Thesis submitted in fulfillment for the requirements for the degree Doctor Technologiae: Environmental Health in the Faculty of Applied Sciences, Cape Peninsula University of Technology, 2012 / Water related issues such as water treatment and distribution have become extremely important all over the world due to population growth, growing urbanization, health and environmental pollutions. Contamination of water bodies especially in Africa with antibiotic resistant bacteria strains is a cause for concern. Escherichia coli O157 H:7, and various strains of non O157 E. coli and Acinetobacter spp. are known for antibiotic resistance. Both bacteria are environmental organisms found coexisting together with high potentials of exchange of resistance genes. Despite the stress conditions confronting these bacteria in water, food and the human body, in the form of disinfectants, antibiotics, salts and the innate immunity, they appear to develop adaptive mechanisms that enable them survive and cause infection. This therefore necessitates the need for investigation of effective virulence factor-targeted control measures. Culture of 62 water samples on Brilliance E. coli/coliform selective medium (BECSM, Oxoid), Eosin Methylene Blue (EMB) agar, or Baumann’s enrichment medium (BEM) and Leeds Acinetobacter Medium (LAM) for the isolation of E. coli and Acinetobacter spp. was carried out. Isolates were investigated for virulence factors, antibiotic resistance and transformation of resistance genes. The effect of oxidative stress exerted by 0.3% Crystal violet, 0.3% Bile salt, 4.0% NaCl, and 8% ethanol on some of the multi-drug resistant strains as well as the effect of stem back extracts of Curtisia dentata on verotoxin production by the verotoxic strains was also investigated. Out of the 69 isolates of E. coli (including O26:H11, O55, O111:NM, 72 O126, O44, O124, O96:H9, O103:H2, O145:NM and O145:H2.) and 41 isolates of Acinetobacter spp. with 26 (53.06%) of the E. coli and 6 (14.63%) of the A. haemolyticus isolates producing verotoxins, and no A. lwoffii isolate produced the toxins. Twenty five - 25(35.23%), 14(20.30%) and 28(40.58%) of the E. coli isolates were positive for VTx1&2, Vtx1 and Vtx2 respectively, 49(71.015%), were positive for extended-spectrum beta-lactamases (ESBLs), 7(77.78%) for serum resistance, 57(82.61%) for cell surface hydrophobicity, 48(69.57%) for gelatinase production and 37(53.62%) for haemolysin production. While transformation occurred among the E. coli and Acinetobacter isolates (transformation frequency: 13.3 x 10-7- 53.4-7), there was poor curing of the plasmid genes, a confirmation of presence of stable antibiotic resistant genes (DNA concentration between 42.7-123.8 μg) and intra-genetic transfer of multidrug resistant genes among isolates. Oxidative stress due to chemicals, salts, alcohol or freeze-thawing (blow temperature stress) exerted various degrees of lethality on E. coli isolates with some bacterial strains losing their potential to express virulence factors with time. There was however, generally insignificant (t test; P≤0.05) lethal effect against all the A. haemolyticus isolates, but crystal violet exerted the highest lethal effect on some individual isolates followed by ethanol, bile salt and NaCl. Isolates from wastewater demonstrated the highest rate of resistance compared to isolates from river water. The cell kill index (CKI) increased as temperature stress (-5; -18; and -28ºC) increased with time. But the rate of loss of expression of virulence factors or viability was slower in isolates from wastewater and abattoir compared to those from river water. Sixty percent of the E. coli isolates