Department of Biochemistry and Microbiology

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The Department of Biochemistry and Microbiology is committed to advancing knowledge of the molecular and microbial processes that underpin life, health, and the environment. Its teaching and research activities span a wide range of topics, including enzymology, genetics, molecular biology, immunology, microbial ecology, and biotechnology. This collection provides access to the department’s scholarly contributions, including research articles, theses, dissertations, and examination question papers. The resources here reflect the department’s role in addressing local and global challenges in areas such as disease management, food security, environmental sustainability, and industrial biotechnology. By making this material available through the institutional repository, the department seeks to support open access to knowledge, encourage academic collaboration, and preserve the intellectual output of staff and students for future generations.

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Parasite prevalence, nutritionally-related blood metabolites and pre-slaughter stress response in Nguni, Bonsmara and Angus steers raised on veld
(University of Fort Hare, 2007) Ndlovu, Thulile
The effects of month on body weight, body condition scores, internal parasite prevalence and on nutritionally related blood metabolites were studied in Angus, Bonsmara and Nguni steers raised on sweet veld. Pre-slaughter stress was also determined using catecholamines, cortisol, dopamine, packed cell volume and serum creatinine levels. The blood chemical constituents evaluated included glucose, cholesterol, total protein, creatinine, urea, globulin, albumin, calcium, phosphorus, magnesium, aspartate amino transferase (AST), alkaline phosphatase (ALP) and creatinine kinase (CK). The Nguni steers maintained their body condition throughout the study period whereas Angus steers had the least body condition scores. Parasite levels were high during the rainy season and low during the dry season. The predominant internal parasites were Haemonchus (39.3%), Trichostrongylus (37.8%), Cooperia pectinita (25.5%), Fasciola gigantica (16.3%) and Ostertagia ostertagi (11.2%). The Nguni had the least parasite infestation levels and had high PCV levels. Nguni had higher levels of cholesterol and glucose (2.86 and 4mmol/l, respectively) than the other two breeds. Nguni and Bonsmara steers had higher (P<0.05) mineral levels. There were significant breed and month differences for glucose, cholesterol, creatinine, calcium, albumin, phosphorus, albumin-globulin ratio and ALP levels. Bonsmara was more susceptible to transport and pre-slaughter stress as it had the highest (P<0.05) levels of adrenalin (10.8nmol/mol), noradrenalin (9.7nmol/mol) and dopamine (14.8nmol/mol) levels, whereas the Nguni had the least levels of adrenalin (6.5nmol/mol), noradrenalin (4.6nmol/mol) and dopamine (4nmol/mol) levels. In conclusion, Nguni steers were better adapted to the local environmental conditions.
The impact of wastewater quality on receiving water bodies and public health in buffalo city and Nkonkobe municipalities
(2007) Osode, Augustina Nwabuje; Water -- Pollution -- Eastern Cape; Water quality management -- South Africa -- Eastern Cape
The Eastern Cape Province of South Africa is composed mainly of rural areas where the communities still rely heavily on surface water sources for their domestic, irrigation and recreational water needs. This fact places major importance on effluents discharged from wastewater treatment plants into the surrounding surface water bodies to adhere to stringent water quality standards. The chemical and microbiological quality of the effluents must therefore be closely controlled and diligently monitored so that these needs can be met without the communities in the region being put at risk of contracting waterborne diseases. This study evaluated the efficiency of the various wastewater treatment plants for the removal of chemical and microbiological contaminants in order to establish the relationship between the quality of the final effluents and that of the receiving water bodies. To this end, four wastewater treatment plants in the region, i.e. Alice and Fort Beaufort (which both serve the Nkonkobe municipal region), Dimbaza and East London (which both serve the Buffalo City municipal region) were investigated. Wastewater samples were taken monthly from the individual plants analysed from the 6th August 2003 to the 24th March 2004. Wastewater samples were physicochemically characterised according to biological oxygen demand (BOD), chemical oxygen demand (COD), dissolved oxygen (DO), pH, phosphate, residual chlorine, temperature, total nitrogen and total suspended solids (TSS). Student’s t-test was used to compare the physicochemical parameters in the effluent and receiving water body samples. The targeted pathogenic microorganisms under investigation in this study were Salmonella typhimurium, Shigella dysenteriae and Vibrio cholera. Standard methods were applied in all aspects of the analyses for the isolation and detection of these microorganisms and also for the identification of the general microbiological quality of the effluent and the receiving water bodies. Polymerase chain reaction (PCR) was used to confirm the presence of the target microorganisms. The risk assessment was conducted based on the outcome of molecular characterisation of isolates to study the impact of target microorganisms on the health of the communities.
