Thomas Eugene Cloete

Distribution of Indigenous Bacterial Pathogens and Potential Pathogens Associated with Roof-Harvested Rainwater

ABSTRACT The harvesting of rainwater is gaining acceptance among many governmental authorities in countries such as Australia, Germany, and South Africa, among others. However, conflicting reports on the microbial quality of harvested rainwater have been published. To monitor the presence of potential pathogenic bacteria during high-rainfall periods, rainwater from 29 rainwater tanks was sampled on four occasions (during June and August 2012) in a sustainable housing project in Kleinmond, South Africa. This resulted in the collection of 116 harvested rainwater samples in total throughout the sampling period. The identities of the dominant, indigenous, presumptive pathogenic isolates obtained from the rainwater samples throughout the sampling period were confirmed through universal 16S rRNA PCR, and the results revealed that Pseudomonas (19% of samples) was the dominant genus isolated, followed by Aeromonas (16%), Klebsiella (11%), and Enterobacter (9%). PCR assays employing genus-specific primers also confirmed the presence of Aeromonas spp. (16%), Klebsiella spp. (47%), Legionella spp. (73%), Pseudomonas spp. (13%), Salmonella spp. (6%), Shigella spp. (27%), and Yersinia spp. (28%) in the harvested rainwater samples. In addition, on one sampling occasion, Giardia spp. were detected in 25% of the eight tank water samples analyzed. This study highlights the diverse array of pathogenic bacteria that persist in harvested rainwater during high-rainfall periods. The consumption of untreated harvested rainwater could thus pose a potential significant health threat to consumers, especially children and immunocompromised individuals, and it is recommended that harvested rainwater be treated for safe usage as an alternative water source.

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.

Domestic Rainwater Harvesting: Microbial and Chemical Water Quality and Point-of-Use Treatment Systems

Quality of the essential commodity, water, is being compromised by contaminants originating from anthropogenic sources, industrial activities, agriculture, etc. Water scarcity and severe droughts in many regions of the world also represent a significant challenge to availability of this resource. Domestic rainwater harvesting, which involves collection and storage of water from rooftops and diverse surfaces, is successfully implemented worldwide as a sustainable water supplement. This review focuses on chemical and microbial qualities of domestic rainwater harvesting, with a particular focus on sources of chemical pollution and major pathogens associated with the water source. Incidences of disease linked to consumption and utilization of harvested rainwater are also discussed. In addition, various procedures and methods used for disinfection and treatment of harvested rainwater, such as implementation of filter systems (activated carbon, slow sand filtration, etc.), heat treatment, and chlorination, among others, are also presented.

Current molecular and emerging nanobiotechnology approaches for the detection of microbial pathogens

Waterborne microbial diseases are escalating worldwide increasing the need for powerful and sensitive diagnostics tools. Molecular methodologies, including immunological and nucleic acid-based methods, have only recently been applied in the water sector. Advances in nanotechnology and nanomaterials have opened the door for the development of new diagnostic tools with increased sensitivity and speed, and reduced cost and labor. Quantum dots, flo dots, gold nanoparticles, magnetic nanoparticles, carbon nanotubes, nanowires, and nanocantilevers, with their unique optical and physical properties, have already been applied in nanodiagnostics. Nanobiotechnology, once remaining technical and practical problems has been addressed, will play an important role in the detection of microbial pathogens.

Prevalence of virulence genes associated with pathogenic Escherichia coli strains isolated from domestically harvested rainwater during low and high rainfall periods