showed various levels of resistance to different antibiotics (ampicillin (10 μg), cefuroxime, cephalexin, ceftazidime and tetracycline (30 95 μg in each case)) (multidrug resistance index (MDRI) values 4.20-5.60%). Relative inhibition zone diameters (RIZD) of C. dentata extracts against E. coli serotypes ranged between 8-28% (MIC, 100-2500 μg/ml), while against A. lwoffii and A. haemolyiticus, the RIZD values ranged between 10-28% (MIC, 100-850 μg/ml) and 6-28% (MIC 150-2500 μg/ml) respectively. However, higher MICs (MIC, 70-2500 mg/ml) were recorded for isolates with high MDRI values. Extracts demonstrated inhibitory action against the expression of both Vtx1 and Vtx2 genes in E. coli, A. haemolyticus and A. lwoffii. Saponins, tannins, glycosides, anthraquinones, flavonoids, steroids, phenols quinones, anthocyanins, amines and carboxylic acids were present in C. dentata. Ethanol root bark extracts consistently showed the highest DPPH radical scavenging activity (62.43%), total phenol content (TPH) (57.62 26 mg GAE/g) and reducing power (RP) (41.32%), followed by those of the stem bark and leaf extracts with the respective values of 54.68%, 37.77 mg GAE/g and 21.83%. The extracts also induced the leakage of Na+ and K+ 107 ions from both test bacteria. Detection of virulence factors, antibiotic resistance genes and transformation among these isolates is a very significant outcome that will influence approaches to proactive preventive and control measures and future investigations. Resistant verotoxic A. haemolyticus could further complicate treatment in verotoxic food-borne or nosocomial infections. Induction of cationic leakage by extracts of C. dentata is an indication of one of its mechanism of action on bacterial cells. The plant can therefore be a good source of antibiotic substances for composition as antioxidants or antimicrobials with novel mechanism of action for the treatment of verotoxic bacterial infections.

水資源缺乏引起的一系列問題在世界上已建得到廣泛關注，因此，確保提供潔淨衛生的水在保護人類健康和環境方面起著重要作用。近來，光催化作為頗有前景的替代方法被廣泛應用殺菌除污。二氧化鈦是目前研究最多應用最廣的光催化劑。基於紫外光譜照射，催化劑表面產生活性氧化物種，這些物種具有強氧化性能殺滅細胞。 / 本文首次研究了母體菌種大腸桿菌BW25113和它的同源單基因缺陷變異體對光催化殺菌的靈敏度差異。母體菌種和變異菌種表現出不同的耐受性。基於此，能幫助發掘出重要的變種。通過生物化學方法，可以檢測出不同菌種的生理性特徵。結合其他方法，可以進一步揭示光催化殺菌的生理性機理。 / 首先，我們篩選出了兩種重要的變異體。一種是大腸桿菌JW1081，即脂肪酸變異體，該菌種缺乏脂肪酸合成調節的關鍵基因。一種是大腸桿菌JW3942，即乙酰輔酶A變異體，該菌種缺乏乙酰輔酶A合成調控得到關鍵激酶。我們發現脂肪酸變異體對光催化處理的耐受性稍低，而乙酰輔酶A變異體則耐受性較高。 同時發現，溫度可以調節細胞膜的不飽和酸和飽和酸的比例。因此，我們提出脂肪酸和乙酰輔酶A是光催化殺菌中的重要影響因子。 / 更進一步研究發掘了細胞內酶和光催化產生的活性氧物種間的關係。大腸桿菌JW3914，即過氧化氫酶變異體，是發現的另一個重要的變異體。通過對母體和變異體的淬滅劑實驗，主要的殺菌活性氧物種是光催化產生的雙氧水，而不是羥基自由基。細胞體內的過氧化氫酶誘導在母體菌體內發現，而未在變異體內檢測到。 / 本課題採用母體/單基因變異體的研究方法，為全面深刻理解光催化殺菌的深層機理提供一種全新的研究思路。 / Many problems associated with the lack of clean, fresh water worldwide are well known. Developing countries will particularly be affected by water availability problems and there will be further pressure on water demand resulting from economic development and population growth. Therefore, the provision of safe and clean water plays a key role in protecting human health and the environment. Recently, photocatalytic oxidation (PCO) has been widely accepted as a promising alternative method of water disinfection. Titanium dioxide (TiO₂) has been investigated extensively and is the most widely used photocatalyst. Upon the irradiation of UVA lamp, reactive charged and oxidative species are generated on TiO₂ surface and can inactive the bacterial cells. / In this study, the photocatalytic performances of a parental strain (E.coli BW25113) and its isogenic single-gene deletion mutant strains have been investigated for the first time. These bacterial strains exhibited different sensitivies towards photocalytic inactivation. Based on