Biodiversity of salmonella strains isolated from selected water sources and wastewater discharge points in the Eastern Cape province of South Africa
(University of Fort Hare, 2008) Mafu, Nwabisa
In this study, the diversity of forty Salmonella isolates from selected drinking water and wastewater sources in the Eastern Cape Province of South Africa was assessed using parameters such as protein and lipopolysaccharide profile analysis, DNA fingerprinting and antibiotic susceptibility profile as test indices. Wastewater samples from Amalinda, Shornville and Fort Hare wastewater plants, and water samples from Gogogo and Tyume rivers were collected on ice and transported to the laboratory of the department of Microbiology and Biochemistry, University of Fort Hare for processing. The DNA dendograms of Salmonella and the applied UPGMA revealed 4 similarity groups of the strains. Most of the strains recovered from Amalinda, Shornville, Fort Hare wastewater plants, Gogogo and Tyume rivers show a high percentage of genetic similarity. On the other hand, protein dendograms of Salmonella isolates revealed 2 similarity groups which varied widely. Also, the lipopolysaccharide dendograms revealed three similarity groups with the first similarity groups showing a very high relatedness between strains from different water sources. The second similarity group included 16 strains which formed a rather homogenous group, and the third similarity group formed a distinct group. Of the seven antibiotics and sulfonamides tested against the Salmonella species, five namely, neomycin, chloramphenicol, kanamycin, streptomycin and cotrimoxazole were significantly inhibitory, while the bacteria showed considerable resistance to doxycycline and sulphamethoxazole. Our results based on restriction digestion, SDS/PAGE and dendogram construction show that there is a high similarity between the forty Salmonella strains studied, and that these methods are valuable tools for evaluating the relatedness of Salmonella species. Our observations have proffered a veritable reference point on the diversity of Salmonella strains in the studied area.
Evolutionary development and functional role of plant natriuretic Peptide (pnp)-b
(University Of Fort Hare, 2009) Hove, Runyararo Memory
Plant natriuretic peptides (PNP) are novel peptides which, like in vertebrates, have been shown to have a function associated with water and salt homeostasis. Two PNP-encoding genes have been identified and isolated from Arabidopsis thaliana, namely; AtPNP-A and AtPNP-B. In this study, the focus was on PNP-B, which has not been extensively studied. Bioinformatic analysis was done on the AtPNP-B gene. This included the bioinformatic study of its primary structure, secondary structure, tertiary structure, transcription factor binding sites (TFBS) and its relation to other known proteins. The AtPNP-B gene was shown to be a 510 bp long, including a predicted 138 bp intron. AtPNP-B was also shown to have some sequence similarity with AtPNP-A and CjBAp12. The TFBS for AtPNP-B and OsJPNP-B were compared and they comprised of TFBS that are related to water homeostasis and pathogenesis. This suggested two possible functions; water stress and homeostasis and a pathogenesis related function for PNP-B. Following bioinformatic analysis, the heterologous expression of the AtPNP-B was attempted to investigate whether the AtPNP-B gene encoded a functional protein and to determine the functional role of PNP-B. However, expression was unsuccessful. An evolutionary study was then carried out which revealed that there were some plants without the intron such as, rice, leafy spurge, oilseed rape, onion, poplar, sugar cane, sunflower and tobacco. These plants would therefore be used for expression and functional studies in the future. The evolutionary studies also revealed that PNP-B had a relationship with expansins and the endoglucanase family 45. Other PNP-B related molecules were also obtained from other plant genomes and therefore used in the construction of a phylogenetic tree. The phylogenetic tree revealed that AtPNP-B clustered in the same group as CjBAp12 while AtPNP-A had its own cluster group. There were also other PNP-B like molecules that clustered in the same group as expansins (α- and β-). Thus, we postulate that, like PNP-A, PNP-B also has a possible function in water and salt homeostasis. However, due to the clustering of AtPNP-B into the same group as CjBAp12, a possible role of PNP-B in pathogenesis-related response is also postulated.
Evolutionary Development and Functional Role of Plant Natriuretic Peptide (PNP)-B
(University of Fort Hare, 2009-02) Hove, Memory Runyararo
Plant natriuretic peptides (PNP) are novel peptides which, like in vertebrates‚ have been shown to have a function associated with water and salt homeostasis. Two PNP-encoding genes have been identified and isolated from Arabidopsis thaliana, namely; AtPNP-A and AtPNP-B. In this study, the focus was on PNP-B, which has not been extensively studied. Bioinformatic analysis was done on the AtPNP-B gene. This included the bioinformatic study of its primary structure, secondary structure‚ tertiary structure‚ transcription factor binding sites (TFBS) and its relation to other known proteins. The AtPNP-B gene was shown to be a 510 bp long, including a predicted 138 bp intron. AtPNP-B was also shown to have some sequence similarity with AtPNP-A and CjBAp12. The TFBS for AtPNP-B and OsJPNP-B were compared and they comprised of TFBS that are related to water homeostasis and pathogenesis. This suggested two possible functions; water Stress and homeostasis and a pathogenesis related function for PNP-B. Following bioinformatic analysis, the heterologous expression of the AtPNP-B was attempted to investigate whether the AtPNP-B gene encoded a functional protein and to determine the functional role of PNP-B. However, expression was unsuccessful. An evolutionary study was then carried out which revealed that there were some plants without the intron such as, rice, leafy spurge, oilseed rape, onion, poplar, sugar cane‚ sunflower and tobacco. These plants would therefore be used for expression and functional studies in the future. The evolutionary studies also revealed that PNP-B had a relationship with expansins and the endoglucanase family 45. Other PNP-B related molecules were also obtained from other plant genomes "and therefore used in the construction of a phylogenetic tree. The phylogenetic tree revealed that AtPNP-B clustered in the same group as CjBAp12 while AtPNP-A had its own cluster group. There were also other PNP-B like molecules that clustered in the same group as expansins (a- and ß—).
