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Item Assessment of bioflocculant production by two marine bacteria isolated from the bottom sediment of marine Algoa Bay(University of Fort Hare, 2015) Ntozonke, Ncedo; Okoh, Anthony I; Nwodo, Uchechukwu UBioflocculants are polymers, mostly, of microbial origin which floc out suspended particles from liquid medium. The ability of these biopolymers to remove suspended particles from solutions is termed bioflocculation, and the efficiency of flocculation activities depends on the characteristics of the flocculants. In comparison with conventionally used flocculants, bioflocculants have the advantage of being safe (no toxic effects known), biodegradable and harmlessness to the environment. The study assessed production of bioflocculant by two marine bacteria from the bottom sediment of marine environment. The 16S rDNA was used for identification, and the two bacteria species were identified as Enterococcus hirae and Bacillus thuringiensis. Factors affecting the production and activity of the bioflocculants produced by these two organisms were studied. The bacteria optimally produced bioflocculant with fructose (91.7%) and urea (91%) as sole carbon and nitrogen sources respectively. Mg2+ (87%) and Ca2+ (86%), likewise, served as best cation sources on the production of the bioflocculant at pH 5(93%). Additionally, the flocculating activity of the bioflocculant increased with the addition of Mg2+ (81%) and Na+ (81%), and the highest flocculating activity was at pH 5 of the kaolin clay. The Fourier transform infrared spectroscopy (FTIR) shows that the bioflocculant is a glycoprotein. The second bacterium (Bacillus thuringiensis) produced bioflocculant optimally when the media had mixed nitrogen sources (Urea, ammonium chloride and tryptone (67%)) and glucose (85.65%) as a sole carbon source, also Ca2+ (74.6%) was the best cation that induced the production of bioflocculant. After purification, the bioflocculant flocculated optimally in alkaline pH 12 (81%) in the presence of Mn2+ (73%) and Ca2+ (72.8%). Chemical analysis of the bioflocculant revealed it to be a polysaccharide. Both bioflocculants flocculate efficiently and can be used to replace synthetic flocculants in water treatment, wastewater, in downstream processing, and processing of food and chemicals and other industrial uses of flocculants. Challenges though (i) are to develop conditions for large scale production of the bioflocculant, (ii) to do further characterization of the both bioflocculants (iii) to assess the bioflocculants for treatments of water/wastewater, and to apply it in various industrial processes.Item Isolation and characterisation of lignocellulose degrading bacteria from Tyume River in the Eastern Cape Province, South Africa(University of Fort Hare, 2015) Tembisa, Papiyana Ayavuya; Okoh, Anthony IThis study focuses on the isolation and characterization of bacteria from lignocellulosic biomass obtained from the sediments of the Tyume River in Alice, Eastern Cape and to determine those bacterial isolates with good potential for modification and decomposition of lignocellulosic biomass for industrial application. Several bacterial isolates were recovered and screened for ability to degrade various lignocellulosic materials. Nine of the isolates were positive for lignocellulolytic activity. Four isolates were cellulase positive and six were xylanase positive. Moreover, one isolate (SB1) was positive for both xylanase and cellulase activities and showed the best hydrolysis zone on solid media. This isolate was then chosen as the best and identified molecularly. The 16S rDNA sequence analysis indicated that SB1 was a Bacillus cereus species. Factors affecting the cellulose and xylanase enzyme production by the organisms were studied. The organisms produced the enzymes maximally at earlier hours of incubation (12-30 hr) and optimally at acidic pH (3-5) and at moderate temperatures (35-45ºC). SB1 appears to hold promise in the decomposition of lignocellulosic wastes.Item Assessment of the flocculating efficiency of bioflocculant produced by bacillus sp. Aemreg4 isolated from Tyhume river, Eastern Cape, South Africa(University of Fort Hare, 2016) Ntsangani, Nozipho; Mabinya, L V; Okoh, Anthony IBioflocculants are flocculating substances produced by microorganisms during growth and have recently received considerable attention from researchers; due to their biodegradability, non-toxicity and lack of secondary pollution from degradation intermediates. This study evaluated the efficiency of bioflocculant produced by Bacillus sp. AEMREG4 isolated from Tyhume River. The bacterial identification was through 16S rDNA sequencing; nucleotide sequences were deposited in GenBank as Bacillus sp. AEMREG4 with an Accession number KP406729. The optimum culture conditions for bioflocculant production were an inoculum size of 4% (v/v) and starch as well as yeast extract as sole carbon and nitrogen sources respectively. The addition of CaCl2 enhanced the flocculating activity, at a wide range of pH 4-10 and the highest flocculating activity was reached at an initial pH 8 (80%). A bioflocculant yield of 0.78 g was recovered from 1 L of culture broth. The optimum flocculating activity of 78% was reached at the lowest bioflocculant dosage of 0.1 mg/ml and the presence of divalent cations (Ca2+, Mn2+ and Mg2+) as well as a trivalent cation (Al3+) enhanced flocculating activity. The purified bioflocculant retained more than 70% flocculating activity when subjected to heating at 100 °C for 1 h and maximum flocculating activity of 83% was achieved at both acidic and basic pH values of 3 and 10 respectively. Chemical analysis showed that the bioflocculant is predominantly polysaccharide. The Fourier transform infrared (FTIR) spectrum revealed the presence of carboxyl, hydroxyl and methoxyl groups as the functional moieties and the scanning electron microscopy (SEM) imaging of the purified bioflocculant showed its morphological structure as rod-shaped which contributes to its high flocculating efficiency. The high flocculation activity displayed by this bioflocculant indicates its potential suitability for industrial application.Keywords: Bioflocculant, Bacillus sp. AEMREG4, flocculating activity.Item A preliminary study on the effects of elevated CO2 on aphid resistance of Tugela Dn and the population dynamics of the Russian wheat aphid (Homoptera: Aphididae), Diuraphis noxia(University of Fort Hare, 2015) Mundondo, Daphine; Bradley, GFood security is of major importance due to the increasing world population with 8.9 billion people expected by 2050 (Cohen, 2003). Diuraphis noxia (RWA), have caused aggravating, massive losses to wheat farmers in many areas of the world. If unchecked, RWA are able to destroy plants resulting in major economic impacts (Botha, 2013). Due to ineffective use of other control methods, the Small Grains Institute in Bethlehem, South Africa, have therefore developed resistant cultivars to the known RWA subtypes over the past decades through intensive breeding programmes (Tolmay et al., 2006). Climate change has however become a major factor threatening food security especially with the observed increase in CO2 from less than 300 ppm in pre-industrial period to the current 385 ppm and is predicted to reach 550 ppm by 2050 (IPCC, 2007; Meehl et al., 2007). Elevated CO2 concentration may affect individual species of a community hence the need to understand the wheat-aphid interactions. In this study, population growth rates and virulence of RWA SA1 at ambient (385 ppm) and elevated (450 ppm) CO2 concentration were evaluated on two wheat cultivars: Tugela Dn (resistant) and Scheepers (susceptible). Fluorescence microscopy techniques using aniline blue were used to investigate feeding related damage caused by RWA SA1 through an examination of callose deposition at the two CO2 concentration. A two-dimensional gel electrophoresis method was developed in order to determine the effect of RWA SA1 on the wheat cultivars proteome at the two CO2 concentration. