamination through livestock. Because this complex problem requires a wide range of expertise, during the project we will strength our academic team to include more disciplines, particularly specialists in child health and social sciences. The tools for identifying hazards from livestock will be made widely available at the end of the project and UK expertise in the microbiological laboratory techniques will be shared with Kenyan collaborators. The experience gained will be used to build up contacts and develop a plan and team for a larger-scale study of livestock hazards, water contamination, and diarrhoeal disease risk in several countries.
description: Bacteroides host strains were isolated using the method previously described in Payán et al. (2005) and Gómez-Doñate et al. (2011), when human and cattle specific Bacteroides spp. strains were isolated from municipal wastewater slaughterhouses wastewater. Because of the absence of these facilities in the studied area (rural Siaya county, Kenya), the authors adapted the method and collected samples from pooled faecal material from school pit latrines (human samples) and from cattle faeces from shoreline of surface water sources (cattle samples). The data was collected in rural areas of the Siaya County (western Kenya). Coordinates of the exact location of the faecal samples collected for the recover and isolation of the new Bacteroides spp. hosts can be found in the file “LocationOfFaecalSampling.csv”. Faecal samples were collected on 7th and 8th of June of 2018. Recover and isolation of Kenyan Bacteroides spp. hosts from human and cattle faeces occurred in the Kenyan Medical Research Institute laboratories located in Kysian (Kisumu) between 10th and 21st of June 2018. This data can be found in the files: • “IsolationOfBacteroidesHostsWeek10-6-18StepA.csv”; • “IsolationBacteroidesHostsWeek10-6-18StepB.csv”; • “IsolationBacteroidesHostsWeek17-6-18StepA.csv”; • “IsolationBacteroidesHostsweek17-6-18StepB.csv”. Detection of faecal indicator bacteria and bacteriophages from environmental samples (water sources; and from a KIWASCO (Kisumu Water and Sewerage Company) wastewater plant influents and effluents) occurred during the following dates 18-21/06/2018; 05-08/11/2018; and 13/06/2019. Location of these sampling sites alongside detected levels can be found in the file “DetectionOfFIB&MSTmarkers.csv”. Detection bacteriophages infecting Bacteroides spp. hosts were performed according to standard methods (ISO 10705-4). Detection of faecal indicator bacteria (total coliforms, Escherichia coli (ISO 9308-1) and intestinal enterococci (ISO 7899-2:2000)) and somatic coliphages (ISO 10705-2) were performed in order to compare detection levels of bacteriophages infecting the recently isolated Bacteroides spp. Kenyan hosts. Collection/Generation/Transformation Methods Sampling of human and cattle faecal material Cattle faecal samples were collected from cattle fresh stools located on the shorelines of 10 water sources (see file: Location of Faecal Sampling.xls). In each location, using a sterile wooded spatula, a small amount of cattle faeces (approximately 5 grammes) was scooped from five different stools. These 25 grammes pooled-sample was then put in 100 ml sterile plastic pots and placed into a cooler box (at approximately 4°C) containing ice packs. Human faecal samples were collected from pit latrines of primary schools which have been previously authorized the research team to do so. A metal spoon was attached to a wooden pole. The spoon was cleaned using paper wipes and alcohol at 70%. In each school, a technician used the pole to scoop approximately 5 grammes of faecal samples from 5 pit latrines. This 25 grammes pooled sample was then put in 100 ml sterile plastic pots and placed in a cooler box (at approximately 4°C) containing ice packs. The spoon was cleaned between each sampling. Samples were transported to the KEMRI laboratories in Kysian within 4 hours and stored in the fridge for maximum of 24 hours until been processed. Isolation of potential cattle and human Kenyan Bacteroides spp. Hosts Bacteroides bile esculine agar (BBEa) (Livingston, 1978) was used for the recovery of Bacteroides from faecal samples. The BBEA composition in 1 l was as follows: bile, 20.0 g; Tryptone soy agar, 40.0 g; esculine, 1.0 g; ammonium ferric citrate, 0.5 g; and haemin, 10 ml of a 0.1% (w/v) solution, made up in NaOH 0.02%. The medium was sterilized at 121°C for 15 min. The pH was adjusted to 7.0 ± 0.5 by aseptically adding Chloric acid (HCl 35%). First day: Five grammes samples were withdraw from each of the 25 grammes polled collected samples, placed in a centrifuge tube (50ml) containing 45 mL of Ringer’s ¼ strength solution and mixed in a vortex, producing 10 ‘master’ faecal solutions. 1ml of each master solutions were serially diluted in 9mls Ringer’s ¼ strength solution (10-1,10-2). 