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Browsing Biosciences by Author "Chihomvu, Patience"

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    Biochemical and molecular characterization of heavy metal resistant bacteria isolated from the Klip River, South Africa
    (Vaal University of Technology, 2014) Chihomvu, Patience; Stegmann, P., Dr.; Pillay, M., Prof.
    The Klip River has suffered severe anthropogenic effects from industrial, agricultural, mining and domestic activities. As a result harmful contaminants such as heavy metals have accumulated in the river, causing microorganisms inhabiting the environment to develop mechanisms to protect them from the harmful effects of the contaminants. The current study deals with the isolation and characterization of heavy metal resistant bacteria isolated from the Klip River Catchment. Water and sediment samples were collected from 6 sites of the Klip River, and the Vaal Barrage (control). In-situ parameters, such as pH, turbidity, salinity, conductivity, temperature and dissolved oxygen were determined. Lead, iron, cadmium, nickel, zinc and copper concentrations of water were determined by atomic absorption spectroscopy. For bacterial analysis sediment and water samples were collected in sterile glass jars and bottles respectively. Heavy metal resistant bacterial isolates were screened on heavy metal constituted Luria Bertani (LB) agar. Biochemical profiles of the isolates were constructed using the API 20E® strips, antibiotic susceptibility tests were done and growth studies were carried out using spectrophotometric methods. The isolates were identified using 16SrDNA sequencing and alignment. A partial sequence of the copper resistance gene pcoA was amplified from strains Lysinibacillus sp. KR25 [KJ935917], and Escherichia coli KR29 [KJ935918]. The pcoR gene was amplified from E. coli (KR29) and the partial sequence for the chromate resistance gene chrB, was amplified from Pseudomonas sp. KR23 [KJ935916]. The gene fragments were then sequenced and translated into protein sequences. The partial protein sequences were aligned with existing copper and chromate resistance proteins in the Genbank and phylogenetic analysis was carried out. The physico-chemical properties of the translated proteins were predicted using the bioinformatics tool Expasy ProtParam Program. A homology modelling method was used for the prediction of secondary structures using SOPMA software, 3D-protein modelling was carried out using I-TASSER. Validation of the 3D structures produced was performed using Ramachandran plot analysis using MolProbity, C-score and TM-scores. Plasmid isolation was also carried out for both the wild type strains and cured derivatives and their plasmid profiles were analysed using gel electrophoresis to ascertain the presence of plasmids in the isolates. The cured derivatives were also plated on heavy metal constituted media. Antibiotic disc diffusion tests were also carried out to ascertain whether the antibiotic resistance determinants were present on the plasmid or the chromosome. The uppermost part of the Klip River had the lowest pH and thus the highest levels of heavy metal concentrations were recorded in the water samples. Turbidity, salinity and specific conductivity increased measurably at Site 4 (Henley on Klip Weir). Sixteen isolates exhibiting high iron and lead resistance (4 mM) were selected for further studies. Antibiotic susceptibility tests revealed that the isolates exhibited multi-tolerances to drugs such as Ampicillin (10 μg/ml), Amoxcyllin (10 μg/ml), Cephalothin acid (30 μg/ml), Cotrimoxazole (25 μg/ml), Neomycin (30 μg/ml), Streptomycin (10 μg/ml), Tetracycline (30 μg/ml), Tobramycin (10 μg/ml) and Vancomycin (30 μg/ml). Growth studies illustrated the effect of heavy metals on the isolates growth patterns. Cadmium and chromium inhibited the growth of most of the microorganisms. The following strains had high mean specific growth rates; KR01, KR17, and KR25, therefore these isolates have great potential for bioremediative applications. Using 16SrDNA sequencing the isolates were identified as KR01 (Aeromonas hydrophila), KR02 (Bacillus sp.), KR04 (Bacillus megaterium), KR06 (Bacillus subtilis), KR07 (Pseudomonas sp), KR17 (Proteus penneri), KR18 (Shewanella), KR19 (Aeromonas sp.), KR22 (Proteus sp.), KR23 (Pseudomonas sp.), KR25 (Lysinibacillus sp.), KR29 (Escherichia coli), KR44 (Bacillus licheniformis) and KR48 (Arthrobacter sp.). Three heavy metal resistance genes were detected from three isolates. The pcoA gene was amplified from strains Lysinibacillus sp KR25, and Escherichia coli KR29; pcoR gene from E. coli KR29 and the chrB gene, from Pseudomonas sp. KR23. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E.coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element which was not detected using gel electrophoresis. The translated protein sequence for pcoA_25 showed 82% homology with the copper resistant protein form Cronobacter turicensis [YP003212800.1]. Sequence comparisons between the pcoR partial protein sequence found in E. coli KR29 showed 100% homology with 36 amino acids (which was 20% of the query cover) from a transcriptional regulatory protein pcoR found in E. coli [WP014641166.1]. For the chrB partial protein sequence detected in Pseudomonas sp. (KR23), 97% of the query sequence showed 99% homology to a vitamin B12 transporter btuB in Stenotrophus sp. RIT309.