Comparative study of the antibiotic producing potentials of three freshwater Actinomycetes belonging to the Saccharopolyspora and Actinosynnema genera.
(University of Fort Hare, 2010) Sibanda, Timothy
Crude extracts of three actinomycetes species belonging to Saccharopolyspora (TR 046 and TR 039) and Actinosynnema (TR 024) genera were screened for antibacterial activities against a panel of several bacterial strains. The extracts showed antibacterial activities against both Gram-negative and Gram-positive test bacteria with inhibition zones ranging from 8 to 28 mm for extract obtained from TR 046; 8 and 15 mm for extract obtained from TR 039 and 10 to 13 mm for extract obtained from TR 024. The minimum inhibitory concentrations ranged from 0.078 to 10 mgml-1 for extract obtained from TR 046; 5 and >10 mgml-1 for extract obtained from TR 039 and 1.25 and 5 mgml-1 for extract obtained from TR 024. The bactericidal activity of extract obtained from TR 046 was evaluated against 5 test bacteria with different susceptibilities to the extract by time-kill assay. The extract showed strong bactericidal activity against Bacillus pumilus (ATCC14884) reducing the bacterial load by 104 cfuml-1 and 102 cfuml-1 at 4 × MIC and 2 × MIC respectively after 6 hr of exposure. It also showed good bactericidal activity against Proteus vulgaris (CSIR 0030) achieving a 0.9log10 and 0.13log10 cfuml-1 reduction at 5 mgml-1 (4 × MIC) and 1.25 mgml-1 (2 × MIC) respectively after 12 hr of exposure. The extract was however weakly bactericidal against two environmental bacterial strains Klebsiella pneumoniae and Staphylococcus epidermidis, and against Pseudomonas aeruginosa (ATCC 19582), the extract showed bacteriostatic activity across all MIC ranges used. The test actinomycetes appear to have immense potential as a source of new antibacterial compound(s).
Prevalence and risk factors for Helicobacter pylori transmission in the Eastern Cape Province application of immunological molecular and demographic methods
(University of Fort Hare, 2010) Dube, Callote
Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative motile curved rod that inhabits the gastric mucosa of the human stomach. The organism chronically infects billions of people worldwide and is one of the most genetically diverse of bacterial species. Infection with the organism potentially induces chronic gastritis and peptic ulcer disease. In addition, H. pylori plays a role in the etiology of gastric cancer and gastric MALT lymphoma. The risk of infection is increased in those living in the developing world, which has been ascribed to precarious hygiene standards, crowded households, and deficient sanitation common in this part of the world. Thus, the aim of this study was to identify the risk factors in the transmission of H. pylori in our environment, i.e. in Nkonkobe Municipality in the Eastern Cape Province, South Africa. Faecal samples were collected from 356 apparently healthy subjects, consisting of 168 males and 188 females aged from 3 months to 60 years (Mean = 31 years). A standardized questionnaire was applied, it described demographic characteristics including age, sex, household hygiene, socioeconomic status, area of residence, duration of stay in the area, sharing bath water, sharing tooth brush, habit of sucking thumb, medication currently being taken or medication taken within the past three months, source of water, type of toilet used, education and occupation. A sandwich-type enzyme immunoassay amplification technology (Amplified IDEIA TM Hp StAR TM , Oxoid, UK) was used to analyze the faecal samples for the detection of H. pylori antigens using monoclonal antibodies specific for H. pylori antigens. To assess the possibility of faecal oral route with tap water as an intermediary link, PCR targeting the ureC (glmM), a highly conserved gene in H. pylori was carried out to detect H. pylori DNA in faecal samples of already positive samples by HpSA test as well as in direct tap water used by the H. pylori positive subjects. QIAamp DNA stool mini kit was used to extract DNA from faecal samples. Tap water samples were then obtained using sterile bottles from areas inhabited by H. pylori positive subjects as determined by HpSA test and PCR. DNA extraction from water samples was done using UltraCleanTM Water DNA Isolation Kit (0.22μm) according to the manufacturer’s instructions. PCR with primers specific for H. pylori glmM gene was carried out with both positive and negative controls incorporated. Fisher’s exact test was used to assess the univariate association between H. pylori infection and the possible risk factors. Odds ratio (OR) and the corresponding 95% confidence interval (CI) were calculated to measure the strength of association using EPI INFO 3.41 package. P values of < .05 were required for significance. The precision rate of the diagnostic tests used was also determined. H. pylori antigen was detected in 316 of the 356 subjects giving an overall prevalence of 88.8%. Prevalence increased with age from 75.9% in children < 12 years age to 100% in the age group from 13 years to 24 years, also 100% prevalence of H. pylori was recorded in young adults aged 25-47 years and subjects aged 60 years (P < .05). H. pylori prevalence was higher in females than in males. Of 188 females who participated in the study, H. pylori antigen was detected in 172 (91.5%) versus 144 (85.7%) of 168 males (P > .05). Interestingly, H pylori antigen was detected more often (100%) in the high socioeconomic group than in those of low socioeconomic group (85.9%). Sixteen (66.7%) of twenty four faecal samples that had previously tested positive for the organism by HpSA test were confirmed positive by PCR. However none of the treated tap water samples tested positive for the organism by PCR. The present study revealed a high prevalence of H. pylori in faecal samples of asymptomatic individuals in the Nkonkobe Municipality, an indication of active infection. The obtained results also revealed that direct treated tap water might not be playing a crucial role in the oral transmission of H. pylori in the studied population.