100 µl of each dilution were then pipetted onto BBE agar and spread using a sterile glass spreader. Incubated anaerobically at 37ºC for 24 hours. Second day: Dark colonies were then picked off using sterile toothpicks and plated for pure culture onto duplicate BBE agar (in Petri plates). These are then incubated at 37ºC for 24hrs under both aerobic and anaerobic conditions. Anaerobic conditions were achieved using anaerobic jars and anaerobic sachets (Anaerogen®). Note: Plates were divided into 4 to allow multiple colonies to be plated on each plate.Third day: Only isolates that grew under strict anaerobic conditions were Gram stained and further tested (e.g. isolates 5 and 7 in above photo). Cell material was transferred to a drop of water on a microscope slide ready for Gram staining using a sterile loop. Once Gram staining were complete, cell morphology and Gram stain characteristics were inspected under microscope. Only Gram-negative obligate anaerobic rods were further processed. Gram-negative strict anaerobes were then sub cultured in agar plates (BBE plates and BPRM plates) and incubated anaerobically at 37ºC for 24 hours. Fourth day: Isolated colonies were then picked (using sterile cotton swabs) from the plates and inoculated into BPRM broth (with additives) in 12ml screw capped glass tubes. Tubes were incubated overnight at 37 ºC. The screw capped glass tubes containing the BPRM broth and isolates were filled up to the top and caps secured (avoiding an air bubble at the top of the tube). Also a blank was made up (i.e. tube containing just BPRM broth and additives) as shown in photo below. After overnight incubation the tubes that were displaying good growth (turbidity) were selected. Overnight cultures that presented good growth were mixed with a cry protector (Bovine serum) in a ratio of 1:1 (v/v). These homogenate were mixed avoiding bubble formation and distributed into vials in aliquots of approximately 1.0 ml and store at -20 ºC. Vials contained the hosts and were ready for use in posterior bacteriophage detection from water sources. Detection of Faecal indicator bacteria (FIB), somatic coliphages and phages infecting Bacteroides spp. from water sources and from KIWASCO wastewater treatment plant. Water samples were collected from drinking water sources in the Siaya County and from KIWASCO wastewater treatment plant (Harambee Road, Kisumu, Kenya). They were collected from a depth of 15 cm below the surface using 1 litre sterile polyethylene containers (Fisher Scientific, UK). Water and wastewater samples were transported to the KEMRI laboratories in Kysian within 4 hours and stored in the fridge for maximum of 24 hours until been processed Detection of Faecal indicator bacteria (FIB) Detection and enumeration of faecal indicator bacteria (FIB) followed ISO standard methods ISO 9308-1:2014 (Total coliforms and E. coli) and ISO7899/2:2000 (Intestinal enterococci). They were performed using the membrane filtration technique. For each surface water sample and for each FIB group investigated, four volumes (0.1, 1, 10 and 100 ml) were poured into a filtration unit containing approximately 10 ml of quarter-strength Ringer’s (QSR) solution and then filtered through a 0.45μm pore-size cellulose nitrate filter (Thermo Scientific) using a vacuum pump (Fisher®). For wastewater samples, in which bacterial concentrations were higher, 10-2, 10-3, 10-4 and 10-5 ml dilutions were filtered. Then, for total coliforms and E coli analysis, filters were placed onto coliform chromogenic agar (CCE) agar (Difco®) in Ø 55mm petri dishes (Fisher). Plates were then incubated upside down for 24 ±2 hours at 37.0 ±0.5°C. Colonies that showed shades of dark-blue to violet were counted as E. coli, while those that appeared pink to red-coloured were recorded as presumptive coliform (Total coliforms) that are not E. coli (ISO 9308-1:2014). For the intestinal enterococci, filters were placed onto Slanetz and Bartley agar (Oxoid®) in Ø 55mm petri dishes (Fisher). Plates were then incubated upside down for 48 ±2 hours at 37.0 ±0.5 °C, and raised colonies that showed shades of red, maroon and pink were counted as presumptive intestinal enterococci (ISO 7899/2:2000). Results were expressed as colony-forming units (CFU) per 100 mL. Initially the 100 ml sample was considered to compute the FIB levels. In case this sample returned Too Numerous To Count (TNTC) results, then a smaller volume, respectively 10ml; 1ml and 0.1 ml, were taken in consideration to compute FIB levels. Detection of Somatic coliphages Water samples were assayed for somatic coliphages using the host bacterium E. coli WG-5. Basically, on the day of the assay, a cryogenic vial 2 ml (Thermo Scientific, Nalgene) containing 1 ml of the WG-5 working culture was removed from a -20°C freezer and left to defrost. The working culture was then added into a 100 ml Schott bottle containing 50 ml of Modified Scholten’s broth (MSB), but before