this, it can help reveal some important mechanism from the mutations. Biotic factors were confirmed by physiological biochemical measurement. / Firstly, we screened out the potential mutation fabF⁻ mutant (E. coli JW1081, carrying the mutation of fabF759(del)::kan) and coaA⁻ mutant (E. coli JW3942, carrying the mutation of coaA755(del)::kan). The isogenic fabF⁻ mutant is slightly more susceptible, and coaA⁻ mutant is less susceptible to photocatalytic inactivation. Through conditioning temperature, it adjusts the ratio of unsaturated to saturated fatty acid (FA) of cell membrane. We propose that FA profile and coenzyme A level significantly affect photocatalytic inactivation of bacteria. Moreover, we show photogenerated electrons (e⁻) can directly inactivate the cells of E. coli. / Furthermore, we report the relationship between the bacterial intracellular enzyme and the reactive charged and oxidative species (ROSs) generated during photocataltic inactivation. The katG⁻ mutant, E. coli JW3914, carrying the mutation of katG729(del)::kan is another important mutation. The parental and katG⁻ mutant strains reveal that photogenerated H₂O₂ but not OH[subscript free] is another important reactive oxygen species to inactivate bacteria. The inducible catalase (CAT) corresponding to H₂O₂can be detected in parental strain but not in katG⁻ mutant. / The research methodology using parental/single-gene deletion mutant strains is expected to shed light on fully understanding of the fundamental mechanism of photocatalytic inactivation of E. coli. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Gao, Minghui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 130-177). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.v / Table of contents --- p.ix / List of Figures --- p.xiii / List of Plates --- p.xvii / List of Tables --- p.xviii / List of Equations --- p.xix / Abbreviations --- p.xxi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Water crisis --- p.1 / Chapter 1.2 --- Traditional disinfection methods --- p.3 / Chapter 1.2.1 --- Chlorination --- p.4 / Chapter 1.2.2 --- Ozonation --- p.6 / Chapter 1.2.3 --- Ultraviolet irradiation --- p.8 / Chapter 1.2.4 --- Multiple disinfectants --- p.10 / Chapter 1.3 --- Advanced oxidation process (AOPs) --- p.10 / Chapter 1.3.1 --- Hydrogen Peroxide/Ozone (H₂O₂/O₃) --- p.11 / Chapter 1.3.2 --- Ozone/Ultraviolet Irradiation (O₃/UV) --- p.12 / Chapter 1.3.3 --- Hydrogen Peroxide/ Ultraviolet Irradiation (H₂O₂/UV) --- p.12 / Chapter 1.3.4 --- Fenton's --- p.Reaction / Chapter 1.4 --- Solar photocatalytic disinfection (SPC-DIS) --- p.14 / Chapter 1.4.1 --- Photocatalyst-TiO₂ --- p.31 / Chapter 1.4.2 --- Irradiation sources --- p.35 / Chapter 1.4.3 --- TiO₂ photocatalytic process --- p.35 / Chapter 1.4.4 --- The role of photogenerated reactive charged and oxidative species (ROSs) --- p.38 / Chapter 1.5 --- Bacteria --- p.40 / Chapter 1.5.1 --- E. coli BW25113 --- p.40 / Chapter 1.5.2 --- E. coli Keio Collection --- p.41 / Chapter 1.5.3 --- Bacterial defense mechanism towards oxidative stresses --- p.44 / Chapter 1.6 --- Photocalytic applications --- p.53 / Chapter 1.7 --- Significance of the project --- p.55 / Chapter 2. --- Objectives --- p.58 / Chapter 3. --- Genetic studies of the roles of fatty acid and coenzyme A in photocatalytic inactivation of Escherichia coli --- p.61 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials and methods --- p.65 / Chapter 3.2.1 --- Photocatalyst --- p.65 / Chapter 3.2.2 --- Bacterial nutrient --- p.66 / Chapter 3.2.3 --- Bacterial strains --- p.67 / Chapter 3.2.4 --- Photocatalytic inactivation --- p.69 / Chapter 3.2.5 --- Fatty acid profile --- p.72 / Chapter 3.2.6 --- Fluorescent measurement of bacterial coenzyme A content --- p.74 / Chapter 3.2.7 --- The role of photogenerated electrons (e⁻) to bacterial inactivation --- p.74 / Chapter 3.2.8 --- Transmission Electron Microscopic (TEM) --- p.75 / Chapter 3.2.9 --- Photoelectrochemical measurement --- p.77 / Chapter 3.3 --- Results --- p.77 / Chapter 3.3.1 --- Photocatalytic inactivation --- p.77 / Chapter 3.3.2 --- Effects of pre-incubation at different temperatures --- p.80 / Chapter 3.3.3 --- Fatty acid profile --- p.83 / Chapter 3.3.4 --- Fluorescent measurement of bacterial coenzyme A content --- p.84 / Chapter 3.3.5 --- The role of electron (e⁻) in photocataytic inactivation --- p.84 / Chapter 3.3.6 --- Transmission electron microscopy (TEM) --- p.89 / Chapter 3.3.7 --- Photocurrent measurement --- p.90 / Chapter 3.4 --- Discussion --- p.90 / Chapter 3.5 --- Conclusions --- p.96 / Chapter 4 --- Genetic and physiological studies of the role of Catalase and H₂O₂ in photocatalytic inactivation of E. coli --- p.98 / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Materials and methods --- p.101 / Chapter 4.2.1 --- Bacterial strains --- p.101 / Chapter 4.2.2 --- Photocatalytic performance --- p.102 / Chapter 4.2.3 --- Scavenger studies --- p.103 / Chapter 4.2.4 --- Effects of different pHs on photocatalytic inactivation --- p.104 / Chapter 4.2.5 --- Measurement of bacterial catalase activity and H₂O₂ --- p.104 / Chapter 4.2.6 --- Transmission electron microscopy (TEM) --- p.105 / Chapter 4.2.7 --- Atomic absorption spectrophotometer (AAS) --- p.105 / Chapter 4.3 --- Results and discussion --- p.106 / Chapter 4.3.1 --- Photocatalytic performance --- p.106 / Chapter 4.3.2 --- Scavenger studies --- p.108 / Chapter 4.3.3 --- Contribution of hydrogen peroxide (H₂O₂) --- p.111 / Chapter 4.3.4 --- Effects of different pHs on photocatalytic inactivation --- p.114 / Chapter 4.3.5 --- Bacterial catalase (CAT) activity --- p.116 / Chapter 4.3.6 --- Destruction model of bacterial cells --- p.118 / Chapter 4.4 --- Conclusions --- p.120 / Chapter 5. --- General conclusions --- p.122 / Chapter 6. --- Prospectives --- p.125 / Chapter 7. --- Appendix --- p.127 / Chapter 8. --- References --- p.130

Biofilm formation may increase survival and persistence of Escherichia coli in the highly variable conditions of soil environments, though it remains unknown the extent variation in biofilm formation affects survival. We asked what genetic traits influence biofilm formation in phylogroup D E. coli isolates from surface soils, and are they associated with the soil environment? Biofilm density was analyzed and compared with soil environment characteristics. Isolates produced more biofilm per unit growth at 15°C than 37°C. Biofilm formation was greater in soil isolates than fecal isolates and in soils with moisture and higher calcium and pH levels. A GWAS analysis found variants involved in cell envelope formation and structure were associated with biofilm formed at 37°C, and stress response and iron acquisition variants were associated with biofilm formed at 15°C. Motility variants were associated with a negative effect on biofilm formed and adhesion variants associated with a positive effect. / National Science Foundation (NSF) award no. DEB-1453397 to P.W.B. / ND-EPSCoR

