Department of Biochemistry and Microbiology

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Antibiogram profiling of Escherichia Coli pathotypes isolated from Kat River and Fort Beaufort abstraction water.
(University of Fort Hare, 2014) Nontongana, Nolonwabo
Escherichia 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.
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..
Assessment of bioflocculant production by two marine bacteria isolated from the bottom sediment of marine Algoa Bay
(University of Fort Hare, 2015) Ntozonke, Ncedo; Prof. Okoh, A I
Bioflocculants 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.
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
Bioflocculants 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.
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.
Assessment of the quality indices and prevalence of Escherichia coli pathotypes in selected rivers of Osun state, Southwestern Nigeria
(2015) Titilawo, Osuolale Yinka; ;
Surface 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.
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.
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
Staphylococcus 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.
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).
A comparative study of the in vitro antidiabetic properties, cytotoxicity and mechanism of action of Albuca bracteata and Albuca setosa bulb extracts
(University of Fort Hare, 2015) Odeyemi, Samuel Wale; Bradley, G.
The search for cheap, non toxic and readily available antidiabetic drugs has been a challenge for researchers and the pharmaceutical industries. Diabetes mellitus is a metabolic disease characterized by defects in the synthesis of insulin and/or insensitivity to the action of insulin at the target cells. The disease has been on the increase mostly in developing countries where large proportions of the population have little access to good medical care due to either accessibility or non availability of synthetic drugs. This has led to the use of medicinal plants to treat diabetes because it is safe, cheap and with few side effects. There is little scientific evidence on the dosages, active compounds, mechanisms of action and toxicity of these traditionally used plants. Two of the most frequently used plants; Albuca setosa and Albuca bracteata were investigated in this study. The qualitative analysis of different extractions of these plants revealed the presence of phenolics, alkaloids, tannins and saponins. The antioxidant properties of aqueous, acetone and methanollic extracts of Albuca setosa and Albuca bracteata were investigated using models such as Diphenyl-1-Picrylhydrazyl (DPPH), 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Ferric ion reducing antioxidant potential (FRAP), Nitric Oxide and Hydrogen Peroxide (H2O2). Both plants revealed inhibitions against DPPH in a concentration - dependent manner with Albuca setosa (0.330 mg/ml) showing higher activity than Albuca bracteata (0.647 mg/ml) determined from the IC50. The aqueous extract of Albuca setosa showed a higher inhibition against DPPH radical compared to the Albuca bracteata aqueous extract at all concentrations investigated. The isolated saponins from Albuca bracteata had a higher DPPH scavenging activity than the crude methanolic extract of the plant in a concentration - dependent manner but are significantly different from each other at 0.4, 0.6 and 1.0 mg/ml only. The IC50 of the saponins was also observed to be higher than the crude extracts and standards. The Albuca setosa aqueous extract showed a higher percentage inhibition of ABTS radicals than Albuca bracteata at all the concentrations investigated. Overall, the Albuca setosa aqueous extract (0.0809 mg/ml) showed maximum activity against ABTS radicals. The iron reducing power was significantly higher (P < 0.05) in the methanolic extract of both plants compared to the aqueous counterpart. Overall, the Albuca bracteata aqueous extract (0.344 mg/ml) showed maximum activity as indicated by the IC50. The aqueous extracts of both plants also revealed percentage inhibitions in a concentration - dependent manner against NO2. The aqueous extract of Albuca bracteata bulb was more active against nitric oxide and hydrogen peroxide inhibition. In this study, the cytotoxicity of the extracts was evaluated at a high dose of 100 μg/ml on Chang liver cells and determined using MTT, crystal violet, glucose consumption, lactate production and lactate dehydrogenase release and FRAP. The aqueous extracts of both Albuca setosa and Albuca bracteata were non-toxic on Chang liver cells at the concentrations investigated. The MTT revealed that the aqueous extract of Albuca setosa bulb had the optimum cell viability of 108.09% while the acetonic extract of Albuca bracteata showed the least cell viability (37.72%) compared with the control. The crystal violet test also revealed the acetone extract of Albuca bracteata to have the least percentage of cell viability at 31.47%, while the aqueous extract of Albuca setosa showed the maximum cell viability at 112.5%.The aqueous extracts of both plants showed higher percentage cell density on the second day of incubation from the proliferation assay. All the tested samples were observed to consume more glucose than the blank except for the methanollic and acetone extracts of Albuca bracteata bulb. The aqueous and methanolic