Surveillance of invasive vibrio species in discharged aqueous effluents of wastewater treatment plants in the Eastern Cape province of South Africa.
(University of Fort Hare, 2010) Igbinosa, Etinosa Ogbomoede
Vibrio infections remain a serious threat to public health. In the last decade, Vibrio disease outbreaks have created a painful awareness of the personal, economic, societal, and public health costs associated with the impact of contaminated water in the aquatic milieu. This study was therefore designed to assess the prevalence of Vibrio pathogens in the final effluents of wastewater treatment plants (WWTPs) in the Eastern Cape Province, as well as their abilities to survive the treatment processes of the activated sludge system either as free cells or as plankton-associated entities in relation to the physicochemical qualities of the effluents. Three wastewater treatment facilities were selected to represent typical urban, sub-urban and rural communities, and samples were collected monthly from August 2007 to July 2008 from the final effluent, discharge point, 500 meter upstream and downstream of the discharge points and analysed for physicochemical parameters, Vibrio pathogens prevalence and their antibiogram characteristics using both culture based and molecular techniques. Physicochemical parameters measured include pH, temperature, electrical conductivity, salinity, turbidity, total dissolved solid (TDS), dissolved oxygen (DO), chemical oxygen demand (COD), nitrate, nitrite and orthophosphate levels. Unacceptably high levels of the assayed parameters were observed in many cases for COD (<10 - 1180 mg/l), nitrate (0.08 - 13.14 mg NO3- as N/l), nitrite (0.06 - 6.78 mg NO2- as N/l), orthophosphate (0.07-4.81 mg PO43- as P/l), DO (1.24 - 11.22 mg/l) and turbidity (2.04 -159.06 NTU). Temperature, COD and nitrite varied significantly with season (P < 0.05), while pH, EC, salinity, TDS, COD, and nitrate all varied significantly with sampling site (P < 0.01; P < 0.05). In the rural wastewater treatment facility, free-living Vibrio densities varied from 0 to 3.45 × 101 cfu ml-1, while the plankton-associated Vibrio densities vary with plankton sizes as follows: 180 μm (0 – 4.50 × 103 cfu ml-1); 60 μm (0 – 4.86 × 103 cfu ml-1); 20 μm (0 – 1.9 × 105 cfu ml-1). The seasonal variations in the Vibrio densities in the 180 and 60 μm plankton size samples were significant (P < 0.05), while the 20 μm plankton size and free-living vibrios densities were not. Molecular confirmation of the presumptive vibrios isolates revealed V. fluvialis (36.5 %), as the predominant species, followed by V. vulnificus (34.6 %), and V. parahaemolyticus (23.1%), and V. metschnikovii (5.8 %) (detected using only API 20 NE), suggesting high incidence of pathogenic Vibrio species in the final effluent of the wastewater facility. Correlation analysis suggested that the concentration of Vibrio species correlated negatively with salinity and temperature (P < 0.001 and P < 0.002 respectively) as well as with pH and turbidity (P < 0.001), in the final effluent.
Assessment of antibiotic production by some marine Streptomyces isolated from the Nahoon Beach
(University of Fort Hare, 2010) Ogunmwonyi, Isoken Nekpen Henrietta
Rapidly emerging strains of bacteria resistant to most advanced antibiotics have become issues of very important public health concern. Research currently directed towards marine actinomycetes presents a vast potential for new compounds that could be able to safely and effectively target resistant species. In this regard, ten putative Streptomyces strains isolated from the Nahoon beach were selected and assessed for antibiotic production and activity against a wide range of bacteria including reference strains, environmental strain and clinical isolates. The ethyl acetate extracts of the putative Streptomyces isolates showed activities against at least 6 and up to 26 of the 32 test bacteria. Inhibition zones were found to range between 9-32 mm diameters at a concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) of the crude extracts ranged from 0.039 - 10 mg/ml and the least minimum bactericidal concentration (MBC) demonstrated was 0.625 mg/ml against a reference strain Staphylococcus aureus ATCC 6538. Time kill kinetics of all extracts revealed bacteristatic and bactericidal activities. Average Log reductions in viable cell counts for all the extracts ranged from 0.86 Log10 and 3.99 Log10 cfu/ml after 3 h interaction and 0.01 Log10 and 4.86 Log10 after 6 h interaction at MIC, 2 × MIC, 3 × MIC and 4 × MIC concentrations. Most of the extracts were speedily bactericidal at 3 × MIC and 4 × MIC resulting in over 50 % elimination of most of the test bacteria within 3 h and 6 h interaction. The partial characterization of the crude extracts by IR spectral analysis revealed possibility of terpenoid, long chain fatty acids and secondary amine derivatives compounds in the extracts. It is therefore recommended that further investigation should address the relationship between the structure of the active component of the extracts and the broad spectrum activity, as well as a rapid method for large scale production and purification and whether this group of antibiotics has any application in managing human infectious disease..