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuestTM Basic 2D Gel analysis software. Populations of RWA SA1 increased significantly on the two wheat cultivars at both CO2 concentration. Although the population growth rate for RWA SA1 on both cultivars was generally exponential at all treatments, growth at elevated CO2 concentration was noticeably faster with populations increasing 3 fold in 14 days as compared to the 2 times at ambient CO2 concentration. Hence, both cultivars provided a better quality host for RWA SA1 at 450 ppm than 385 ppm. There was no significant difference between RWA SA1 population on Tugela Dn and on Scheepers at elevated CO2 concentration on day 14 after infestation which means there was a change in the resistance mechanism in Tugela Dn at this condition. Approximately 70% of the total leaf showed chlorosis by 21 days of aphid infestation for both cultivars although the susceptible cultivar was more vulnerable. There was low callose deposition in the controls (uninfested plants) but heavy callose in infested plants due to aphid feeding. A proteomics approach was used as a pilot study to investigate whether it would be possible to identify the changes in the resistance mechanism during aphid infestation under elevated CO2 levels. The major changes in the proteome of the control group (uninfested Tugela Dn at ambient versus elevated CO2 concentration) occurred in the early events (day 1-7) in the molecular weight range of approximately 25 kDa to 55 kDa are mainly within the basic to neutral pH range. This was suggested to be a result of mechanisms to adjust to the CO2 concentration. Elevated CO2 results in instant higher photosynthetic rates and C:N ratios as well as changes in expression levels of SA-dependant defense genes (Lindroth 1995; Hughes and Bazzaz, 2001; Sun et al., 2013). Because most of these changes are directly regulated by proteins, it is expected that the most differential protein expression will occur immediately after the atmospheric changes (early events) as was shown in the study. Infested plants under elevated and ambient conditions showed that the stress conditions gave rise to differentially regulated proteins within the wheat proteome. Most changes occurred elevated CO2 levels. It can be suggested that the changes were a result of differentially regulated plant defence proteins which fall in this range (25 kDa - 80 kDa) such as peroxidases, chitinases and β-1.3-glucanases as well as protein kinases, heat-shock proteins and photosynthetic proteins. These results indicate that there has been changes in the resistance due to elevated CO2 because of the evident changes in the proteome. If so, then the results will be similar to those documented by Louw (2007) where up-regulation was due to putative storage proteins, proteins involved in photosynthesis, heat shock proteins and defense proteins. Of course, the pI value and molecular mass of the proteins and the identification of the proteins in these spots, must be determined in future work to specifically identify whether these suggestions are authentic. However, Louw (2007) also reports that the susceptible Betta wheat cultivar, displayed a defence response similar to the HR although it was unable to up-regulate specific defensive proteins against RWA infestation but proteins for broad resistance. Although the changes in the proteins in infested Tugela Dn under elevated CO2 concentration were not accurately identified, the defense mechanism is similar to that portrayed by the susceptible Betta wheat cultivar which shows that the resistance mechanism had been overcome. Because this was a pilot study and preliminary results were obtained due to limited funding and time constraints, suggestions were made on how to further develop the method to obtain statistically significant results.Item Phytochemical analysis and antibacterial properties of aqueous and ethanol extracts of Brachylaena elliptica (Thurb.) dc. and Brachylaena ilicifolia (Lam.) Phill & Schweick(University of Fort Hare, 2015) Sagbo, Idowu Jonas; Afolayan, A J; Bradley, GResistance of human pathogenic bacterial strains results in selective pressure against known antibiotic. However, plant derived compounds that possess antibacterial potential are currently being investigated for treatment of wound infections in diabetic patients as they are inexpensive and non-toxic. Hence, this dissertation was designed to evaluate two medicinal plants (Brachylaena elliptica and Brachylaena ilicifolia) traditionally used in the treatment of various diseases such as diabetes, and its secondary complications in diabetic patients. The in vitro antioxidant activity of both plants were evaluated using DPPH (1, 1-diphenylhydrazl), ferric reducing power, ABTS (2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), NO (nitric oxide) and H2O2 (hydrogen peroxide) techniques. The antibacterial test and Minimum inhibitory concentration (MIC) was determined by agar dilution method against 5 bacteria strains (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogene, Proteus vulgaris and Proteus mirabilis) infecting wounds in diabetic patients using amoxicillin and ciprofloxacin as positive control. The phytochemical analyses were assessed using standard published methods. Identification of bioactive components in essential oils of both plants were assessed using GCMS. The aqueous and ethanol extracts of both plants were also evaluated to identify bioactive components using LC-MS. The results of the phytochemical analysis revealed the presence of phenols, tannins, flavanoids, flavanols, proanthocyanidins, saponins and alkaloids in both plants. Both plants indicated strong antioxidant activities which might be due to the presence of bioactive compounds. The aqueous and ethanol leaf extracts of both plants demonstrated appreciable broad spectrum activities against these wound pathogens with MIC ranging between 5 and 0.3 mg/ml. The GC-MS analysis of the essential oils of both plants revealed the presence of monoterpenes, oxygenated sesquiterpenes, phenolics and esters. The LC-MS analysis of the aqueous and ethanol leaf extracts of both plants showed that both plants are rich in alkaloids, terpenes, terpenoids, monoterpernoids, and flavanoids. Conclusively, this study has partially justified the ethnomedicinal use of B. elliptica and B.licifolia leaves for the treatment of various diseases, including diabetes and wound infections caused by bacteria in diabetic patients. These may be attributed to the presence of antioxidant compound such as phenols, flavanoids, saponins, tannins, alkaloids and other phytochemical compounds.Item Characterization of some virulence and antibiotic resistance genes of Staphylococcus aureus isolated from cases of Bovine Mastitis in Nkonkobe Municipality, Eastern Cape Province, RSA(University of Fort Hare, 2015) Pekana, Abongile; Green, E; Okoh, Anthony I; Ndip, R NStaphylococcus aureus is one of the predominant causative agents of mastitis disease in dairy herds. Mastitis disease has a negative impact in the economic losses in the dairy sector across the globe. The aim of this study is to detect some of the virulence genes in the S. aureus isolated from 400 milk samples of subclinical and clinical mastitis dairy cows in Fort Hare dairy farm and Middle Drift dairy farm in Alice in the Eastern Cape province of South Africa. In addition antibiotic resistance pattern and antibiotic resistance genes were investigated. Gram-staining, oxidase test, catalase test and API Staph kit were preliminary biochemical tests used for the identification of S. aureus isolates. The MALDI-TOF-MS was also used for further identification. Polymerase chain reaction was performed of genes encoding antibiotic resistance as well as clumping (clfA), coagulase (coa) gene, toxic shock syndrome (tsst), exfoliative toxin A and B (eta and etb), and the gene segment encoding the immunoglobulin G binding region and X region of protein gene spa. A total of 20 (5%) S. aureus strains obtained from 400 milk samples from the two farms were subjected to 16 antibiotics for antibiotic susceptibility testing. In Middle Drift dairy farm 11 (5.5%) isolates were obtained from 200 samples and 9 (4.5%) isolates were obtained in Fort Hare dairy farm from 200 samples. A large percent of the isolates were resistant to penicillin G (60%), followed by trimethoprim (60%) and tetracycline (60%), trimethoprim-sulfamethaxazole (55%), telithroprim (55%) and doxycycline (45%). Most of