ABSTRACT The possible health risks associated with the consumption of harvested rainwater remains one of the major obstacles hampering its large-scale implementation in water limited countries such as South Africa. Rainwater tank samples collected on eight occasions during the low- and high-rainfall periods (March to August 2012) in Kleinmond, South Africa, were monitored for the presence of virulence genes associated with Escherichia coli. The identity of presumptive E. coli isolates in rainwater samples collected from 10 domestic rainwater harvesting (DRWH) tanks throughout the sampling period was confirmed through universal 16S rRNA PCR with subsequent sequencing and phylogenetic analysis. Species-specific primers were also used to routinely screen for the virulent genes, aggR, stx, eae, and ipaH found in enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli, respectively, in the rainwater samples. Of the 92 E. coli strains isolated from the rainwater using culture based techniques, 6% were presumptively positively identified as E. coli O157:H7 using 16S rRNA. Furthermore, virulent pathogenic E. coli genes were detected in 3% (EPEC and EHEC) and 16% (EAEC) of the 80 rainwater samples collected during the sampling period from the 10 DRWH tanks. This study thus contributes valuable information to the limited data available regarding the ongoing prevalence of virulent pathotypes of E. coli in harvested rainwater during a longitudinal study in a high-population-density, periurban setting.

APPLICATION OF NANOTECHNOLOGY IN ANTIMICROBIAL COATINGS IN THE WATER INDUSTRY

Biofouling is a concern in the water industry due to the impact it has on maintainence of system functioning and the cost involved for prevention. Mechanical and chemical methods such as the application of biocides currently used to control biofouling are not always effective. The need for alternative methods for the prevention of biofouling therefore exists. Self-cleaning and antimicrobial surfaces, such as antimicrobial and antifouling coatings, have already shown the potential to control biofouling. A new contribution to this field is the application of nanotechnology in the design and fabrication of coatings. Nanomaterials may prohibit biofouling either by repelling microorganisms through hydrophobic nanostructures or killing of microorganisms in direct contact with surfaces containing nanobiocides. This review focuses on the different approaches and techniques to fabricate antimicrobial polymeric surfaces, the application of specific nanoparticles and nanomaterials, and nanoenabled antimicrobial coatings such as paints.

Dynamics of indigenous bacterial communities associated with crude oil degradation in soil microcosms during nutrient-enhanced bioremediation.

Bacterial population dynamics were examined during bioremediation of an African soil contaminated with Arabian light crude oil and nutrient enrichment (biostimulation). Polymerase chain reaction followed by denaturing gradient gel electrophoresis (DGGE) were used to generate bacterial community fingerprints of the different treatments employing the 16S ribosomal ribonucleic acid (rRNA) gene as molecular marker. The DGGE patterns of the nutrient-amended soils indicated the presence of distinguishable bands corresponding to the oil-contaminated–nutrient-enriched soils, which were not present in the oil-contaminated and pristine control soils. Further characterization of the dominant DGGE bands after excision, reamplification and sequencing revealed that Corynebacterium spp., Dietzia spp., Rhodococcus erythropolis sp., Nocardioides sp., Low G+C (guanine plus cytosine) Gram positive bacterial clones and several uncultured bacterial clones were the dominant bacterial groups after biostimulation. Prominent Corynebacterium sp. IC10 sequence was detected across all nutrient-amended soils but not in oil-contaminated control soil. Total heterotrophic and hydrocarbon utilizing bacterial counts increased significantly in the nutrient-amended soils 2 weeks post contamination whereas oil-contaminated and pristine control soils remained fairly stable throughout the experimental period. Gas chromatographic analysis of residual hydrocarbons in biostimulated soils showed marked attenuation of contaminants starting from the second to the sixth week after contamination whereas no significant reduction in hydrocarbon peaks were seen in the oil-contaminated control soil throughout the 6-week experimental period. Results obtained indicated that nutrient amendment of oil-contaminated soil selected and enriched the bacterial communities mainly of the Actinobacteria phylogenetic group capable of surviving in toxic contamination with concomitant biodegradation of the hydrocarbons. The present study therefore demonstrated that the soil investigated harbours hydrocarbon-degrading bacterial populations which can be biostimulated to achieve effective bioremediation of oil-contaminated soil.

Furanone-containing poly(vinyl alcohol) nanofibers for cell-adhesion inhibition.