Investigation of antidiabetic properties, mechanisms of action and toxicology of Strychnos Henningsii (Gilg) bark.
(University of Fort Hare, 2010) Oyedemi, Sunday Oyewole
The apparent reversal of trend from modern drugs to herbal medicine is partly due to the fact that synthetic drugs have always shown adverse reactions and other undesirable side effects. Hence, the use of medicinal plants for the treatment of diseases such as diabetes is very common especially in the rural areas. Majority of these plants are used based on the experience and indigenous knowledge without identification of the therapeutic agents. There is enormous wealth of medicinal plants in the world yet many of them have not been discovered or studied scientifically to substantiate their ethno-medicinal usages. Ethnobotanical study has been the method often used to search for locally important plant species for the discovery of crude drugs with low side effects. An ethnobotanical survey was conducted on the medicinal plants commonly used for the management of diabetes mellitus in Nkonkobe Municipality, Eastern Cape of South Africa. Information was obtained through structured questionnaire administered to traditional healers and herbalists in the region. The study revealed 15 species of plants belonging to 13 families. Strychnos henningsii and Leonotis leonorus among others were repeatedly mentioned by the traditional healers as the two mostly used plants for the management of diabetes mellitus. The infusion and decoction of the roots, leaves and barks of these plants are the methods of preparation. The antioxidant potential of aqueous bark extract of S. henningsii was investigated both in vivo and in vitro using spectroscopic method. The antioxidant activity of the extract against hydrogen peroxide (H2O2), 2,2′-azinobis[3- ethylbenzothiazoline6-sulfonic acid] diammonium salt (ABTS), as well as reducing power was concentration dependent. The extract exhibited lower and average scavenging activities against 1,1diphenyl2picrylhydrazyl (DPPH) and nitric oxide (NO) radicals with IC50 value of 0.739 and 0.49 mg/ml respectively. The administration of the plant extract at 250, 500 and 1000 mg/kg significantly increased the activities of the antioxidant enzymes in the hepatotoxic rats induced with carbon tetrachloride. On the other hand, the stem bark extract had lower effect on lipid peroxidation level except at the dose of 250 mg/kg. The effect of oral administration of S. henningsii extract was evaluated in normal Wistar rats for 28 days. The observed result indicated non- toxic effect of sub-acute administration of plant extract to the animals except at certain doses. This is because, there was no apparent damage to some haematological and biochemical parameters used in assessing organ specific toxicity. However, the alterations observed on platelet, white blood cells and its differentials imply parameter and dose selective toxicity when repeatedly consumed on daily basis at the doses investigated. This study also investigated the antidiabetic activities of the extract at the doses of 125, 250 and 500 mg/kg body weight in diabetic rats induced with streptozotocin -nicotinamide for 15 days. The extract appreciably (P <0.05) reduced the blood glucose level, feed and water intake while the best result was obtained at 250 mg/kg. Similarly, the level of triacylglycerol at the three doses investigated was significantly decreased. In addition, the glucose tolerance was reduced to near normal level after 90 min at certain doses. The clinical significance of the extract on some biochemical and haematological parameters lessen both hepatic and renal damages. Anaemic condition in diabetic animals was also improved after plant extract administration. However, no significant effect was observed in white blood cells and some of its differentials. The extract demonstrated strong glucose utilization in 3T3-L1 cells with a response of 278.63% of the control at 12.5μg/ml while that of Chang liver cells was 103.54%. The cytotoxicity result revealed non-toxic effects of the extract to both cell lines. Treatment of 3T3 L1 cells with the extract did not reduce lipid accumulation. The extract inhibited the activity of α- glucosidase and α- amylase in a concentration dependent manner with IC50 values of 38μg/ml and 60.9 μg/ml respectively. The percentage protein antiglycation of S. henningsii was 18.4, 38.2 and 61.2% for 0.25, 0.5 and 1 mg/ml respectively while aminoguanidine a known inhibitor of protein glycation was 87.2% at 1 mg/ml. The FRAP assay values of the extract was 357.05 μmol Fe (II)/g. The findings from this study support the folkloric usage of this plant for the management of diabetes mellitus in the region.