the isolates were sensitive to several (50-85%) antibiotics. Of the twenty isolates tested 12 samples contained the penicillin antibiotic resistance gene (blaZ gene), 8 samples contained at least one aminoglycoside-modifying enzyme gene (AME gene); the (aac(6’)/aph(2’’) gene and no amplification occurred for aph(3’)-IIIa and ant(4’)-Ia) genes. In the case of the tetracycline antibiotic resistance gene (tetK and tetM), 2 samples contained tetM and a single sample contained tetK gene. No amplification was observed for the erythromycin antibiotic resistance genes (ermA, ermB, ermC, Mef and msrA). All the samples tested were negative for the expression of toxic syndrome gene (tsst), etb, and Immunoglobulin G binding region. However, amplification of the clumping factor was observed in 7 (35%) isolates of S. aureus, exfoliative toxin (eta) expressed 4(20%) isolates; coagulase gene (coa) yielded six DNA bands of six differences sizes from 16 (80%) isolates. A total of four different bands size were expressed for the spa X region from 12 (60%) isolates. The data obtained in this study suggests that poor hygienic practices and inadequate management practices are responsible for the increase in Staphylococcus aureus isolation. The high resistance of S. aureus to antibiotics and the distribution of virulence genes contribute in bovine mastitis in these farms may cause health problems in the community consuming raw milk purchased from these farms.Item Isolation and molecular characterization of Bacillus cereus from cow’s raw milk(University of Fort Hare, 2015) Lukanji, Zinathi; Ndip, R N; Green, E; Okoh, Anthony I; Ndip, R NBacillus cereus is a group of ubiquitous facultative anaerobic spore forming Gram-positive rods commonly found in soil. It has been detected and implicated in several contaminated food products and raw milk in dairy farms and it causes foodborne gastroenteritis by producing several toxins. This study is aimed at characterizing virulence determinants of B. cereus from cow‟s raw milk. A total of 400 raw milk samples were collected in Fort Hare Dairy Trust and Middledrift Dairy Farm; and cultured on Polymyxin pyruvate Egg-Yolk Mannitol Bromothymol Agar (PEMBA) for 48 hours at 37°C. DNA was isolated from the isolates and 16S rDNA was amplified and sent to Central Analytical Laboratory for sequencing. The gyrB gene of B. cereus was also used to confirm the identity of the isolates. Antibiotic susceptibility profiles of the isolates together with virulence genes were investigated. Multilocus Sequence typing was used to investigate the genetic relatedness of some selected isolates. Furthermore, spores of the isolates were produced, harvested and their concentrations determined. All (100%) of the isolates were identified as having a 96-99% similarity to B. cereus, B. thuringiensis and B. anthracis using sequencing; while gyrB gene was observed in all (100%) of the isolates. Three virulence genes nheA, nheB, nheC encoding for non haemolysin enterotoxin were amplified in all (100%) the isolates. All (100%) of the isolates were susceptible to doxycycline, gentamycin, tetracycline, ciprofloxacin, chloramphenicol and streptomycin. Resistance to rifampicin and penicillin G was predominant with equal rate of 100%, while susceptibility to erythromycin, clindamycin and doxycycline ranged from 60% to 100%. The selected isolates were related and are descendants of the same ancestor. All (100%) the isolates produced spores. The B. cereus isolates contain virulence genes, has multiple antibiotic drug resistance and produce spores, which poses a health risk to the public and cannot be used as probiotics.Item Evaluation of the quality indices of the final effluents of two wastewater treatment plants in buffalo City Metropolitan Municipality in the Eastern Cape Province(University of Fort Hare, 2015) Osuolale, Olayinka O; Okoh, Anthony IWaste waters can be sources of pollution to surface water and the environment with severe implications for public health. Most treatment plants in the Buffalo City Municipality in the Eastern Cape Province discharge their treated effluent into the surface waters which directly and indirectly impacts on the quality of surface waters in the region. The objective of this study was to determine the microbiological and physicochemical qualities of the final effluents of two wastewater treatment plants in the Buffalo City Municipality in the Eastern Cape Province of South Africa over a period of 12 months (September 2012 to August 2013). The qualities of the final effluents of WW-Ama Wastewater Treatment Plant with respect to phosphate (3.9 mg/l - 20.6 mg/l), free chlorine (0.05 mg/l - 0.71 mg/l), chemical oxygen demand (COD) (4.7 mg/l - 211 mg/l), and faecal coliform (0 - 2.92 × 104 CFU/100 ml) were not in compliance with the permissible limits set for effluent discharged to surface water by South Africa guidelines for effluent discharge. Other physicochemical parameters like biological oxygen demand (BOD) (2.2 mg/l - 9.0 mg/l), total dissolve solid (TDS) (253 mg/l - 336.3 mg/l) and turbidity (4.8 NTU - 43.20 NTU) with no SA regulatory set limits were compared to other regulatory standards and they do not comply with the limits. Also, at the second WWTP’s, the WW-Dim Treatment Plant effluent quality for free chlorine (0.06 mg/l - 7.2 mg/l), BOD (0.1 mg/l - 7.4 mg/l), and turbidity (4.02 NTU - 24.3 NTU) also did not comply. For microbiological qualities, counts of presumptive E. coli and Vibrio ranged between 0 - 2.92 × PROFESSOR ANTHONY I. OKOHAntibiogram of the bacterial isolates were determined using the disk diffusion method. A total of 107 confirmed E. coli and 100 confirmed Vibrio spp. were used for this assay. Results of antibiotic sensitivity test revealed that 63.6% of the E. coli isolates were resistance to ampicillin while 49.5% were resistant to tetracycline and cephalothin. The least resistances were observed against gentamicin (3.7%) and cefotaxime (1.9%). No resistance was observed against meropenem. For the Vibrio spp, resistance was most frequently observed against tetracycline (38%) ampicillin (26%), chloramphenicol (16%), cefotaxime (14%), trimethoprim-sulfamethoxazole (13%) and the least resistance observed was against ciprofloxacin (1%). This study demonstrates that poorly treated wastewater effluent can be a source of eutrophic water with high nutrient levels and pathogenic bacteria and enteric viruses as well as antibiotic resistance determinants that could impact negatively on human health. The finding of this study also suggests that WWTPs have to be properly monitored and controlled to ensure compliance to set guidelines. This could be attained through the application of appropriate treatment processes, which will help to minimize possible dangers to public environment health.Item Assessment of the quality indices and prevalence of Escherichia coli pathotypes in selected rivers of Osun state, Southwestern Nigeria(University of Fort Hare, 2015) Titilawo, Osuolale Yinka; Okoh, Anthony I; Obi, Larry CSurface waters are important freshwater sources used for domestic, industrial, agricultural and recreational activities, and the availability of good quality freshwater is indispensable for preventing water-borne diseases and improving quality of life especially in communities that lack pipe-borne water. Water samples were collected from ten rivers at different locations in Osun State, Southwestern Nigeria. A total of 12 physicochemical parameters, counts of total coliforms (TC) and Escherichia coli isolates were determined using standard analytical procedures. Confirmed Escherichia coli isolates (n=300) were assessed for the presence of 10 virulence genes (VGs) associated with Escherichia coli strains causing intestinal and extra-intestinal infections. The recovered Escherichia coli isolates were elucidated for their antibiogram profiling by disk diffusion method and the resistant isolates were further profiled for their genotypic antimicrobial resistance by polymerase chain reaction technique. The physicochemical qualities ranged as follows: pH (6.9 - 7.6), temperature (26 – 29 ºC), turbidity (2.28 – 9.46 NTU), electrical conductivity (229 – 581 μS/cm), nitrate (0.03 – 0.05 mg/L), nitrite (0.00 – 0.01 mg/L), sulphate (3.33 – 20.33 