The 3(2H) furanone derivative 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) was investigated for its antimicrobial and cell-adhesion inhibition properties against Klebsiella pneumoniae Xen 39, Staphylococcus aureus Xen 36, Escherichia coli Xen 14, Pseudomonas aeruginosa Xen 5 and Salmonella typhimurium Xen 26. Nanofibers electrospun from solution blends of DMHF and poly(vinyl alcohol) (PVA) were tested for their ability to inhibit surface-attachment of bacteria. Antimicrobial and adhesion inhibition activity was determined via the plate counting technique. To quantify viable but non-culturable cells and to validate the plate counting results, bioluminescence and fluorescence studies were carried out. Nanofiber production was upscaled using the bubble electrospinning technique. To ascertain that no DMHF leached into filtered water, samples of water filtered through the nanofibrous mats were analyzed using gas chromatography coupled with mass spectrometry (GC-MS). Scanning electron microscopy (SEM) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) were used to characterize the electrospun nanofibers.

Immobilized furanone derivatives as inhibitors for adhesion of bacteria on modified poly(styrene-co-maleic) anhydride)

The ability of brominated furanones and other furanone compounds with 2(3H) and 2(5H) cores to inhibit bacterial adhesion of surfaces as well deactivate (destroy) them has been previously reported. The furanone derivatives 4-(2-(2-aminoethoxy)-2,5-dimethyl-3(2H)-furanone and 5-(2-(2-aminoethoxy)-ethoxy)methyl)-2(5H)-furanone were synthesized in our laboratory. These furanone derivatives were then covalently immobilized onto poly(styrene-co-maleic anhydride) (SMA) and electrospun to fabricate nonwoven nanofibrous mats with antimicrobial and cell-adhesion inhibition properties. The electrospun nanofibrous mats were tested for their ability to inhibit cell attachment by strains of bacteria commonly found in water ( Klebsiella pneumoniae Xen 39, Staphylococcus aureus Xen 36, Escherichia coli Xen 14, Pseudomonas aeruginosa Xen 5, and Salmonella tymphimurium Xen 26). Proton nuclear magnetic resonance spectroscopy ((1)H NMR), electrospray mass spectroscopy (ES-MS), and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) were used to confirm the structures of the synthesized furanones as well as their successful immobilization on SMA. To ascertain that the immobilized furanone compounds do not leach into filtered water, samples of water, filtered through the nanofibrous mats were analyzed using gas chromatography coupled with mass spectroscopy (GC-MS). The morphology of the electrospun nanofibers was characterized using scanning electron microscopy (SEM).

Microbial and Physico-chemical Characteristics Associated with the Incidence of Legionella spp. and Acanthamoeba spp. in Rainwater Harvested from Different Roofing Materials

The incidence of Legionella and Acanthamoeba spp. was correlated to microbial indicator analysis and physico-chemical characteristics of rainwater harvested from catchment areas constructed from galvanized zinc, Chromadek®, and asbestos, respectively. Quantitative PCR (qPCR) analysis indicated that no significant difference (p > 0.05) in copy numbers of Legionella spp. and Acanthamoeba spp. was recorded in tank water samples collected from the respective roofing materials. However, significant positive Spearman (ρ) correlations were recorded between the occurrences of Legionella spp. gene copies vs. nitrites and nitrates (p = 0.05) in all tank water samples. Significant positive correlations were also established between Acanthamoeba spp. vs. barium (p = 0.03), magnesium (p = 0.02), sodium (p = 0.02), silicon (p = 0.05), arsenic (p = 0.03), and phosphate (p = 0.01), respectively. Additionally, while no significant correlations were observed between Legionella spp. vs. the indicator bacteria (p > 0.05), positive correlations were observed between Acanthamoeba spp. vs. total coliforms (p = 0.01) and Acanthamoeba spp. vs. Escherichia coli (p = 0.02), respectively. Results obtained in the current study thus indicate that the incidence of Acanthamoeba and Legionella spp. in harvested rainwater was not influenced by the roofing material utilized. Moreover, it is essential that the microbial quality of rainwater be assessed before this water source is implemented for potable and domestic uses as untreated harvested rainwater may lead to legionellosis and amoebae infections.