this, a 2 ml sample of MSB was removed and pipetted into a 4.5 ml cuvette (Kartell®) and used to “zero” the spectrophotometer (Corning colorimeter 253). The host was then incubated in an incubator (Gallenkamp cooled Incubator) at 37.0±0.5C for approximately two to three hours until the correct optical density was reached (0.33 OD at 600 nm). The inoculum culture was then placed on cooler packs in isothermal boxes and used within 3 hours (Note: after two hours of incubation, at every 15-30 minutes 2 ml samples were pipetted into a cuvette, to check when the correct optical density (OD) was reached). Meanwhile, a Schott bottle containing 50ml of semi-solid Modified Scholten’s agar (ssMSA; 0.8% agar number 1) was removed from the fridge, melted in a microwave and placed in a water bath (Fisher® brand) at approximately 50°C. After equalizing its temperature, additives (calcium chloride and naladixic acid) were added to the melted semi-solid agar, which was hand agitated and distributed into plastic sterile test tubes (15ml, Sterilin®). The test tubes were placed back in the water bath, prior to use. Naturally occurring phages present in faecally contaminated surface waters were used as a positive control throughout the study. Finally, using previously calibrated pipettes, water sample aliquots of 1 ml (2x) were mixed with 1.0 ml of WG-5 working culture into sterile test tubes containing 2.5 ml of molten ssMSA. The mixture was briefly mixed in a min vortex (Fisher® FB 15212985) and poured onto Ø 90mm Petri dishes containing Modified Scholten’s agar (MSA; 1.6% agar number 1, Oxoid® LP0011). Petri dishes were left to set for approximately 5-15 minutes and incubated (Gallenkamp Cooled Incubator) upside down at 37.0±2.0°C for 18±2 hours. Following incubation, zones of lysis (plaques) were enumerated and the results expressed as plaque forming units (PFU) per 100ml Detection of bacteriophages infecting Bacteroides spp. Water samples were assayed for bacteriophages infecting Bacteroides fragilis by using a previously isolated human-specific host strain GB-124 (Payan et al., 2005; Ebdon et al., 2007) and the recently isolated potential kenyan hosts (human:WAA.1, TIB. 1, TIA.1, ONA.3, LU.2; cattle SIN19, KN1,KN2,KN3, and KN8). In brief, on the day before the assay, a 2 ml cryogenic vial (Thermo Scientific, Nalgene), containing 1 ml of each of the above host working cultures was removed from -20°C storage and left to defrost. The working cultures were then added to 10 ml (11x) of complete Bacteroides phage recovery medium broth (BPRMB) in a 12 ml Pyrex screw-topped test tube, excluding all air. Also, another Pyrex screw-topped test tube was filled with BPRMB only, as a negative control and blank. Both tubes were incubated overnight at 37.0 ± 2.0°C. On the day of the assay, the tube containing complete BPRMB was only used to “zero” the spectrophotometer (Novaspec II, Pharmacia LKB). Also, inoculum cultures were prepared by adding 1 ml of overnight Bacteroides hosts cultures to 11 ml of pre-warmed BPRMB in a 12 ml Pyrex test tube, excluding all air and incubated at 37.0 ± 2.0°C until the correct optical density (OD=0.33 at 620nm) was reached (Note: after 1.5 hours of incubation, optical density was checked every 20-30 minutes). When the correct OD was reached, the tubes containing the inoculum cultures were placed on cooler packs in isothermal boxes and used within three hours. Meanwhile, a Schott bottle containing 100 ml of semi-solid Bacteroides phage recovery medium agar (ssBPRMA; 0.8% agar number 1) was removed from the fridge, melted in a microwave and placed in a water bath (Fisher® brand FB 60303) at approximately 50°C. After equalizing its temperature, additives were added to the melted semi-solid, which was hand agitated and distributed into 12 plastic sterile test tubes (15 ml, Sterilin®). The test tubes were placed back in the water bath prior to use. Finally, using previously calibrated pipettes, sample aliquots of 1 ml water were mixed with 1 ml of Bacteroides spp. hosts working cultures into the test tubes containing 2.5 ml of melted ssBPRMA. The mixture was briefly mixed min vortex (Fisher® FB 15212985) and poured onto Ø 90mm Petri dishes containing Bacteroides phage recovery medium agar (BPRMA; 1.6% agar number 1). Petri dishes were left to set for approximately 5-15 minutes, placed upside down in an anaerobic jar (Oxoid® AG0031A) containing an anaerobic sachet (Anaerogen, Oxoid®) and incubated (Gallekamp PLUS II incubator) at 37.0±2.0°C for 18±2 hours. Following incubation, zones of lysis (plaques), where bacteriophages had lysed the Bacteroides spp. hosts cells, were enumerated and expressed as plaque-forming units (PFU). Petri dishes containing GB-124 reference phage were used as a positive control. Note: Full descriptions of agar and diluents components are described in section Fieldwork and laboratory instrumentation, chemicals and agar media.