mg/L), chloride ions (7.83 – 27.33 mg/L), dissolved oxygen (4.23 – 5.57 mg/L), total dissolved solids (56 – 184 mg/L), total hardness (78 – 519 mg/L) and alkalinity (50.67 – 146.67 mg/L). Statistical analysis showed that pH, temperature, electrical conductivities, nitrates, nitrites, chloride, dissolved oxygen, total dissolved solid, total hardness and alkalinity were significantly different (P < 0.05), whereas turbidity and sulphate were not significantly different (P ˃ 0.05) from each parameter with respect to sampling sites. While the VG lt for enterotoxigenic E. coli had the highest prevalence of 45%, the enteropathogenic E. coli genes eae and bfp were detected in 6% and 4% of the isolates respectively. The VGs stx1 and stx2 specific for the enterohemorrhagic E. coli pathotypes were equally detected in 7% and 1% of the isolates respectively. Also, the VG eagg harboured by enteroaggregative pathotype and diffusely-adherent E. coli VG daaE were detected in 2% and 4% of the isolates respectively and enteroinvasive E. coli VG ipaH was not detected. In addition, the VGs papC for uropathogenic and ibeA for neonatal meningitis were frequently detected in 19% and 3% of isolates respectively. While all the isolates tested were susceptible to imipenem, meropenem, amikacin and gatilofloxacin, others were variously susceptible, and resistant as follows; ciprofloxacin (96%), kanamycin (95%), neomycin (92%), streptomycin (84%), chloramphenicol (73%), nalidixic acid (66%), nitrofuratoin (64%), gentamycin (63%), doxycycline (58%), cefepime (57%), tetracycline (49%) and cephalothin (42%). Conversely, all the isolates were resistant to sulphamethoxazole, and high levels of resistance were equally observed against amoxycillin (59%), ampicilin (57%) and cefuroxime (40%). Cefepime, cephalothin, cefuroxime, nalidixic acid, nitrofuratoin, chloramphenicol and tetracycline were not significantly different in their effect against the isolates from all locations (P > 0.05), whereas the resistance profile of the isolates against gentamycin, ciprofloxacin, sulphamethoxazole, ampicillin and amoxicillin were significantly different (P < 0.05). Amikacin, kanamycin, streptomycin, meropenem, imipenem and gatilofloxacin were statistically excluded from the analysis since all tested isolates showed total susceptibility to these antimicrobials. The multiple antibiotic resistance indexing ranged from 0.50 to 0.80 for all the sampling locations and exceeded the threshold value of 0.2. Prevalence and distributions of the 19 resistance determinants assessed were obtained as follows; [sulfonamides (sulI (8%), sulII (41%)], [beta-lactams; (ampC 22%; blaTEM, (21%), blaZ (18%),], [tetracyclines (tetA (24%), tetB (23%), tetC (18%), tetD (78%), tetK (15%), tetM, (10%)], [phenicols; (catI (37%), catII (28%), cmlA1 (19%)] and [aminoglycosides; (aacC2 (8%), aphA1 (80%), aphA2 (80%), aadA (79%) and strA (38%)]. The Pearson chi square exact test revealed many strong significant associations among ampC, blaTEM, blaZ and tetA genes with some determinants screened. In the same vein, a grand total of 366 resistance gene fingerprints were spotted across the sampling locations and among the resistant pathotypes, the modal prevalent gene prints were found among the ETEC strains in 148 (40%), being the predominant pathotype observed, followed by UPEC strains 80 (22%) while the lowest was the least occurring EAEC pathotype 14 (4%). While some physicochemical parameters exceeded prescribed standards for drinking water, some fell within. The total coliforms obtained in all the sampling sites were above the acceptable limits. Findings reveal the presence of diarrhoeagenic and non-diarrhoeagenic E. coli in the selected rivers and suggest a potential public health risk as the rivers are important resources for domestic, recreational and livelihood usage by their host communities. The multiple drug resistance indexing signifies isolates and pathotypes of high antimicrobial usage origin. An increase in the antimicrobial resistance signatures towards conventionally used antibiotics as observed in this study necessitates for safe water supply, adequate sanitation facilities and proper surveillance programs towards the monitoring of antimicrobial resistance determinants in water-bodies. Generally, results from this study indicate that the river waters are not suitable for consumption, domestic or recreational use and re-echo the importance of safeguarding the freshwater resources of Southwestern Nigeria.Item Antibiogram profiling of Escherichia coli pathotypes isolated from Kat River and Fort Beaufort abstraction water(University of Fort Hare, 2014) Nontongana, Nolonwabo; Okoh, Anthony IEscherichia coli (E. coli) is a widespread species that includes a broad variety of strains, ranging from highly pathogenic strains causing worldwide outbreaks of severe disease to virulent isolates which are part of the normal intestinal flora or which are well characterized and safe laboratory strains. The pathogenicity of a given E. coli strain is mainly determined by specific virulence factors which include adhesins, invasins, toxins and capsule. The aim of this study was to evaluate the prevalence and antibiogram profiles of E. coli pathotypes previously isolated from Kat River and Fort Beaufort abstraction water. A total of 171 E. coli isolates showed at least one pathogenic determinant among the isolated 278 E. coli. The other 107 isolates were negative for the tested virulence genes. All 278 presumptive isolates tested positive for the UidA gene, and were therefore classified as non-categorized pathogenic E. coli. The 171 pathogenic isolates had at least one characteristic gene of pathogenic E. coli and were identified and classified as enteropathogenic E. coli (6%), enterotoxigenicE. coli (131), uropathogenic E. coli (6), neonatal meningitis E. coli (14), diffusely adherent E. coli (1) and enterohaemrrhagic E. coli (1). Interestingly, no virulence genes were detected for the enteroinvasive E. coli and the enteroaggregative E. coli. The antibiotic resistance profiles for all isolates that were identified as E. coli showed 100% resistance to penicillin G, 98% resistance to ampicillin, 38% resistance to trimethoprim-sulphamethoxazole and 8% resistance to streptomycin. Multiple antibacterial resistance (MAR) was also observed, where 44% of the isolates were resistant to three antibiotics and 8% resistant to four antibiotics. The results of this study showed the Kat River and Fort Beaufort abstraction water are reservoirs of pathogenic strains of E. coli which harbour antibiotic resistance determinants that can cause serious health risks to the people in the surrounding communities.Item Enterococcus pathotypes as reservoirs of antibiotic resistance determinants in the Kat River and Fort Beaufort abstraction waters(University of Fort Hare, 2014) Ntloko, Phindiwe; Okoh, Anthony IIn this study, 400 presumptive Enterococcus isolates previously recovered from Kat River and Fort Beaufort Abstraction water dam were subjected to molecular confirmation and pathotyping. Two hundred and seventy-four (68%) of these isolates were confirmed to be enterococci species. Confirmations studies were polymerase chain reaction (PCR) based, using enterococci specific primers targeting the tuf gene. The confirmed enterococci isolates were further differentiated into their pathotypes, the targets of which were: E. faecalis, E. avium, E. hirae, E. casseliflavarus and E. gallinarum using well documented species specific primer sequences. E. faecalis accounted for 20% of the isolates, followed by E. avium (16%), E. hirae (13%), E. casseliflavarus (5%) and E. gallinarum (3%). Furthermore, all the confirmed isolates were analysed for antibiotic susceptibilities using a panel of nine different antibiotics, namely vancomycin, linezolid, ciprofloxacin, ampicillin, gentamicin, chloramphenicol, tetracycline, erythromycin, penicillin, and those that were resistant were assayed for the presence of relevant antibiotic resistance genes. All the 274 isolates were found to harbour vanA resistance gene confirming their phenotypic resistance to the vancomycin. Similarly, 60% (109/180) of the isolates showed phenotypic resistance to erythromycin which was further confirmed by the presence of ermA genes