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    The effects of solar irradiated Salmonella Typhimurium and campylobacter jejuni on the proliferation and activation of macrophages in vitro
    (Vaal University of Technology, 2019-12) Chihomvu, Patience; Ssemakalu, Cornelius Cano, Dr.; Ubomba-Jaswa, Eunice, Dr.; Pillay, Michael, Prof.
    Salmonella enterica serovar Typhimurium and Campylobacter jejuni are the leading causes of Salmonellosis and Campylobacteriosis that is characterised by gastroenteritis. These waterborne diseases can be easily prevented by home water treatment methods such as solar disinfection (SODIS). The SODIS process involves placing microbiologically unsafe water in clear plastic or glass bottles and exposing them to direct sunlight for approximately six to eight hours. SODIS kills microbes through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating. The result is microbiologically safe water. Continuous drinking of SODIS treated water may confer some immunological effects on the consumer. These immunological effects have not been thoroughly explored. Therefore, the objectives of this study were to firstly, characterise the effects of solar irradiation on the viability of S. Typhimurium and C. jejuni; secondly, to determine the cytotoxicity and modulation of cell death of solar irradiated S. Typhimurium and C. jejuni on macrophages. Thirdly, to analyse the chemokine and cytokine profiles of macrophages infected with solar irradiated S. Typhimurium and C. jejuni. Lastly, to analyse the host-cell interactions of macrophages infected with solar-irradiated and non-solar irradiated S. Typhimurium and C. jejuni using a proteomic approach. In all the experiments, S. Typhimurium and C. jejuni were (i) heat/chemically treated, (ii) solar and non-solar irradiated for 4 and 8 hours. A murine macrophage cell line RAW264.7 was co-cultured with the differentially treated bacteria species for 3 and 24 hours. Appropriate controls were included. The impact of solar irradiated S. Typhimurium and C. jejuni on intracellular growth, proliferation, cytotoxicity, and apoptosis on macrophages was assessed. Intracellular growth of the both bacterial species was assessed with the gentamicin protection assay, and cytotoxicity was determined by Lactate Dehydrogenase Assay (LDH). The macrophages treated with solar irradiated S. Typhimurium and C. jejuni showed no intracellular growth after 48 hours post-infection. However, the non-irradiated S. Typhimurium survived within the macrophages and were highly toxic to the macrophages (average cytotoxicity of 91%±32). The non-solar irradiated C. jejuni were metabolically active but non-culturable, whereas the solar-irradiated C. jejuni was metabolically inactive. Thus, solar irradiated C. jejuni showed a lower percentage cytotoxicity (2.57% ± 0.32%) in comparison to non-solar irradiated C. jejuni at 24 hours post-infection (p.i.) (30.28% ± 0.05%). Flow cytometric analysis showed that the non-irradiated S. Typhimurium brought about a statistically significant increase in the percentage of necrotic cells (48% ± 2.99%), whereas bacteria irradiated for 8 hours produced a lower percentage of necrotic cells (25% ± 5.87%). The heat/chemical attenuated samples had the lowest percentage of necrotic cells (21.15% ± 5.36%) at 24 h p.i. Macrophages treated with solar irradiated and non-solar irradiated C. jejuni did not induce necrosis, but apoptotic cell death. At 24 h p.i., the highest proportion of apoptotic cell death was observed in macrophages treated with non-solar irradiated C. jejuni whereas the solar irradiated C. jejuni showed a lower percentage of apoptotic cell death. Therefore, there is great possibility that S. Typhimurium and C. jejuni could become avirulent after SODIS treatment and this could prevent gastroenteritis in consumers of SODIS-treated water. The activation of macrophages infected with solar irradiated S. Typhimurium and C. jejuni was also assessed in this study. The production of nitric oxide (NO) was determined using the Greiss