extracts of Albuca setosa bulbs produced the highest lactate with 120.2 μg/ml and 113.7 μg/ml respectively. The acetone extracts of both Albuca setosa and Albuca bracteata revealed toxicity with a higher lactate dehydrogenase release compared to the control. The aqueous extracts of both Albuca setosa and Albuca bracteata bulbs showed reducing power of 18.7 and 22.1 μmol/l of FeSO4 respectively. The in vitro antidiabetic potentials investigated revealed that the aqueous extracts of both plants inhibited the activity of α- glucosidase but have weak inhibition against α- amylase. The acetone extract of Albuca bracteata showed the highest inhibition against the alpha amylase followed by the acetone extract of Albuca setosa. Both plant extracts also showed weak inhibition against the dipeptidylpeptidase-IV (DPP-IV) at the concentration investigated. The glucose uptake in HepG2 for both aqueous and methanol extracts of Albuca setosa were not significantly different from the control but the MTT result of the treatments in HepG2 for both Albuca setosa and Albuca bracteata aqueous extracts revealed low cell density compared to the control. The aqueous extract of Albuca setosa demonstrated glucose utilization in L6 cells with a response of 188.93% of the control at 25 μg/ml while the aqueous extract of Albuca bracteata demonstrated glucose utilisation in L6 with a response of 89.17% of the control. The MTT of the treatments in L6 revealed no sign of cytotoxicity, as the cell density of all the treatments were not significantly different from the control. The aqueous extracts of both Albuca setosa and Albuca bracteata also showed high glucose utilization in 3T3-L1 cells with 123.21% and 131.56% respectively. In conclusion, Albuca setosa possesses better in vitro strong antidiabetic activity compared to Albuca bracteata. The mechanism of glucose utilization in L6 and 3T3-L1 of Albuca setosa and Albuca bracteata may be due to the activation of some molecules in the insulin signaling pathway, while the in vitro antidiabetic activity of Albuca bracteata may also involve its inhibitory effect on α-glucosidase. The antioxidant properties of both plants may also play an important role in ameliorating the complications of diabetes. These findings therefore support the folkloric usage of these plants for the management of diabetes mellitus.
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.
Evaluation of bioflocculant-producing potential of bacillus pumilus strain isolated from Tyume River in the Eastern Cape Province of South Africa
(University of Fort Hare, 2015) Makapela, Busisiwe
A bioflocculant is a kind of metabolite produced by microorganisms during their growth. Most bioflocculants are mainly composed of high polymers such as extracellular polysaccharides, glycoprotein, protein and nucleic acids. Bioflocculants promote flocculation by forming bridges between suspended particles in solutions resulting in precipitation of the suspended particles. Generally, when suspended particles are flocculated into large flocs, they settle down thus resulting in a clarified solution and can easily be removed. In this study, a bacterial strain named F45 was investigated for its ability to produce a bioflocculant. Source samples for isolation of bioflocculant-producing microorganisms were collected from the Tyume River, Eastern Cape Province of South Africa. The collected samples included water samples, rock scrapings and sediment samples. In total, 144 isolates were obtained from all the samples in which 13 were found to be bioflocculant producers as evidenced by the ability of the produced metabolite to flocculate kaolin suspension. Notable among these isolates was a bacterial strain F45 which was obtained from rock scrapings and whose bioflocculant exhibited a flocculating activity above 60%. Identification of the F45 strain revealed it to have 95% similarity to Bacillus pumilus strain ZAP 028. The optimum culture conditions for bioflocculant production by this strain were inoculum size of 4% (v/v), maltose as carbon source, multiple nitrogen source composed of yeast extract, urea and ammonium sulphate. The highest flocculating activity was recorded at an initial pH of 7. A bioflocculant yield of about 0.289 g/l was recovered from the fermented broth. The purified bioflocculant was a white powder which showed high flocculating activity (96.5%) against kaolin suspension at a dosage of 0.1 mg/ml. All the cations tested stimulated the flocculating activity of the purified bioflocculant except for Fe3+ which only showed a low flocculating activity of 21%. Thermal stability test of the purified bioflocculant proved it to be stable as it could retain more than 90% of its activity after being heated at 100ºC for 1 hour. Fourier-transform infrared (FTIR) spectroscopy analysis revealed that purified bioflocculant contained hydroxyl groups, carboxyl groups and uronic acid. The bioflocculant was composed of sugar (75.4%), protein (5.3%) and uronic acid (15.4%). Scanning electron microscopy (SEM) revealed a dendritic bioflocculant structure and elemental analysis of the purified bioflocculant showed that the weight fractions of elements C, N, O, S and P were 22.71%, 11.56%, 41.60%, 0.51% and 7.98% respectively. The results obtained suggest that this bioflocculant could be utilized as an alternative for harmful synthetic flocculants in various industrial applications.