in these isolates. The presence of antibiotic resistant bacteria in surface waters poses a risk to public health.Item Identification of agricultural and industrial pollutants in the Kat River, Eastern Cape and their effect on agricultural products found along the river banks(University of Fort Hare, 2015) Mutingwende, Nhamo; Bradley, GThere is growing concern that commonly used Pharmaceuticals and Personal Care Products (PPCPs) and pesticides are entering and contaminating drinking water supplies. The use of targeted quantitation of PPCP has been well established but there is an emerging trend to also screen for and identify unexpected environmental pollutants. Chemicals like pesticides hormones and antibiotics are especially of interest because of proven endocrine disrupting effects and a possible development of bacterial resistance. Powerful screening methods are required to detect and quantify the presence of these compounds in our environment. PPCP encompass a wide range of pollutants, including Endocrine Disrupting Compounds (EDC), pesticides, hormones, antibiotics, drugs of abuse, x-ray contrast agents and drinking water disinfection by-products to name a few. In order to properly assess the effects of these compounds on our environment, it is necessary to accurately monitor their presence. The diversity of chemical properties of these compounds makes method development challenging. LC/MS/MS is able to analyse polar, semi-volatile, and thermally labile compounds covering a wide molecular weight range. The new AB SCIEX TripleTOF™5600 LC/MS/MS was used to profile environmental samples for unexpected pollutants, to identify and characterise the chemical composition and structure of the pollutants, and to quantify (based on intensity) the concentration in collected water samples. Liquid Chromatography coupled to tandem Mass Spectrometry (LCMS/ MS) is able to analyse polar, semi-volatile, and thermally labile compounds covering a wide molecular weight range, such as pesticides, antibiotics, drugs of abuse, x-ray contrast agents, drinking water disinfection by-products etc. More recently there is a growing interest from environmental researchers to also screen for and identify non-targeted compounds in environmental samples, including metabolites and degradates, but also completely unexpected pollutants. The new AB SCIEX TripleTOF™5600 LC/MS/MS system is capable of performing highly sensitive and fast MS scanning experiments to search for unknown molecular ions while also performing selective and characteristic MS/MS scanning for further compound identification and, therefore, is the instrument of choice for this challenging task. General unknown screening workflows do not use a target analyte list and compound detection is not based on any prior knowledge, including retention times and information on possible molecular and fragment ions. Therefore, acquired chromatograms are very rich in information and can easily contain thousands of ions from both any compounds present in the sample as well as from the sample matrix itself. Thus, powerful software tools are needed to explore such data to identify the unexpected compound. Water samples were collected both upstream and downstream of two WWTPs (Seymour and Fort Beaufort) and were directly injected on the AB SCIEX TripleTOF™5600 LC/MS/MS after being filtered. 15 sample points along the Kat River, ranging from a point as close to the source as possible to a point just before it joins the Great Fish River were used. The samples collected from the source were used as the control in each of the experiments, the assumption being the closer you get to the source, the less contaminated the water would be for the analysis of pesticides. Points were selected where the Kat River crosses the R67 or on farms where the river was accessible using farm roads. Samples were collected from October 2013 to November 2014.The Peak view software and Analyst software were used in the analysis of PPCPs. The XIC Manager allows you to manage large lists of compounds and perform automatic extracted ion chromatogram (XIC) calculations and review results operations. The results were displayed in the chromatogram pane and the XIC table (see results). The results reported here in this thesis indicate that there is contamination in the Kat River water due to both pesticides and PPCPs. The results also indicate that the food products are also contaminated and hence both the Kat River agricultural produce and its water need to be closely monitored for both pesticide and PPCPs contaminants. Further studies to investigate the quantitative levels of pesticides and PPCPs in the Kat river water to determine if the concentration levels of the detected pesticides are below the reported Maximum Residues Limits will be explored in the future.Item Prevalence and antibiogram of some swine associated Shiga toxin producing Escherichia coli Serogroups and Salmonella species in Nkonkobe Municipality, Eastern Cape Province, South Africa(University of Fort Hare, 2015) Iwu, Chinwe Juliana; Okoh, Anthony IGastrointestinal illnesses have continually become a global public health issue. Exposure to zoonotic food borne pathogens such as Salmonella and diarrhoegenic E. coli either by direct or indirect contact through the consumption of food producing animals is likely an important mode of infection to humans. More so, the use of antibiotics in farm animals similar to those used in humans can select for resistance in bacteria frequently harboured by them. These resistant strains can be passed on to humans through contaminated meat products and water leading to resistant infections with consequences such as prolonged illnesses, treatment failures, and increased morbidity and mortality. In animals, these can lead to reduced productivity. Monitoring the level of resistance among bacteria from animal isolates will help in generating data that could be used to create awareness of their presence in the environment and aid in preventing a potential epidemic in the community. In this study, we investigated the prevalence and antimicrobial resistance profile of Escherichia coli serogroups and Salmonella species in faecal samples collected from pigs in Nkonkobe Municipality in the Eastern Cape Province, South Africa between April – July, 2014. A total of 310 presumptive Shiga toxin producing Escherichia coli (STEC) were confirmed as E. coli spp using polymerase chain reaction (PCR) technique by amplification of the uidA gene, out of which 179 (58%) were confirmed positive. Approximately, serogrougs O157:H7, O145 and O26 made up 24% (n=43), 8% (n=14) and 20% (n=35) of the E. coli population respectively. Only E. coli O26 was positive for stx2 gene in 31% of the isolates harbouring the gene, while the other serogroups were non-pathogenic. Susceptibility of the isolates to 18 antibiotics was carried out in vitro by the standardized agar disc-diffusion method. All the isolates were susceptible to imipenem. Similarly, a relatively high susceptibility was observed in norfloxacin (83-100%), ciprofloxacin (63-100%), gentamycin (77-100%), and chloramphenicol (77-100%). However, all the isolates were resistant to tetracycline and its long acting counterpart oxytetracycline. Resistances observed against other antimicrobials are as follows: ampicillin (84-91%), streptomycin (14-100%), erythromycin (91-100%), ceftazidime (35%). Multiple antimicrobial resistance patterns and indices ranged from 3 to 12 and 0.2 to 0.7 to respectively. Genes encoding resistances to ampicillin (ampC), streptomycin (strA) and tetracycline (tetA) were frequently detected in 50-100%, 22-29% and 40-86% of the resistant isolates respectively. In the other arm of the dissertation, two hundred and fifty eight presumptive isolates of Salmonella were recovered from the faecal samples of pigs. Specific primers targeting serogroups A, B, C1, C2, and D were used to delineate the isolates into different serogroups using PCR. Only serogroup A (n=48) was detected. These isolates were examined for antimicrobial susceptibility by disc diffusion method using 18 antibiotics. The results showed that a large proportion of the isolates were resistant to tetracycline (100%), oxytetracycline (100%), ampicillin (75%), sulphamethoxazole/trimethoprim (75%) and streptomycin (75%). Majority of the isolates exhibited multidrug resistances with the predominant multiple antibiotic resistance (MAR) phenotype being against eleven antibiotics. A high multiple antibiotic resistance (MAR) index in a range of 0.3- 0.6 was observed. The incidence of genes encoding resistance against tetracycline (tetA), streptomycin (stra), and ampicillin (ampC) were 54%, 44% and 61% respectively. These findings reveal that pigs within the Nkonkobe Municipality in the Eastern Cape Province could harbour Shiga toxins and multidrug resistant serogroups of E. coli as well as resistant Salmonella which could be transmitted to humans through the food chain. To ensure public health safety, continuous monitoring and sufficient sanitation in swine industries must be ensured.Item Evaluation of some wastewater treatment facilities in Chris Hani and Amathole district municipalities as potential sources of Escherichia coli in the environment(University of Fort Hare, 2014) Mazwi, Sinazo Nomathamsanqa; Okoh, Anthony IAccess to clean and safe water is essential for the survival of human beings. Pollution of freshwater sources constitutes a major problem hindering access to safe water for drinking and other domestic uses. Wastewater effluent discharges often impact the microbiological qualities of surface waters with its attendant health and environmental problems. This study evaluated the microbiological qualities of the discharged effluents of four selected wastewater treatment plants in Amathole and Chris Hani District Municipalities of the Eastern Cape Province over a twelve-month sampling period. Microbiological analysis (faecal coliform, Escherichia coli and Escherichia coli O157:H7) was done using standard methods and polymerase chain reaction method was used to confirm identities ofbacterial isolates. Presumptive bacteria counts ranged as follows: faecal coliforms 0 to 1.6 × 103 CFU/100 ml, E. coli 0 to 1.4 × 103 CFU/100 ml and E. coli O157:H7 0 to 9.6 × 102 CFU/100 ml. Forty eight percent (305/626) of the presumptive E. coli isolates were confirmed using species-specific uidA gene which code for β-glucuronidase enzyme in E. coli. Antibiotic susceptibility profile of the isolate using a panel of 10 antibiotics shows 100% (150/150) resistance to antibiotics rifampicin and penicillin G while 49.3% (74/150) of the isolates and 46.7% (70/150) were susceptible to streptomycin and cefotaxime respectively. Multiple antibiotic resistance phenotypes (MARP) of the isolates showed resistance to two or more test antibiotics while the calculated multiple antibiotic resistance index (MARI) for the tested isolated is 0.49. The detection of potentially pathogenic E. coli in the final effluents suggestspotential danger to the receiving water bodies where the effluents are discharge. The high MARI valued obtained in this study indicates that the isolates are form environment where the tested antibiotics are being used and may further lead to the spread of multiple antibiotics resistance among other pathogens that may be present in the same environment.Item Assessment of the prevalence of faecal coliforms and Escherichia coli o157:h7 in the final effluents of two wastewater treatment plants in Amahlathi Local Municipality of Eastern Cape Province, South Africa(University of Fort Hare, 2014) Ajibade, Adefisoye Martins; Green, E; Okoh, Anthony IThe production of final effluents that meet discharged requirements and guidelines remain a major challenge particularly in the developing world with the resultant problem of surface water pollution. This study assessed the physicochemical and microbiological qualities of two wastewater treatment works in the Eastern Cape Province of South Africa in terms of the prevalence of faecal coliforms and Escherichia coli O157:H7 over a five month period. All physicochemical and microbiological analyses were carried out using standard methods. Data were collected in triplicates and analysed statistically using IBM SPSS version 20.0. The ranges of some of the physicochemical parameters that complied with set guidelines include pH (6.7 – 7.6), TDS (107 – 171 mg/L), EC (168 – 266 μS/cm), Temperature (15 – 24oC), NO3- (0 – 8.2 mg/L), NO2- (0.14 – 0.71 mg/L) and PO4 (1.05 – 4.50 mg/L). Others including Turbidity (2.64 – 58.00 NTU), Free Cl (0.13 – 0.65 mg/L), DO (2.20 – 8.48 mg/L), BOD (0.13 – 6.85 mg/L) and COD (40 – 482 mg/L) did not comply with set guidelines. The microbiological parameters ranged 0 – 2.7 × 104 CFU/100 ml for FC and 0 – 9.3 × 103 for EHEC CFU/100 ml, an indication of non-compliance with set guidelines. Preliminary identification of 40 randomly selected presumptive enterohemorrhagic E. coli isolates by Gram’s staining and oxidase test shows 100% (all 40 selected isolates) to be Gram positive while 90% (36 randomly selected isolates) were oxidase negative. Statistical correlation between the physicochemical and the microbiological parameters were generally weak except in the case of free chlorine and DO where they showed inverse correlation with the microbiological parameters. The recovery of EHEC showed the inefficiency of the treatment processes to effectively inactivate the bacteria, and possibly other pathogenic bacteria that may be present in the treated wastewater. The assessment suggested the need for proper monitoring and a review of the treatment procedures used at these treatment works.Item Quality indices of the final effluents of two sub-urban-based wastewater treatment plants in Amathole District Municipality in the Eastern Cape Province of South Africa(University of Fort Hare, 2014) Gcilitshana, Onele; Okoh, Anthony I; Zhou, LWorldwide, water reuse is promoted as an alternative for water scarcity, however, wastewater effluents have been reported as possible contaminants to surface water. The failure of some wastewater treatment processes to completely remove organic matter and some pathogenic microorganisms allows them to initiate infections. This manifests more in communities where surface water is used directly for drinking. To assess water quality, bacteria alone cannot be used as it may be absent in virus-contaminated water. This study was carried out to assess the quality of two wastewater treatment plant effluents from the Eastern Cape Province of South Africa. Physicochemical parameters and microbiological parameters like faecal coliforms, adenovirus, rotavirus, hepatitis A virus, norovirus and enterovirus were evaluated over a projected period of one year. Physicochemical parameters were measured on site using multiparameters, faecal coliforms enumerated using culture-based methods and viruses are detected using both conventional and real-time PCR. Physicochemical parameters like electrical conductivity, turbidity, free chlorine and phosphates were incompliant with the standards set by the Department of Water affairs for effluents to be discharged. Faecal coliform counts were nil for one plant (WWTP-R) where they correlated inversely (P < 0.01) with the high free chlorine. For WWTP-K, faecal coliforms were detected in 27% of samples in the range of 9.9 × 101 to 6.4× 104 CFU/100ml. From the five viruses assessed, three viruses were detected with Rotavirus being the most abundant (0-2034176 genome copies/L) followed by Adenovirus (0–275 genome copies/L) then Hepatitis A virus (0–71 genome copies/L) in the WWTP-K while none of the viruses was detected in WWTP-R. Species B, species C and Adv41 serotypes were detected from the May 2013 and June 2013 samples where almost all parameters were incompliant in the plant. The detection of these viruses in supposedly treated effluents is suggestive of these being the sources of contamination to surface water and therefore renders surface waters unsafe for direct use and to aquatic life. Although real-time PCR is more sensitive and reliable in detection of viruses, use of cell-culture techniques in this study would have been more efficient in confirming the infectivity of the viruses detected, hence the recommendation of these techniques in future projects of this nature.Item Studies on the antimicrobial, antioxidant and antiproliferative potential of the ethyl acetate extract and compounds of Peltophorum africanum(University of Fort Hare, 2014) Okeleye, Benjamin