Assessment of the prevalence of virulent escherichia coli strains in the final effluents of wastewater treatment plants in the Eastern Cape Province of South Africa
(University of Fort Hare, 2010) Osode, Augustina Nwabuje
Escherichia coli (E. coli) is a common inhabitant of surface waters in the developed and eveloping worlds. The majority of E. coli cells present in water are not particularly pathogenic to humans; however, there are some present in small proportion that possess virulence genes that allow them to colonize the digestive tract. Pathogenic E. coli causes acute and chronic diarrheal diseases, especially among children in developing countries and in travelers in these locales. The present study, conducted between August 2007 and July 2008, investigated the prevalence and distribution of virulent E. coli strains as either free or attached cells in the final effluents of three wastewater treatment plants located in the Eastern Cape Province of South Africa and its impact on the physico-chemical quality of the receiving water body. The wastewater treatment plants are located in urban (East Bank Reclamation Works, East London), peri-urban (Dimbaza Sewage Treatment Works) and in rural area (Alice Sewage Treatment Works). The effluent quality of the treatment plants were acceptable with respect to pH (6.9-7.8), temperature (13.8-22.0 °C), dissolved oxygen (DO) (4.9-7.8 mg/L), salinity (0.12-0.17 psu), total dissolved solids (TDS) (119-162 mg/ L) and nitrite concentration (0.1-0.4 mg/l). The other physicochemical parameters that did not comply with regulated standards include the following: phosphate (0.1-4.0 mg/L); chemical oxygen demand (COD) (5-211 mg/L); electrical conductivity (EC) (237-325 μS/cm) and Turbidity (7.7-62.7 NTU). Results suggest that eutrophication is intensified in the vicinity of the effluent discharge points, where phosphate and nitrate were found in high concentrations. Presumptive E. coli was isolated from the effluent samples by culture-based methods and confirmed using Polymerase Chain Reaction (PCR) techniques. Antibiogram assay was also carried out using standard in vitro methods on Mueller Hinton agar. The viable counts of presumptive E. coli for the effluent samples associated with 180 μm plankton size ranged between 0 – 4.30 × 101 cfu/ml in Dimbaza, 0 – 3.88 × 101 cfu/ml in Alice and 0 – 8.00 × 101 cfu/ml in East London. In the 60 μm plankton size category E. coli densities ranged between 0 and 4.2 × 101 cfu/ml in Dimbaza, 0 and 2.13 × 101 cfu/ml in Alice and 0 and 8.75 × 101 cfu/ml in East London. Whereas in the 20 μm plankton size category presumptive E. coli density varied from 0 to 5.0 × 101 cfu/ml in Dimbaza, 0 to 3.75 × 101 cfu/ml in Alice and 0 to 9.0 × 101 cfu/ml in East London. The free-living presumptive E. coli density ranged between 0 and 3.13 × 101 cfu/ml in Dimbaza, between 0 and 8.0 × 101 cfu/ml in Alice and between 0 and 9.5 × 101 cfu/ml in East London. Molecular analysis successfully amplified target genes (fliCH7, rfbEO157, ial and aap) which are characteristic of pathogenic E. coli strains. The PCR assays using uidA-specific primer confirmed that a genetic region homologous in size to the E. coli uidA structural gene, including the regulatory region, was present in 3 of the E. coli isolates from Alice, 10 from Dimbaza and 8 from East London. Of the 3 E. coli isolates from Alice, 1 (33.3%) was positive for the fliCH7 genes and 3 was positive for rfbEO157 genes. Out of the 10 isolates from Dimbaza, 4 were positive for fliCH7 genes, 6 were positive for the rfbEO157 genes and 1 was positive for the aap genes; and of the 8 isolates from East London, 1 was positive for fliCH7 genes, 2 were for the rfbEO157 genes, 6 were positive for the ial genes. Antimicrobial susceptibility profile revealed that all of the E. coli strains isolated from the effluent water samples were resistant (R) to linezolid, polymyxin B, penicillin G and sulfamethoxazole. The E. coli isolates from Dimbaza (9/10) and East London (8/8) respectively were resistant to erythromycin. All the isolates were found to be susceptible (S) to amikacin, ceftazidime, ciprofloxacin, colistin sulphate, ceftriaxone, cefotaxime, cefuroxime, ertapenem, gatifloxacin, gentamycin, imidazole, kanamycin, meropenem, moxifloxacin, neomycin, netilmicin, norfloxacin and tobramycin. The findings of this study revealed that the Alice wastewater treatment plant was the most efficient as it produced the final effluent with the least pathogenic E. coli followed by the Dimbaza wastewater treatment plant. In addition, the findings showed that the wastewater treatment plant effluents are a veritable source of pathogenic E. coli in the Eastern Cape Province watershed. We suggest that to maximize public health protection, treated wastewater effluent quality should be diligently monitored pursuant to ensuring high quality of final effluents.
Molecular and phenotypic characterization of Helicobacter pylori isolates from patients with gastroduodenal pathologies in the Eastern Cape Province of South Africa.
