In-situ multi-mode extraction (iMME) sampler for a wide-scope analysis of chemical and biological targets in water in urbanized and remote (off-the-grid) locations.

Chemical pollution (including chemicals of emerging concern - CECs) continues to gain increasing attention as a global threat to human health and the environment, with numerous reports on the adverse and sometimes devastating effects upon ecosystems the presence of these chemicals can have. Whilst many studies have investigated presence of CECs in aquatic environments, these studies have been often focused on higher income countries, leaving significant knowledge gaps for many low-middle income countries. This study proposes a new integrated powerless, in-situ multi-mode extraction (iMME) sampler for the analysis of chemicals (105 CECs) and biological (5 genes) markers in water in contrasting settings: an urbanized Avon River in the UK and remote Olifants River in Kruger National Park in South Africa. The overarching goal was to develop a sampling device that maintains integrity of a diverse range of analytes via analyte immobilization using polymeric and glass fibre materials, without access to power supply or cold chain (continuous chilled storage) for sample transportation. Chemical analysis was achieved using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Several mobile CECs showed low stability in river water, at room temperature and typical 24 h sampling/transport time. It is therefore recommended that, in the absence of cooling, environmental water samples are spiked with internal standards on site, immediately after collection and analyte immobilization option is considered, in order to allow fully quantitative analysis. iMME has proven effective in immobilization, concentration and increased stability of CECs at room temperature (and at least 7 days storage) allowing for sample collection at remote locations. The results from the River Avon and Olifants River sampling indicate that the pristine environment of Olifants catchment is largely unaffected by CECs common in the urbanized River Avon in the UK with a few exceptions: lifestyle chemicals (e.g., caffeine, nicotine and their metabolites), paracetamol and UV filters due to tourism and carbamazepine due to its persistent nature. iMME equipped with an additional gene extraction capability provides an exciting new opportunity of comprehensive biochemical profiling of aqueous samples with one powerless in-situ device. Further work is required to provide full integration of the device and comprehensive assessment of performance in both chemical and biological targets.

Spatiotemporal Investigation of Antibiotic Resistance in the Urban Water Cycle Influenced by Environmental and Anthropogenic Activity.

With increasing emergence of antimicrobial resistant bacteria (ARB) and the risk this poses to public health, there are growing concerns regarding water pollution contributing to the spread of antimicrobial resistance (AMR) through inadequate amenities and the rapid rate of urbanization. In this study, the impact of different anthropogenic factors on the prevalence of AMR in the urban water cycle in Stellenbosch, South Africa (SA) was examined. Carbapenem, colistin, gentamicin and sulfamethoxazole resistant Gram-negative bacteria were recovered by selectively culturing aqueous, biofilm and sediment samples from sites impacted to varying degrees by informal settlements, residential, industrial, and agricultural activities, as well as a municipal wastewater treatment works (WWTW). A metagenomic approach determined community profiles and dominant AMR genes at various sites, while carbapenem resistant colonies were characterized using whole genome sequencing (WGS). Isolates recovered from agricultural sites exhibited relatively high levels of resistance to carbapenems and colistin, whereas sites impacted by domestic run-off had a higher prevalence of resistance to gentamicin and sulfamethoxazole, corresponding to usage data in SA. Similar microbial taxa were identified in raw sewage, sites downstream of informal settlements, and industrial areas that have limited waste removal infrastructure while WWTW were seen to reduce the prevalence of ARB in treated wastewater when operating efficiently. The results indicate the multiple complex drivers underpinning environmental dissemination of AMR and suggest that WWTW assist in removing AMR from the environment, reinforcing the necessity of adequate waste removal infrastructure and antibiotic stewardship measures to mitigate AMR transmission. IMPORTANCE The results from this study are of importance as they fill a gap in the data available on environmental AMR in South Africa to date. This study was done in parallel with co-investigators focusing on the prevalence of various antimicrobials at the same sites selected in our study, verifying that the sites that are influenced by informal settlements and WWTW influent had higher concentrations of antimicrobials and antimicrobial metabolites. The various locations of the sample sites selected, the frequency of the samples collected over a year, and the different types of samples collected at each site all contribute to informing how AMR in the environment might be affected by anthropogenic activity.

Comparison of phytoplankton control measures in reducing cyanobacteria assemblage of reservoirs found in the arid region of Southern Africa.