Ifeoluwa; Ndip, Roland N; Mkwetshana, Noxolo TCells are constantly exposed to a variety of oxidizing agents, some of which are necessary for life. Oxidants produced in excess can cause an imbalance, leading to oxidative stress, especially in chronic bacterial, viral, and parasitic infections. This can result to damage of biomolecules such as lipids, proteins, and DNA, hence, an increased risk for cancer. Plants have a long history of use in the treatment of cancer. Plant secondary metabolites have proved to be an excellent reservoir of new medical compounds. Fruits, vegetables, and whole grains contain a wide variety of antioxidant phytochemicals, such as phenolics and carotenoids, and may help protect cellular systems from oxidative damage and also may lower the risk of chronic diseases. Peltophorum africanum, a member of the family Fabaceae (Sond) is also known as the African weeping wattle and is used in traditional medicine in South Africa. This study investigated the antimicrobial, antioxidant and antiproliferative potential of the ethyl acetate extract and compounds of Peltophorum africanum in order to validate its pharmacological use. The study assessed the in vitro antimicrobial activity of ethyl acetate extract (EAE) of Peltophorum africanum stem bark and its fractions by the agar well and macrodilution methods. The toxicity on a normal human liver cell (Chang liver cell) and antiproliferation of human breast (MCF-7), colon (HT-29) and cervical (HeLa) cancer cell lines were determined using the CellTiter-Blue cell viability assay and the mechanism of action delineated using the Nucleic Acid and Protein Purification Nucleospin® Tissue Kit, Scanning Electron Microscopy (SEM), Propidium iodide (PI) and Acridine orange (AO) double-staining techniques, the Cleaved Caspase 3 (Asp 175) Alexa Fluor® 488 Antibody and the Coulter® DNA PrepTM Reagents Kit. Purification and identification of the compounds from EAE and fractions as well as the morphological alteration of bacteria, yeast and cancer cells were determined using thin layer chromatography, infrared spectra fingerprint and GC-MS analysis, micro-dilution and scanning electron microscopy with energy-dispersive X-ray analysis. In vitro antioxidant activity of EAE was determined by means of radical scavenging and ferric reducing power analysis using 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2`-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) kit, hydrogen peroxide (H2O2), iron (iii) chloride (Fe3+) and nitric oxide (NO). To assess the likely effects of secondary metabolites on the activities observed; total proanthocyanidins, phenolics, flavonols, and flavonoids were determined using standard phytochemical methods. Data were analyzed by one way analysis of variance (ANOVA; SPSS Version 17.0, 2011), regression analysis (MINITAB, version 12 for windows), probit analysis test (software NCSS, 2007) and GraphPad Prism4 software package. The p-values < 0.05 were considered significant. Marked activity of the extract was observed against Plesiomonas shigelloides ATCC 51903, with MIC and MLC values of 0.15625 and 0.3125mg/mL, respectively. The extract was both bactericidal (MICindex ≤ 2) and bacteriostatic/fungistatic (MICindex > 2) in activity. Lethal dose at 50 (LD50) showed 82.64 ± 1.40 degree of toxicity at 24 hrs, and 95 percentile of cell death dose activity ranged from log 3.12 ± 0.01 to 4.59 ± 0.03. The activity of the eight fractions tested ranged from 1.0 ± 0.5 to 3.7 ± 1.6 mg/mL (IC50) and from 2.1 ± 0.8 to 6.25 ± 0 mg/mL (IC90) (Chapter 3). Due to the effect of compounds present in the crude extract and fractions, the P. aeruginosa treated with EAE had a reduction of sodium from 5.55 % (untreated) - 1.50 %. For C. albicans, pottasium was reduced from 4.16 % (untreated) - 0.76 % (T1). Remarkable morphological alterations were observed including deformation of the germ tubes and perforation of the cell wall (Chapter 4). Extract scavenging activity of 88.73± 6.69 % (25 μg mL-1), 53.93±1.09 % (25 μg mL-1) were recorded for H2O2 and NO respectively with proanthocyanidins (92.18±4.68 mg/g) occurring more (p < 0.05) in the extract compared to all other phenolics compounds (Chapter 5). Significant reduction in cell viability of the cells was noted as the MCF-7 cells were reduced from 100 - 54.33±1.84 % after 72 hrs of treatment with 5 μg/mL of EAE (P. value < 0.05). TEt10 was cytotoxic against human normal cells (chang liver cell) at EC50 of 37 μg/mL and 74 μg/mL after 24 and 48 h of treatment respectively. Marked antiproliferative activity of 13.2 μg/mL (EC50) was observed when HeLa cells were treated for 48 h. Internucleosomal DNA of MCF-7, HT-29 and HeLa cells randomly fragmented into an uninterrupted spectrum of sizes, complemented by the intercalation of nucleic acid-specific fluorochromes by PI and AO spotting two phases of apoptosis; early (EA) and late (LA) apoptosis. Distinctive ultramorphological changes observed include; cell shrinkage, membrane blebbing, and typical cell induced death. The study also recorded 705.102 ± 28.56 % TEt10 caspase-3 activity compared to curcumin 592.857 ± 165.76 % (positive control) and untreated (negative control; 100 ± 15.81 %) cells. Percentage HeLa cell with Sub-G1 DNA phase increased from 0.13 ± 0.06 % (negative control) to 13.8 ± 3.04 % compared to curcumin (8.17 ± 2.20 %) after treatment with TEt10. The compounds identified in the fractions including Colchicine, N-(trifluoroacetyl)methyl-N-deacetyl-, Lupeol and .gamma.-Sitosterol may be responsible for the induction of apoptosis observed and could be further studied in vivo as a potential template for new anticancer treatment (Chapter 6 & 7).Item Molecular study of mycobacterium tuberculosis complex (MTBC) DNA from Port Elizabeth(University of Fort Hare, 2014) Londiwe, Bhembe Nolwazi; Green, E; Ndip, R N; Okoh, Anthony IMycobacterium tuberculosis complex (MTBC) is a causative agent of tuberculosis (TB) in humans and animals. The burden of tuberculosis in South Africa is worsened by the concurrent epidemic of HIV. The dynamic of TB epidemics has been investigated and yet little data has been given about the Eastern Cape, particularly Port Elizabeth. The study aimed to investigate the prevalence of drug resistant MTBC and to determine the mutations causing resistance in Port Elizabeth. One hundred and ninety (190) DNA samples isolated from sputum specimen in humans suspected of having TB were amplified using the Seeplex® MTB Nested ACE detection assay. To differentiate Mycobacterium tuberculosis complex (MTBC) members for surveillance purposes a multiplex polymerase chain reaction (PCR) method was done based on genomic regions of differences such as RD1, RD1mic, RD2seal, RD4, RD9 and RD12. Target genes known to confer resistance to first and second-line drugs were amplified and the amplicons sequenced using Big Dye Terminator DNA sequencing kit v3.1 (Applied Biosystems, UK). The patient’s demographic profiles were obtained from the National Health Laboratory Service (NHLS). All hundred and ninety DNA samples tested positive for MTBC using the Seeplex® MTB Nested ACE assay. Results show a high prevalence of extensive drug resistant TB in Port Elizabeth, Eastern Cape Province. One hundred and eighty four (184) DNA isolates were used in the identification of different MTBC species. We ended up working with 184 DNA isolates because we ran out of DNA, and we could not go back to isolate DNA from the affected individuals due to the fact that some patients died, while some have been released to go to their homes. From the 184 DNA isolates 45 (24.5%) isolates were identified to be M. tuberculosis, 94 isolates (51.1%) to be M. bovis BCG and 3 isolates (1.6%) to be M. cannetti. Sequencing results show the position of mutation in each DNA isolate; however in the study we got resistance to MDR to be 100% and 42% pre-XDR while 58% was XDR. These results raise an alarm for the prevalence MDR in MTBC from Port Elizabeth. This is a serious health concern which calls for a need to strategise on the identification of extensive drug resistant TB patients from multi-drug resistant TB patients and ensure monitoring of their treatment.Item Genetic and phenotypic characterisation of foodborne bacteria isolated from ready-to-eat foods in