(University of Fort Hare, 2011) Tanih, Nicoline Fri
Helicobacter pylori is an important human pathogen known to chronically infect billions of people worldwide, causing a number of gastric related diseases. Prevalence of this organism is very high in Africa and has been reported to vary between and even within countries. H.pylori eradication using the two antibiotics regimen and a proton pump inhibitor often fails due to increasing drug resistance. Studies in South Africa have demonstrated the presence of this organism in the study area. This study investigated the prevalence of H. pylori in the Eastern Cape Province of South Africa; determined the antimicrobial susceptibility patterns of isolates; the molecular basis of the resistance pattern of isolates; the most prevalent genotype and the sequence diversity of the genes involved in virulence. We examined 254 consecutive patients who were referred to Livingstone Hospital, Port Elizabeth with gastric related morbidities between June 2008 to December 2008 for endoscopy and determined the prevalence of infection with respect to age, sex, endoscopic diagnosis, ethnic background and lifestyle. Two gastric biopsies were collected (one from the antrum and the other from the corpus) and the organism was isolated on Columbia agar base. Determination of antimicrobial susceptibility/resistant patterns of the isolates to the current antibiotics employed in treatment were executed. Genotyping was carried out using PCR based approach to determine the prevalence of virulence genes (cagA, vacA and iceA) while the molecular basis of resistance and diversity of virulence genes were determined by sequencing the amplified products. Presumptive isolates were further confirmed by PCR targeting the glmM gene. The overall prevalence of H. pylori was 66.14% (168/254). Prevalence was highest (100%) amongst patients with duodenitis (1/1), gastric cancer (GC) (4/4) and gastric erosion (5/5) as they were all positive for the organism. However, patients with gastritis (75%; 6/8), gastric ulcer (GU) (70.8%; 17/24), duodenal ulcer (DU) (65%; 26/40), non-ulcer dyspepsia (NUD) (64.7%%; 55/85), and gastro- oesophageal reflux disease (GERD) (64%; 29/45) also had high prevalence rates of the organism. The organism was not isolated (0%) from patients with gastro-duodenitis and atypical oesophageal reflux disease respectively. The prevalence of infection was highest amongst the coloured (66.92%; 87/130) and lowest in whites (59.52%; 25/ 42). Susceptibility was determined using the Kirby-Bauer disc diffusion and agar dilution methods. Data was analyzed and marked susceptibility of isolates was observed for ciprofloxacin (100%) and amoxicillin (97.5%). Isolates also demonstrated good activity to clarithromycin (80%) and gentamicin (72.5%). However, marked resistance (95.5%) was observed for metronidazole. The MIC ranged from 0.0625–8 μg/mL. The lowest MIC with a range of 0.0625 - 1μg/mL was recorded for ciprofloxacin while the highest (5–8μg/mL) was noted for gentamicin. Multidrug resistance was a common phenomenom encountered in this study. Thirty-two (17.02%) isolates showed multidrug-resistance to metronidazole and erythromycin (METRERTR). The least resistance pattern was CLARTETRAMXRMETRGENRERTR (O.53%) and ERTR (O.53%). Polymerase Chain Reaction using specific primer sequences was used to identify the presence of virulence genes. cagA was identified in 90% of the strains investigated. Fifty-eight of the 100 strains had the vacA signal sequence genotype s1 and 26 had subtype s2. Combined vacA s1/s2 was detected in 16 of the strains. vacA middle region analysis showed that 8(8%) strains were m1 while 50 were m2. Combined vacA m1/m2 was detected in 36 of the strains. s1m2 (20%) and s2m2 (20%) genotypes were the most common allelic combinations of the vacA gene among our strains. Multiple vacA genotypes were detected in this study, amongst which the most prevalent was s1m1m2 (61%) 28/46. IceA1 was present in 2 (2%) of our strains while iceA2 was present in 58 of all the samples analyzed. Sequenced data indicated that rdxA and frxA truncation was found only in metronidazoleresistant strains. Mutation in the rdxA gene may contribute more significantly than frxA gene to the high level of resistance to metronidazole. Two point mutations (A2142G and A2143G) in the 23SrRNA genes of clarithromycin- resistant strains were detected. The findings from this study support the need to continue monitoring the antibiotic susceptibility of H. pylori in the Eastern Cape Province of South Africa to guide empiric treatment of such infection.
Production and characterization of a bioflocculant from a consortium of bacteria belonging to the halomonas and micrococcus genera.
(University of Fort Hare, 2013) Okaiyeto, Kunle
The physicochemical properties of two bioflocculant producing bacteria; Halomonas sp. Okoh and Micrococcus sp. Leo were investigated. The optimum culture conditions for the individual species were determined. All the growth conditions examined for the individual bacteria were similar. Glucose and ammonium sulphate as sole carbon and nitrogen sources respectively resulted in optimum production of bioflocculant. The flocculating activity of the bioflocculants was stimulated when Al3+ was used as the coagulating aid under acidic medium. The information obtained from individual strains was used to produce a bioflocculant from the consortium of the two bacteria. After purification, the bioflocculant yields from 1L fermentation broths were 1.213 g from Halomonas sp. Okoh, 0.738 g from Micrococcus sp. Leo and 3.51 g from the consortium. The chemical analyses of the purified bioflocculants showed that they were glycoproteins. The thermostability property of the bioflocculants was investigated between 50-100oC and the results revealed that they are heat-stable. Fourier transform infrared revealed the presence of hydroxyl, carboxyl and amino groups in the bioflocculant molecules. Scaning electron microscope (SEM) images showed the structure of each bioflocculant(s) and kaolin clay before and after flocculation. From the results obtained, the idea of using the two strains in consortium for bioflocculant production resulted in an improvement in terms of flocculating activity and yield. The bioflocculants appears to have promise as an alternative to chemical flocculants used in various industrial processes such as wastewater treatment and drinking water purification.