Ecological restorations of reservoirs are implemented worldwide; however, minimal successes are reported and understood for warmer African lakes like Swakoppoort Dam, Namibia. The objectives of the study were (a) to establish the effectiveness of the two control measures in reducing cyanobacteria growths in comparison with untreated control areas and (b) to compare the results generated before and after control measures with the reference Von Bach Dam. During Phoslock® treatment, the average cyanobacteria cells and total phosphate (TP) were 90,521 cells/ml and 0.3 mg/L in the treated area and 55,338 cells/ml and 0.1 mg/L in the control area. During Solar Powered Circulation (SPC) treatment, the average cyanobacteria cells were on average 906,420 cells/ml in the treated areas and 121,891 cells/ml in the control area. The TP on average was 0.3 mg/L during SPC treatment, while during the combined treatment, the average cyanobacteria cells, TP, and total nitrogen (TN) were 18,387,226 cells/ml, 0.27 mg/L, and 2.41 mg/L before and 22,836,511 cells/ml, 0.42 mg/L, and 1.50 mg/L after treatment. This was higher compared to the reference site. PCA triplot indicates no grouping pattern, and the repeated-measures mixed model analyses indicate that treatment had no significant effect on cyanobacteria cells. It was evident that the two control measures were ineffective in reducing cyanobacterial cells. PRACTITIONER POINTS: Key findings of the article: Two phytoplankton control measures were found ineffective to reduce the cyanobacterial cell numbers. High cell numbers of cyanobacteria were recorded at the treatment areas compared to untreated control areas during both treatments. The combined effect of the two control measures was ineffective as more cyanobacterial cells were recorded during the treatment. During control measure treatment, the Swakoppoort Dam was hypertrophic, which could be due to a malfunctioned WWTP upstream. The inefficiency of the control measures could be due to small treatment area, higher nutrients, or treatment period. The implications of the results to water/wastewater practice: The selection of appropriate mitigation measures considering treatment area for dams with high nutrient situated in warmer arid environments. There is a need to understand the trophic relationships, climatic conditions, and the sources of the internal and external nutrients to manage water quality. Focus on point and non-point sources of nutrients as the root causes of the degradation of Swakoppoort Dam water.

Effects of preservation of rumen inoculum on volatile fatty acids production and the community dynamics during batch fermentation of fruit pomace.

Rumen fluid (RF) as inocula is useful for evaluating biomass digestibility and has potential for producing volatile fatty acids (VFA) via the carboxylate platform. However, RF is not readily available, necessitating evaluation of potential preservation methods. Glycerol (50% v/v) and DMSO (5% v/v) were used to preserve rumen inocula for 3 months at -80 °C. Effects of cryo-preservation on digestibility, VFA production and community composition with ß-diversity distance metrics were compared to fresh RF using apple, citrus and grape pomace as substrates. For all substrates, DMSO cryo-preserved rumen digestibility parameters, VFA yield and product distribution were more significantly comparable to fresh RF (P > 0.05) than was glycerol cryo-preserved RF. Similarly, ß-diversity coefficient (unweighted unifrac) between DMSO cryo-preserved RF and fresh RF was 0.250 while the coefficient was 0.359 for the glycerol cryo-preserved RF compared to fresh RF. This showed that a DMSO cryo-preserved RF is less affected by preservation effects and is a more promising alternative to fresh RF.

Valorisation of the invasive species, Prosopis juliflora, using the carboxylate platform to produce volatile fatty acids.

Biomass derived from low-value, high-volume invasive plant species is an attractive, alternative feedstock to produce biofuels and biochemicals. This study aimed to use the carboxylate platform to valorize the invasive leguminous shrub, Prosopis juliflora (Mesquite), by utilizing in vitro rumen fermentations without chemical pretreatment to produce volatile fatty acids. The three fractions of the mesquite: leaves (ProL), stems (ProS) and branches (ProB) were compared regarding chemical composition, neutral detergent fiber (NDF) digestibility at 7 time points and VFA production after 72 h with sugarcane bagasse (SCB) as a reference. NDF digestibility was significantly (P < 0.05) higher in ProL (35.8%) than ProS (30.4%) and ProB (20.9%) compared to SCB (21.9%). VFA concentrations from 20 g biomass L-1 showed significant differences with 8.07, 6.71 and 6.51 g L-1 for ProL, ProS and ProB respectively, while SCB yielded 4.02 g L-1. These concentrations were comparable with other platforms that employ chemically pretreated biomass for VFA production.

Bacterial species diversity as an indicator of dibromonitrilopropionamide (DBNPA) biocide efficacy.

Microorganism growth in industrial systems is controlled through the use of biocides and biodispersants. There is, however, no simple means of determining the efficacy of these control mechanisms, but it is currently tested using complex bacterial culturing techniques. Biolog Ecoplates® have been used to detect bacterial population changes in various communities. These microtitre plates comprise 31 different carbon substrates (in triplicate) with wells. When a sample is added to the wells, bacteria capable of metabolising the relevant carbon sources respire the substrates, causing the tetrazolium dye in the well to turn purple, indicating a positive result. Hypothetically, the higher the microbial diversity, the more substrates will be utilised and vice versa. The objective of this study was to test this hypothesis, using Biolog Ecoplates® as a potential simple indicator to determine the efficiency of a biocide to control microbial growth in cooling water systems by monitoring the changes in the microbial metabolic pattern. This study proved the hypothesis using Biolog Ecoplates®, indicating that the addition of biocides at various concentrations resulted in fewer substrates being utilised, indicative of a decrease in microbial species diversity.

Application of quantitative PCR for the detection of microorganisms in water.