Evaluation of flocculating potentials and characterization of bioflocculants produced by three bacterial isolates from Algoa bay, South Africa
(University of Fort Hare, 2016) Okaiyeto, Kunle
Flocculation has been widely adopted as one of the most effective methods to remove colloidal particles in water or wastewater treatment. Synthetic flocculants are conventionally used because of their high flocculating efficiency and cost-effectiveness. However, they have been reported to have hazardous properties and implicated in some serious health problems including senile dementia and neuro-toxicity, as well as being recalcitrant in the environment. Consequently, efforts are being geared away from the use of synthetic flocculants in water and wastewater treatment. Hence, the need for safe and eco-friendly flocculants has become imperative. Compared with synthetic flocculants, bioflocculants have special advantages such as safety, biodegradability and harmlessness to the environment and humans; attributes which make them potential alternatives in water treatment, downstream as well as fermentation processes. In the current study, the potentials of bacterial isolates recovered from Algoa Bay in the Eastern Cape Province of South Africa for bioflocculant production were investigated. The bacterial isolates were identified by polymerase chain reaction (PCR) as belonging to the Bacillus genus. The analysis of 16S ribosomal deoxyribonucleic acid (rDNA) nucleotide sequence of isolate M72 showed 99% similarity to Bacillus toyonensis strain BCT-7112 and was deposited in the GenBank as Bacillus toyonensis strain AEMREG6 with accession number KP406731. Likewise, the 16S rDNA nucleotide sequences of isolates M69 and M67 showed 98% sequence similarity to Bacillus licheniformis strain W7 and Bacillus algicola strain QD43 respectively; and M67 isolate was subsequently deposited in the GenBank as Bacillus sp. AEMREG7 with accession number KF933697.1. The results of the nutritional requirements and fermentation conditions revealed that optimum inoculum size for REG-6 production was 4% (v/v), while 5% (v/v) and 3% (v/v) were most favourable for MBF-W7 and MBF-UFH production respectively. Glucose was the best carbon source for the production of bioflocculants (REG-6 and MBF-UFH) by Bacillus toyonensis AEMREG6 and Bacillus sp. AEMREG7 respectively, while maltose supported optimum bioflocculant (MBF-W7) production by Bacillus specie. Inorganic nitrogen (NH4NO3) was the favoured nitrogen source for both REG-6 and MBF-W7 production, while mixed nitrogen sources [yeast extract + urea + (NH4)2SO4] supported the maximum production of MBF-UFH. The initial medium pH for REG-6 was 5, while MBF-W7 and MBF-UFH were both maximally produced at the initial pH of 6. After a 96 h cultivation period under optimal culture conditions, 3.2 g of purified REG-6 with a maximum flocculating activity of 77% was recovered from 1 L fermented broth of Bacillus toyonensis AEMREG6. Yields of 3.8 g and 1.6 g pure bioflocculants with the respective highest flocculating activities of 94.9% and 83.2% were also obtained from 1 L, 72 h-fermented broths of Bacillus licheniformis and Bacillus sp. AEMREG7 respectively. Furthermore, all the three bioflocculants (REG-6, MBF-W7 and MBF-UFH), displayed thermal stability within the temperature range of 50 to 100 oC, with strong flocculating activities of over 80% against kaolin suspension over a wide range of pH range (3–11) and relatively low dosage requirements of 0.1-03 mg/ml in the presence of divalent cations in the treatment of kaolin clay suspension and Thyme River waters. Chemical composition analyses of the bioflocculants showed them to be glycoproteins with a predominantly polysaccharide backbones as shown by the following carbohydrate/protein (w/w) ratios: 77.8%:11.5% (REG-6); 73.7%:6.2% (MBF-W7) and 76%:14% (MBF-UFH). Fourier transform infrared spectroscopy (FTIR) revealed the presence of hydroxyl, carboxyl and amide groups which are preferred for effective flocculation. Scanning electron microscopy (SEM) images of the purified bioflocculants showed that they have an irregular, coarse-grained structure connected in netted textures; it also revealed how the bioflocculants connected the scattered kaolin particles firmly together to form bigger flocs which subsequently precipitated out of suspension as a result of gravity. MBF-W7 showed good turbidity removal potential (86.9%) and chemical oxygen demand (COD) reduction efficiency (75.3%) of Thyume River waters. MBF-UFH showed higher flocculating activity for kaolin clay suspension compared to synthetic flocculants (aluminium chloride and iron chloride). The results obtained from this study suggested that the bioflocculants (REG-6, MBF-W7 and MBF-UFH) produced by these bacterial isolates have great potentials to serve as an alternatives to hazardous synthetic flocculants conventionally utilized in various industrial processes including water/wastewater treatment, and stand as attractive candidates for further research and development for industrial-scale application.