Reagent Assay, whereas the production of chemokines, cytokines, and growth stimulating factors by the RAW264.7 cells in vitro was measured using the Luminex 200. The results showed that both solar and non-solar irradiated S. Typhimurium inhibited the production of nitric oxide in the RAW264.7 cells. The heat/chemically attenuated S. Typhimurium induced a significant increase (p<0.0.5) in the production of NO2− in the macrophages when compared to the unstimulated RAW264.7. The chemokine and cytokine levels produced by the macrophages were similar in the solar inactivated S. Typhimurium and the live untreated S. Typhimurium. However, macrophages treated with heat/chemically attenuated S. Typhimurium showed an anti-inflammatory response by inhibiting the production of pro-inflammatory cytokines such as IL-1, IL-1, IL-2, IL-6, and IL-17 in macrophages. The macrophages treated with solar and non-solar irradiated C. jejuni possibly produced an anti-inflammatory effect since the amount of pro-inflammatory cytokines in the samples was significantly reduced during the late infection period (24 h p.i.). This study also analysed the proteomic profiles of macrophages treated with LPS, non-solar irradiated, solar irradiated, heat/ chemical inactivated S. Typhimurium, and C. jejuni. This was carried out using SWATH-mass spectrophotometry-based proteomics. Proteins were extracted from infected macrophages after 24 hours p.i. HILIC-based sample clean-up and digestion, DDA LCMS-MS (spectral library), SWATH LCMS-MS, and data processing were carried out. A total of 15,077 peptides matching to 2,778 proteins were identified at 1% FDR with numerous differentially expressed proteins (DEPs) detected in macrophages treated with lipopolysaccharide (LPS), non-solar irradiated C. jejuni (NS), heat-attenuated C. jejuni (HA) and 4h-solar irradiated (SI4) and 8h-solar irradiated (SI8) C. jejuni, respectively. Pathway analysis revealed that most of the upregulated proteins in macrophages treated with solar irradiated C. jejuni were involved in oxidation-reduction processes, endoplasmic reticulum stress, transport, antigen processing and presentation of exogenous peptide antigens via MHC class I (TAP-dependant) and ATP-biosynthetic processes. The KEGG-pathways also revealed the roles of some upregulated proteins in lysosomal and phagosome pathways. In conclusion, our results revealed that there is coordinated up-regulation of MHC-I processing pathways occurred at 24 h p.i. It is likely that proteins from solar irradiated C. jejuni may undergo proteasomal degradation, and the peptides are transported to the endoplasmic reticulum (ER) and loaded onto MHC-I molecules. Peptide loading results in class I complexes consolidation and transit to the cell surface where antigens can be presented to circulating CD8 + T cells. Additionally, solar irradiated C. jejuni also undergoes degradation in the phagosome. The phagosome has the potential to create antigens that can be expressed on the cell surface of macrophages to stimulate different lymphocytes and induce appropriate immune responses, thus, connecting the innate to adaptive immunity, and this could also have health benefits via the consumption of SODIS treated water. However, proteomic analysis of S. Typhimurium showed no significant differentially expressed proteins in macrophages treated with LPS, non-solar irradiated, and solar irradiated S. Typhimurium. This may be due to an overestimation of the extracted protein. However, DEPs in macrophages treated with heat-attenuated S. Typhimurium showed that macrophages may have adapted an anti-inflammatory M2 phenotype because the IFN-γ signalling pathway was downregulated. This may have contributed to non-expression of the chemokine IFN-γ in RAW264.7 cells. Moreover, proteins such as Hmox1 and Sqstm1 were upregulated, and this is also characteristic of M2 macrophages. This study provided new insights on the effect of solar irradiated Salmonella Typhimurium and Campylobacter jejuni on the proliferation and activation of macrophages in vitro.