A comparative study of the various pretreatment techniques for bioethanol production from Kikuyu Grass
(University of Fort Hare, 2013-12) Mbande, Zukiswa Coceka
Bio-ethanol is by far the most widely used bio-fuel for transportation worldwide. Production of bio-ethanol from biomass is one way to reduce both consumption of crude oil and environmental pollution. There are mainly two processes involved in the conversion of lignocellulosic materials to ethanol production: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars and fermentation of the sugars to ethanol. However, pretreatment is an essential step before the hydrolysis of biomass and subsequent production of bio-ethanol. The current study was focused on four different chemical methods for the pretreatment of Kikuyu grass using mild temperatures. The temperature used was 100°C for various periods compared.
Evaluation of the final effluents of some wastewater treatment plants in Amatole and Chris Hani district municipality of the Eastern Cape Province as sources of vibrio pathogens in the aquatic environment
(2014) Nongogo, Vuyokazi; Okoh, A I
Certain 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.
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, A.I.
In 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.
Prevalence of pathogenic Escherichia coli Strains in the final effluents of four wastewater treatment plants in the Eastern Cape Province of South Africa.
(University of Fort Hare, 2014) Seti, Nozuko Zukiswa
Water is an essential need that stimulates health and well being. Increase in population size and urbanization negatively affect water resources due to high demands of effluent outputs. Wastewater is an important reservoir for Escherichia coli and can present significant acute toxicity if released into receiving water sources without being adequately treated. E. coli is used as indicator organism for the detection of faecal contamination. These strains have been considered to be one of the primary causes of diarrhoeal infections worldwide. The present study was conducted between September 2012 and June 2013 to assess the prevalence of pathogenic E. coli strains in the final effluents of four wastewater treatment plants in Chris Hani and Buffalo City Municipalities in the Eastern Cape Province of South Africa. Standard membrane filtration technique was used for bacteriological analysis and molecular based technique was used for identification of E. coli pathotypes. The results were recorded in colony forming units/100 ml. Faecal coliforms ranged between 0-9.6×10³ CFU/100 ml for the wwtp-Q and E. coli densities ranged between 0-8.4×10³ CFU/100ml. Faecal coliforms ranged between 4×10²-9.7×10³ CFU/100 ml for wwtp-M and E. coli densities ranged between 1.2×10¹-8.4×10³ CFU/100 ml. The wwtp-E showed to have bacterial counts of faecal coliforms ranging between 4.0×10³-8.2×10³ CFU/100 ml and E. coli densities ranging between 3.5×10¹-7.1×10³ CFU/100 ml. The WWTP-K in this study was only assessed for the presence of E. coli. Faecal coliforms were assessed by the other members of the group. This plant showed to have E. coli densities ranging between 0-7.5×10²CFU/100 ml. A total of 200 presumptive E. coli isolates were subjected to screening by conventional PCR in which (29%) of the wwtp-M isolates were positively identified as E. coli, (16%) of the wwtp-K, (22%) of the wwtp-Q and (34%) of the wwtp-E isolates were positively confirmed as E. coli. A total of 100 randomly selected E. coli isolates were characterised into different pathotypes. (16%) of positive isolates were detected as EPEC and 11% were detected as UPEC strains. There was no detection for the ETEC strains. Antibiotic susceptibility patterns of E. coli strains showed high levels of resistance to Penicillin G, Erythromycin, Tetracycline and Sulfamethoxazole. High levels of Susceptibility were observed in antibiotics such as Chloramphenicol, Amoxicillin and Tetracycline. The results of this study reveal that the plants were above the recommended Standard limit of zero CFU/100 ml for effluents meant to be discharge into receiving water sources. This study reveals inadequacy of the plants studied to produce effluents of acceptable quality.
Quality indices of the final effluents of two sub-urban-based wastewater treatment plants in Amatole District Municipality in the Eastern Cape Province of South Africa.
(University of Fort Hare, 2014) Onele, Gcilitshana
Worldwide, 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.
Evaluation of some wastewater treatment facilities in Chris Hani and Amatole district municipalities as potential sources of Escherichia coli in the environment
(2014) Mazwi, Sinazo Nomathamsanqa
Access 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.
Preparation and characterisation of nanocomposites of biomass and montmorillonite clay for use as biofuels
(University of Fort Hare, 2014) Nyamutswa, Lavern Tendayi
The aim of the study was to prepare composites of clay and biomass which burn longer than unmodified biomass. Montmorillonite clay was converted to mono-ionic clay by ion exchange with sodium using sodium chloride solution. The mono-ionic clay was organically modified with an organic surfactant, methyl triphenyl phosphonium bromide. Nanocomposites were then prepared by combining the modified forms of the clay with sawdust. The three forms of clay used for the formation of composites were unmodified montmorillonite, mono-ionic montmorillonite and organically modified montmorillonite. The solution blending method was used to make the nanocomposites. FT-IR and XRD analysis showed that organic surfactant increases the interlayer space of the clay since it is bulkier than the inorganic cations that are naturally present in the interlayer space of montmorillonite. The combination of clay and sawdust resulted in the formation of exfoliated nanocomposites as shown by the absence of peaks in the low 2 theta angle in the x-ray diffraction data of the nanocomposite. The nanocomposite which was made from sawdust and 1% organically modified clay showed the best results in terms of burning time and thermal stability, as well as giving a calorific value closest to unmodified sawdust and the least amount of residue.

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