Prevalence, antibiogram and molecular characterization of methicillin-resistant staphylococcus aureus recovered from treated wastewater effluent and receiving surface water.

Masters Degree. University of KwaZulu-Natal, Durban. 2018.Inadequately treated wastewater effluent serves as a reservoir of potentially pathogenic bacteria and contributes to the spread of these organisms in the environment, including Staphylococcus aureus (S. aureus), a faecal bacterium known to cause pneumonia, septicaemia and skin infections in humans. The presence of S. aureus in water has become problematic as it has been shown to exhibit resistance towards β-lactam antibiotics commonly used to treat infections, including methicillin, leading to the emergence of methicillin-resistant S. aureus (MRSA). The current study aimed to determine the prevalence of MRSA and mecA (known to induce methicillin resistance) in the influent, treated effluent and receiving surface rivers of two wastewater treatment plants (WWTPs) in Durban. The study also evaluated the antibiogram and virulence gene profiles of MRSA isolates recovered from the treated effluent and receiving surface water using the Kirby-Bauer disc diffusion and PCR assays. Genetic fingerprinting was carried out to determine the phylogenetic relationship between isolates with selected antibiogram profiles. The prevalence of MRSA in WWTP1 ranged from 11.45-85.63% (influent), 16.28-39.36% (before chlorination), 2.16-5.07% (after chlorination), 1.06-7.24% (downstream) and 4.95-14.09% (upstream). In WWTP2, the prevalence of MRSA for the influent ranged from 48.25-86.18%, before chlorination; 23.73-93.75%, after chlorination; 4.28-48.82%, downstream; 1.74-19.31% and upstream; 5.90-28.78%. Correlation studies of selected physico-chemical parameters to the prevalence of MRSA was carried out. The real-time PCR assay showed a reduction in the concentration of mecA from the influent to the treated effluent in both WWTPs. The highest resistance was observed towards lincomycin (100%), followed by oxacillin (98.75%), cefoxitin and penicillin (97.50%) and ampicillin (96.25%). Additionally, 72.50%, 66.25%, 52.50%, 40% and 33.75% of the isolates showed resistance against cefozolin, azithromycin, amoxicillin/clavulanic acid, erythromycin and vancomycin, respectively. The following antibiotic resistance genes were detected in resistant isolates: aac(6ꞌ)/aph(2ꞌꞌ) in 56.25%, ermC in 62.50%, msrA in 22.50% and blaZ and tetK in 70%. The virulence genes hla and sea were detected in 57.50% of the isolates, hld in 1.25% and the lukS P/V gene was not detected. Thirteen pulsotypes v (designated A-M) was generated for selected isolates using pulse field gel electrophoresis, correlating them to their respective antibiograms. The study revealed a lower prevalence of MRSA and concentration of mecA in the treated effluent as compared to the influent of both WWTPs. It also revealed that these multi-drug resistant strains, isolated from the treated effluent and receiving surface waters, are potentially pathogenic and could contribute to the spread of disease in the environment. Hence, the need for more stringent monitoring and evaluation of treatment performance of the WWTPs

Rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against HIV

Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV‐endemic regions such as sub‐Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of (OS)GRFT in the best‐performing plants was 223 μg/g dry seed weight. We also established a one‐step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger‐scale process to facilitate inexpensive downstream processing. (OS)GRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole‐cell assays using purified (OS)GRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure (OS)GRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom‐to‐operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases

Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the presence of the cysteine-proteases, putative targets of cystatins, among other enzymatic activities. All barley cystatins, except HvCPI-1 and HvCPI-7, inhibited in vitro mite cathepsin L- and/or cathepsin B-like activities, HvCPI-6 being the strongest inhibitor for both mite species. Transgenic maize plants expressing HvCPI-6 protein were generated and the functional integrity of the cystatin transgene was confirmed by in vitro inhibitory effect observed against T. urticae and B. chilensis protein extracts. Feeding experiments impaired on transgenic lines performed with T. urticae impaired mite development and reproductive performance. Besides, a significant reduction of cathepsin L-like and/or cathepsin B-like activities was observed when the spider mite fed on maize plants expressing HvCPI-6 cystatin. These findings reveal the potential of barley cystatins as acaricide proteins to protect plants against two important mite pests

The burden of sickle cell disease in Cape Town

Background. South Africa has a low incidence of sickle cell disease (SCD). However, its demographics are changing because of immigration from sub-Saharan African countries where SCD is prevalent. Objectives. We aimed to determine the frequency of SCD presenting to the Haematology/Oncology Service at Red Cross War Memorial Children’s Hospital in Cape Town and to measure the associated disease burden. Methods. This was a retrospective cross-sectional study of patients first attending the Haematology Service between January 2001 and June 2010. Results. A total of 58 SCD patients were indentified, with an annual frequency that increased over the study period by 300 - 400%. Up to 93.1% (n=54) were originally from other African countries, mainly the Democratic Republic of Congo (62.1%, n=36). One patient had sickle D-Punjab genotype, and all the other patients had the homozygous sickle cell anaemia genotype (Hb SS). Their haematological parameters demonstrated a normocytic anaemia with high white cell counts. The mean number of clinic visits per patient per year was 22.2 (range 0 - 64), and the mean number of hospital admissions per patient per year was 1.2 (range 0 - 5). All the patients were on antibiotic prophylaxis. The majority had at least one blood transfusion (65.5%, n=38), and a significant proportion required intravenous analgesia on admission (29.3%, n=17) and hydroxyurea treatment (36.2%, n=21). Conclusions. Over the past 10 years the frequency of SCD has increased considerably, imposing a significant burden and new challenges to the health services in Cape Tow

Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection.

BACKGROUND:Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS:Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS:This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform

Evolution of Plant-Made Pharmaceuticals

The science and policy of pharmaceuticals produced and/or delivered by plants has evolved over the past twenty-one years from a backyard remedy to regulated, purified products. After seemingly frozen at Phase I human clinical trials with six orally delivered plant-made vaccines not progressing past this stage over seven years, plant-made pharmaceuticals have made a breakthrough with several purified plant-based products advancing to Phase II trials and beyond. Though fraught with the usual difficulties of pharmaceutical development, pharmaceuticals made by plants have achieved pertinent milestones albeit slowly compared to other pharmaceutical production systems and are now at the cusp of reaching the consumer. Though the current economic climate begs for cautious investment as opposed to trail blazing, it is perhaps a good time to look to the future of plant-made pharmaceutical technology to assist in planning for future developments in order not to slow this technology’s momentum. To encourage continued progress, we highlight the advances made so far by this technology, particularly the change in paradigms, comparing developmental timelines, and summarizing the current status and future possibilities of plant-made pharmaceuticals