Evaluation of incidence of Mycobacterium tuberculosis complex associated with soil, hayfeed and water in three Agricultural facilities in Amathole District Municipality in the Eastern Cape Province, South Africa
(2016) Ntloko, Athini
Mycobacterium bovis and other species of Mycobacterium tuberculosis complex (MTBC) can result to a zoonotic infection known as Bovine tuberculosis (bTB). MTBC has members that may contaminate an extensive range of hosts, including wildlife. Diverse wild species are known to cause disease in domestic livestock and are acknowledged as TB reservoirs. It has been a main study worldwide to deliberate on bTB risk factors as a result some studies focused on particular parts of risk factors such as wildlife and herd management. The objectives of this study were to design questionnaires from commercial farms and smallholding farms; isolate and identify MTBC from collected samples using culture and PCR assays recovered from Fort Hare, Middledrift and Seven star dairy farms; and assessing genotypic drug resistance through detection of mutations conferring resistance to INH and RMP associated with first line treatment for MTBC infection. Questionnaires were administered to thirty (30) smallholding farm owners in the two villages (kwaMasele and Qungqwala) and three (3) three commercial farms ( Fort Hare dairy farm, Middledrift dairy farm and Seven star dairy farm). Detection of M. tuberculosis complex was achieved by Polymerase Chain Reaction using primers for IS6110; whereas a genotypic drug resistance mutation was detected using Genotype MTBDRplus assays. Nine percent (9%) of respondents had more than 40 cows in their herd, while 60% reported between 10 and 20 cows in their herd. Relationship between farm size and vaccination for TB differed from forty one percent (41%) being the highest to the least five percent (5%). The highest number of respondents who knew about relationship between TB cases and cattle location was ninety one percent (91%). Approximately fifty one percent (51%) of respondents had knowledge about wild life access to the farms. Relationship between import of cattle and farm size ranged from nine percent (9%) to thirty five percent (35%). Cattle sickness in relation to farm size differed from forty three (43%) being the highest to the least three percent (3%); while thirty three percent (33%) of respondents had knowledge about health management. Respondents with knowledge about the occurrence of TB infections in farms were forty eight percent (48%). The frequency of DNA isolation from samples ranged from the highest forty five percent (45%) from water to the least twenty two percent (22%) from soil. Fort Hare dairy farm had the highest number of positive samples forty four percent (44%) from water samples; whereas Middledrift dairy farm had the lowest positive from water, seventeen percent (17%). Twelve (22%) out of 55 isolates showed resistance to INH and RMP that is, multi-drug resistance (MDR) and nine percent (9%) were sensitive to either INH or RMP. The mutations at rpoB gene differed from 58% being the highest to the least (23%). Fifty seven percent (57%) of samples showed a S315T1 mutation while only 14% possessed a S531L in the katG gene. The highest inhA mutations were detected in T8A (80%) eighty percent and the least was observed in A16G (17%). The results of this study reveals that risk factors for bTB in cattle and dairy farm workers is a serious issue abound in the Eastern Cape of South Africa; with the possibility of widespread dissemination of multidrug resistant determinants in MTBC from the environment.