Thesis (MSc Food Sc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Over the past two decades, there has been an increase in the use of water sources for irrigation, as well as an increase in Escherichia coli outbreaks linked to fresh produce. The full extent and type of E. coli contamination present in natural water sources is unknown and the contamination sources have also not been confirmed. The aim of this study was to enumerate and characterise E. coli from both irrigation water and potential contamination source sites. Total coliform and E. coli counts found in contamination source sites were as high as log 7.114 and log 6.912 MPN.100 mL-1, respectively. Total coliform and E. coli counts for irrigation sites were lower, with maximum counts of log 5.788 and log 5.768 MPN.100 mL-1, respectively. It was found that more than one third (5/14 = 35.71%) of the irrigation sites had E. coli counts exceeding the guidelines (<1 000 counts.100 mL-1) for ‘safe’ irrigation water for fresh produce (<1 000 counts.100 mL-1) as set by the Department of Water Affairs (DWA) and World Health Organisation (WHO), making the water unsuitable for the irrigation of fresh produce. Phylogenetic subgroups (A0, A1, B1, B22, B23, D1 and D2) and the MALDI Biotyper system (PCA dendrogram) were used to create a fingerprint of each E. coli isolated from the environment. These were then used to link E. coli strains from irrigation water to their most probable contamination origin. Escherichia coli population structure was found in this study, to be better suited for linking E. coli strains from irrigation water to their most likely source, than just applying the phylogenetic grouping. The MALDI Biotyper data in combination with the phylogenetic subgroup assignment was then used to group similar strains and link E. coli from irrigation water to their contamination sources by comparing population structures. Strains isolated from surface and groundwater showed similar distribution patterns, but groundwater strains showed a population structure more indicative of porcine and bovine origin, while surface water showed population characteristics which could not be used to make conclusive links between the irrigation water and suspected contamination sources. When investigating the population structures of individual sample sites, it was found that phylogenetic subgroups A0, A1 and B1 frequently made up the bulk of the E. coli population. It was also found that linking individual irrigation sites to contamination sources was successful, as irrigation site Berg-2 was found to have a similar population structure to contamination source site Plank-1 which represents human pollution from an informal settlement. This led to the conclusion that Berg-2 was being contaminated by human pollution, most probably from an informal settlement. Upon further investigation it was found that Berg-2 is downstream of an informal settlement, proving that E. coli population structure is a successful means of microbial source tracking (MST). Virulence factors of the 153 E. coli isolated during the study were identified and the potential risk associated with using the investigated irrigation water for irrigation of fresh produce, was determined. Two enteropathogenic E. coli (EPEC) strains were isolated from the irrigation water, one from the Plankenburg River water, and the other from a borehole in the Drakenstein area. The latter indicates that borehole water is not as safe as was once thought, and that there are bacterial contaminants finding their way into groundwater. The occurrence of an EPEC strain in river water shows that neither ground nor surface water is guaranteed to be safe, and that treatment of water being used for the irrigation of fresh produce should be implemented. / AFRIKAANSE OPSOMMING: Oor die afgelope twee dekades was daar nie net 'n toename in die gebruik van alternatiewe waterbronne vir besproeiing nie, maar ook 'n toename in uitbrake van Escherichia coli uitbrake wat aan vars produkte gekoppel kan word. Die tipe