Alice, South Africa(University of Fort Hare, 2014) Nyenje, Mirriam E; Green, EzekielFoodborne illnesses following the ingestion of contaminated food are a major public health problem worldwide. They include a broad group of illnesses ranging from mild to chronic or life-threatening; caused by either toxins released from the disease-causing microbes, or by the microbes themselves. Antimicrobial susceptibility data shows an alarming increase in the frequency of antimicrobial resistance of foodborne pathogens, a situation which is worrisome as it decreases the effectiveness of drugs employed to reduce the morbidity and mortality associated with serious and life-threatening infections and thus, compromising human health. This study was therefore designed to assess the occurrence and characterization of bacterial foodborne pathogens in various foods sold in Alice, Eastern Cape Province of South Africa in an effort to throw more light on the inherent risk associated with such foods. The study was conducted during the period of 2011 - 2013. Two university restaurants and eight ready-to-eat food vending sites in Alice Town were selected based on their prominence to the students, workers and rest of the community. Microbiological analysis was conducted on 252 samples which included vegetables, potatoes, rice, pies, beef and chicken stew. The isolates were identified using biochemical tests and confirmation of the two most prevalent organisms (Listeria ivanovii and Enterobacter cloacae) was done using PCR techniques. The antimicrobial susceptibility profile of Listeria ivanovii and Enterobacter cloacae strains were identified using the disc diffusion technique; minimum inhibitory concentration was determined by the broth dilution method and M.I.C. Evaluator test strips. The microtiter plate adherence assay was employed to ascertain the ability of these isolates to adhere to a surface whereas the role of cell surface properties in biofilm formation was assessed using the coaggregation and autoaggregation assays. The architecture of the formed biofilms was examined under the scanning electron microscope. The virulence and resistant genes were also detected and characterised by sequencing the PCR products. Bacterial growth was present in all the food types tested; organisms isolated included: Listeria spp. (22%), Enterobacter spp. (18%), Aeromonas hydrophila (12%), Klebsiella oxytoca (8%), Proteus mirabilis (6.3%), Staphylococcus aureus (3.2%) and Pseudomonas luteola (2.4%). PCR confirmed 30 (97%) isolates as E. cloacae complex while 44% (22/50) tested positive for L. ivanovii. All the strains of E. cloacae (100%) and 96% of L. ivanovii isolates (based on phenotypic identification) were resistant to at least four or more of the antibiotics. In this study, bla-TEM was also detected from 48 (96%) of L. ivanovii and 30 (100%) of E. cloacae strains; further analysis of the bla-TEM demonstrated the occurrence of bla-TEM-1. Of the 56 bla-TEM-1 positive isolates sequenced, 7% (4/56) had mutation of either insertion or substitution of a nucleotide. Two virulence genes (ucaA and hlyA) were detected in E. cloacae isolates and none in L. ivanovii using PCR. Sequence analysis of the hsp60 gene reported the presence of two sub-species for E. cloacae; E. cloacae cluster III (75%) and E. cloacae cluster IV (25%); while analysis of the iap60 gene demonstrated that 55.8% (19/34) were L. ivanovii, 44% (15/34) L. seeligeri and 14.7% (5/34) L. welshemeri. A total of 90% L. ivanovii and 88% E. cloacae strains demonstrated the ability to form biofilms; the coaggregation index ranged from 12 to 77% while the autoaggregation index varied from 11 to 55% for L. ivanovii and 27% to 98% for E. cloacae. The findings of this study indicate that most of the ready-to-eat food samples examined did not meet bacteriological quality standards, thus posing potential risks to consumers. This should draw the attention of the relevant authorities to certify that hygienic standards are improved to curtain foodborne infections. Furthermore, the presence of multi-resistant strains is of major concern as these foods could serve as important vehicles transmitting multi-resistant bacteria and genes to humans. In addition the ability of these pathogens to form biofilms may lead to adherence of these organisms to kitchen utensils and other environments leading to cross-contamination of food processed in these areas and increase resistance of organisms to antimicrobial agents.Item Evaluation of the final effluents of some wastewater treatment plants in Amathole and Chris Hani District Municipality of the Eastern Cape Province as sources of vibrio pathogens in the aquatic environment(University of Fort Hare, 2014) Nongogo, Vuyokazi; Okoh, Anthony ICertain areas in the world still depend on the receiving water bodies as sources of domestic water and for recreational purposes. The discharge of poor quality effluents from wastewater treatment plants can impact negatively on these water bodies, as they can act as vehicles for pathogens to the environment, posing a threat to humans if such water is used without precaution. Vibrio species are amongst those pathogens that can survive wastewater treatment processes, ending up in the environment, hence the aim of this study was to evaluate the final effluents of some wastewater treatment plants as sources of vibrio pathogens. Five wastewater treatment plants (WWTP) located in Amathole and Chris Hani District Municipalities in the Eastern Cape were used in this study. Samples were collected monthly from September 2012 – August 2013 and analysed using the standard membrane filtration technique. Yellow and green colonies on TCBS agar were enumerated as presumptive Vibrio species and expressed as CFU/100ml for each plant. Colonies were later picked based on their phenotypic characteristics, sub-cultured on fresh TCBS agar to ascertain purity. These presumptive isolates were then subjected to Gram staining and Oxidase test. Gram negative and Oxidase positive isolates were selected for further confirmation using Polymerised Chain Reaction (PCR). PCR was also employed for characterisation of Vibrio into three species viz V. parahaemolyticus, V. fluvialis and V. vulnificus. Antibiogram profile of the characterised species was then determined together with the presence of relevant antibiotic resistance genes Vibrio densities for the twelve month period ranged between 0 - 1.48×104 CFU/100ml with two of the plants located in East bank and Queenstown characterized by extremely high counts and one plant( Reeston) with very low counts.Three hundred presumptive Vibrio isolates were screened for identity confirmation. Of these, the dominating species found was V. fluvialis (28.6%) followed by V. vulnificus (28%) and the least was found to be V. parahaemolyticus (11.6%). The remaining unidentified 31.6% were suspected to belong to other Vibrio species not covered within the scope of this study. All the confirmed isolates i.e., V. parahaemolyticus, V. vulnificus and V. fluvialis were susceptible to imipenem, gentamicin and meropenem and resistant to only tetracycline. Between 60-100% of the V. parahaemolyticus isolates, 7.1% to 100 % V. vulnificus isolates and 2.5 to 100 % V. fluvialis showed resistances to polymixin B, sulfamethazole, erythromycin, penicillin G, chloramphenicol, trimethroprim and trimethroprim & sulfamethazole. Antibiotic Resistance Genes that were assessed included dfRA, SXT, floR and Sul2 varying in proportion with each species showing diversity in the Vibrio community. The dfR A gene was detected in all the V. parahaemolyticus isolates while floR gene was not detected in any of the isolates belonging to the three species. The distribution of sul2 gene cut across the species being 1% (1) in V. fluvialis, 3% (1) in V. parahaemolyticus and 4% (3) in V. vulnificus. The SXT gene was only determined in V. parahaemolyticus. It is clear that the final effluents of the selected plants are reservoirs for Vibrio pathogens as well as antibiotic resistance genes in the environment. The isolation of Vibrio from WWTP shows that this pathogen is in circulation in some pockets of the population. Therefore, wastewater treatment plants need to be properly monitored to ensure that they comply with set guidelines.