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.
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.
Evaluation of the quality indices of the final effluents of two wastewater treatment plants in Buffalo City Metropolitan Municipality in the Eastern Cape Province
(2015) Osuolale, Olayinka O.
Wastewaters 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 × 104 CFU/100 ml and 0 - 9.93 × 103 CFU/100 ml for E. coli and Vibrio respectively at the WW-Ama plant and 0 - 1.86 × 104 CFU/100 ml for and 0 - 1.44 × 103 CFU/100 ml for E. coli and Vibrio respectively at the WW-Dim plant. About 41.7% of the samples analysed for E. coli and 16.7% for Vibrio for WW-Ama plant complied with the set limit of 1000 CFU/100 ml while 83.3% (E. coli) and 91.7% (Vibrio) of the WW-Dim samples complied in with the set limit. Also, DNA of the confirmed E. coli and Vibrio isolates were used for confirmation of their identities using species specific polymerase chain reaction (PCR) methods. About 300 confirmed E. coli and Vibrio isolates were pathotyped. The prevalence of the E. coli pathotypes were of the following orders: uropathogenic E. coli (9%), enteroaggregative E. coli (3.7%), neonatal E. coli (1.7%) and enteropathogenic E. coli (0.7%). None of the targeted Vibrio pathotypes i.e Vibrio parahaemolyticus, Vibrio fluvialis and Vibrio vulnificus were detected in either plant suggesting that the confirmed isolates are members of other Vibrio species besides those targeted. The incidences of enteric viruses in the final effluents were also investigated using real - time PCR. Target viruses included enteric viruses Adenovirus, hepatitis A virus (HAV), enterovirus, norovirus and rotavirus. Norovirus, enterovirus and hepatitis A virus were not detected in any sample from the treatment plants, while adenovirus and rotavirus were detected in all the plants with the WW-Ama plant having the highest detection rate and concentration of the viruses. The detection rate for Adenovirus was 67% for the WW-Ama Treatment Works and 17% for the WW-Dim samples; while for Rotavirus, the detection rate was 42% for WW-Ama and 17% for WW-Dim Sewage Treatment Works effluents. Antibiogram 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.
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—).
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.
Genotypic and phenotypic characterization of enterococci from cow dung and environmental water sources in three selected dairy farms in Amathole District.
(University of Fort Hare, 2016) Tanih, Godfred Ngu; Green,E.
Enterococcus species are integral members of the gastrointestinal microfloral of humans, animals, birds, as well as insects. Their presence in water and food has been greatly associated with faecal contamination. This study was aimed at evaluating the incidence of Enterococcus species in cow dung and environmental water sources in three commercial dairy farms. In addition, their antibiotic profiles were determined as well as resistance and virulence genes. Furthermore, the genetic relatedness of the isolates was determined by molecular typing method (RAPD PCR). Three hundred and thirty four water and faecal samples consisting of 117, 116 and 101 were collected from Seven Star Middle Drift and Fort Hare Dairy trusts respectively. Of the 334 samples collected, 289 were of faecal origin and 45 from water sources within the farms. All samples were screened for enterococci using culture base growth media and molecular methods targeting the tuf gene. Speciation was done using species-specific primers and the incidences of various species within the farms determined. Furthermore resistance to antibiotics and multidrug-resistant phenotypes were established using the disk diffusion method. Genes coding for virulence and resistance were also determined. From the samples collected, 313 (289 faecal and 24 water) presumptive enteroccocci were isolated, 305 of 313 (97.45%) were confirmed as Enterococcus of which 239 of 305 (78.38%) were identified as E. hirae, 15 of 305 (4.92 %) as E. faecium, 12/305 (3.93%) as E. durans, 6 of 305 (1.97%) as E. faecalis and 33 of 305 (10.82%) were unidentified. Out of the five virulence genes that were targeted in the study only gelE (71.80% of 219/305) and ace (27.2% 83/305) were present in the isolates. Phenotypic resistance to antibiotics was observed is in all twelve antibiotics tested with multidrug resistance phenotypes detected in some enterococcal isolates most predominant in Seven Star and Middledrift dairy trust. Finally RAPD profiles of the isolates showed high relatedness between the strains from water and cow dung sources in all three commercial dairy farms suggesting possible contamination from cow dung to the water sources or vice versa.

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