E. coli-besmetting wat in natuurlike waterbronne teenwoordig is, is onbekend en die besmettingsbron is ook nog nie bevestig nie. Daarom was die doel van hierdie studie om die voorkomssyfer van E. coli van beide besproeiingswater en potensiële kontaminasiebronne te bepaal, asook om die E. coli te karakteriseer. Totale kolivorme en E. coli-tellings wat in kontaminasiebronne gevind is, het ‘n maksimum van log 7,114 en log 6,912 MPN.100 mL-1 onderskeidelik bereik, terwyl die totale kolivorme en E. coli-tellings vir besproeiingswater laer was, met 'n maksimum van log 5,788 en 5,768 MPN.100 mL-1, onderskeidelik. Dit is bevind dat meer as 1/3 (5/14 = 35,71%) van die besproeiingswaterbronne meer E. coli bevat as wat toegelaat word in die riglyne vir "veilige" besproeiingswater vir vars produkte (<1 000 fekale koliforme.100 mL-1) wat deur die Departement Waterwese (DWA) en die Organisasie vir Wêreldgesondheid (WHO) aanbeveel word. Filogenetiese subgroepe (A0, A1, B1, B22, B23, D1 en D2) en die ‘MALDI Biotyper’-stelsel (PKA dendrogram) is gebruik om unieke profiele vir elke geïsoleerde E. coli te skep. Dié profiele is daarna gebruik om E. coli-stamme van besproeiingswater te koppel aan die mees waarskynlike oorsprong van kontaminasie. Daar is in hierdie studie bevind dat die E. coli-populasiestruktuur beter geskik was vir die koppeling van E. coli-stamme van besproeiingswater na hul mees waarskynlikste bron, as net die toepassing van die filogenetiese groepering. Dit was toe gevind dat E. coli wat uit oppervlak- en grondwater geïsoleer is, soortgelyke verspreidingspatrone het, maar grondwaterstamme se bevolkingstruktuur is meer aanduidend van fekale besmetting deur varke en beeste, terwyl oppervlakwater se bevolkingseienskappe nie duidelik genoeg was om ‘n gevolgtrekking oor moontlike bronne van besmetting te maak nie. Toe die populasiestruktuur van die individuele bemonsteringspunte ondersoek is, is daar bevind dat die filogenetiese subgroepe A0, A1 en B1 dikwels die meeste tot die E. coli bevolking bydra. Daar is ook bevind dat die koppeling van isolate in individuele besproeiingswaterbronne met hul mees waarskynlike bronne van kontaminasie suksesvol was. Besproeiingswater van Berg-2 het 'n soortgelyke populasiestruktuur as Plank-1 wat beskou is as ‘n kontaminasiebron. Dit het gelei tot die gevolgtrekking dat Berg-2 heel waarskynlik deur menslike besoedeling beïnvloed word, soos Plank-1, en dat daar moontlik ook ‘n informele nedersetting by Berg-2 betrokke is. Na verdere ondersoek is gevind dat Berg-2 inderdaad ook stroomaf van 'n ander informele nedersetting geleë is, wat bewys dat die E. coli-populasiestruktuur 'n suksesvolle manier is om E. coli kontaminasie te verbind met besproeiingswater. Patogeniese faktore, wat in E. coli voorkom en maagkieme veroorsaak, is vooraf getoets in elkeen van die 153 E. coli-isolate wat tydens die studie geïdentifiseer is. Twee ‘enteropathogenic’ E. coli (EPEC)-stamme is uit die besproeiingswater geïsoleer: een uit die Plankenburgrivier (Plank-3), en die ander uit 'n boorgat in die Drakenstein-gebied (Boorgat A1). Hierdie inligting dui aan dat boorgatwater nie so veilig is as wat voorheen vermoed is nie, en dat bakteriese kontaminasie wel vookom wat nie alleen die grondwater besmet nie, maar ook daarin oorleef. Die voorkoms van die EPEC-stamme in hierdie studie is ‘n aanduiding dat beide grond- en opppervlakwater ewe gevaarlik kan wees, en dat daar dus geen waarborg vir die veiligheid van die water is nie. Die behandeling van grond- en oppervlakwater, wat vir die besproeiing van vars produkte gebruik word, moet daarom ernstig oorweeg word om moontlike uitbrake van E. coli op vars produkte te verhoed.