The occurrence of microorganisms in water due to contamination is a health risk and control thereof is a necessity. Conventional detection methods may be misleading and do not provide rapid results allowing for immediate action. The quantitative polymerase chain reaction (qPCR) method has proven to be an effective tool to detect and quantify microorganisms in water within a few hours. Quantitative PCR assays have recently been developed for the detection of specific adeno- and polyomaviruses, bacteria and protozoa in different water sources. The technique is highly sensitive and able to detect low numbers of microorganisms. Quantitative PCR can be applied for microbial source tracking in water sources, to determine the efficiency of water and wastewater treatment plants and act as a tool for risk assessment. Different qPCR assays exist depending on whether an internal control is used or whether measurements are taken at the end of the PCR reaction (end-point qPCR) or in the exponential phase (real-time qPCR). Fluorescent probes are used in the PCR reaction to hybridise within the target sequence to generate a signal and, together with specialised systems, quantify the amount of PCR product. Quantitative reverse transcription polymerase chain reaction (q-RT-PCR) is a more sensitive technique that detects low copy number RNA and can be applied to detect, e.g. enteric viruses and viable microorganisms in water, and measure specific gene expression. There is, however, a need to standardise qPCR protocols if this technique is to be used as an analytical diagnostic tool for routine monitoring. This review focuses on the application of qPCR in the detection of microorganisms in water.

Lactobacillus equigenerosi strain Le1 invades equine epithelial cells.

Lactobacillus equigenerosi strain Le1, a natural inhabitant of the equine gastrointestinal tract, survived pH 3.0 and incubation in the presence of 1.5% (wt/vol) bile salts for at least 2 h. Strain Le1 showed 8% cell surface hydrophobicity, 60% auto-aggregation, and 47% coaggregation with Clostridium difficile C6. Only 1% of the cells adhered to viable buccal epithelial cells and invaded the cells within 20 min after contact. Preincubation of strain Le1 in a buffer containing pronase prevented adhesion to viable epithelial cells. Preincubation in a pepsin buffer delayed invasion from 20 min to 1 h. Strain Le1 did not adhere to nonviable epithelial cells. Administration of L. equigenerosi Le1 (1 × 10(9) CFU per 50 kg body weight) to healthy horses did not increase white blood cell numbers. Differential white blood cell counts and aspartate aminotransferase levels remained constant. Glucose, lactate, cholesterol, and urea levels remained constant during administration with L. equigenerosi Le1 but decreased during the week after administration.

Lactobacillus plantarum 24, isolated from the marula fruit (Sclerocarya birrea), has probiotic properties and harbors genes encoding the production of three bacteriocins.

Lactobacillus plantarum 24, isolated from marula fruit grows at pH 4.0 and tolerates acid levels and bile concentrations normally present in the human gastro-intestinal tract. Wistar rats that have been administered L. plantarum 24 showed no signs of discomfort or abnormal behavior. Tissue samples from the liver, spleen and intestine appeared normal. Furthermore, strain 24 harbors the genes encoding plantaricins A, F, and NC8α, a gene encoding immunity to plantaricin, and an ABC transporter similar in sequence to that reported for plantaricin G. At least one antimicrobial peptide within the size range of plantaricins A, F, and NC8α has been detected on a tricine-SDS-PAGE gel. Little is known about the microbial population in marula. This is the first report of a L. plantarum strain from marula fruit with bacteriocin genes and probiotic properties.

The potential of nanofibers and nanobiocides in water purification.

Electrospun nanofibers and nanobiocides show potential in the improvement of water filtration membranes. Biofouling of membranes caused by the bacterial load in water reduces the quality of drinking water and has become a major problem. Several studies showed inhibition of these bacteria after exposure to nanofibers with functionalized surfaces. Nanobiocides such as metal nanoparticles and engineered nanomaterials are successfully incorporated into nanofibers showing high antimicrobial activity and stability in water. Research on the applications of nanofibers and nanobiocides in water purification, the fabrication thereof and recently published patents are reviewed in this article.

Evaluation of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 as probiotics by using a gastro-intestinal model with infant milk formulations as substrate.

Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 produce bacteriocins with activity against a number of Gram-positive and Gram-negative bacteria. Both strains survived intestinal conditions simulated in a gastro-intestinal model (GIM) with infant milk formulations as substrate and prevented the growth of Listeria monocytogenes ScottA. The strains are inhibited by the antibiotics amoxicillin, cefadroxil, roxithromycin and doxycycline, anti-inflammatory medicaments containing meloxicam, ibuprofen and sodium diklofenak, and analgesics containing paracetamol, codeine phosphate and promethazine. Strain 423 is sensitive to vancomycin and does not contain genes encoding gelatinase, cell aggregation substance (AS), adhesion to collagen (Ace), enterococcus surface protein (Esp), Enterococcus faecalis endocarditis antigen (EfaAfs), cytolysin and non-cytolysin (beta-hemolysin III). Genes encoding AS, cytolysin and non-cytolysin (beta-hemolysin III) were amplified from the genome of strain ST4SA. Survival of strains ST4SA and 423 improved when used as combined cultures in the GIM and compared well with the survival of commercially available probiotics subjected to the same conditions.

Adhesion of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 cells under conditions simulating the intestinal tract, and in the presence of antibiotics and anti-inflammatory medicaments.

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.