Doctoral Degrees (Pharmaceutical Sciences)

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The antibiotic treatment of gram negative bacteremia-pharmacokinetics and dynamics.
Rheeders, Malie.; Miller, Raymond Martin.
Abstract available in PDF file.
Design and synthesis of novel pH-responsive fatty acid-based lipids for the development of nano-delivery systems to enhance Vancomycin activity against Methicillin-resistant Staphylococcus aureus (MRSA).
(2020) Makhathini, Sifiso Sakhiseni.; Govender, Thirumala.
The ability of antimicrobials to prevent and treat infections caused by a range of microorganisms, including bacteria, is threatened by the emergence of drug-resistant microorganisms that is associated with high mortality rates globally. Novel nano-drug delivery systems, including lipidbased drug delivery systems, represent an alternative therapeutic approach to combat antimicrobial resistance resulting from conventional dosage forms. Since bacteria are associated with an acidic environment and the bacterial envelope is made up of lipid bilayer, the application of pHresponsive lipid-based nanomaterials for targeted antibiotic delivery is recognized as an active area of research. The aim of this study was to design and synthesize fatty acid-based pH-responsive lipids ( FAL, OLA-SPDA and DMGSAD-lipid) and explore their potential for the preparation of pH-responsive nano-based vancomycin (VCM) delivery systems to treat infectious diseases caused by methicillin-resistant Staphylococcus aureus (MRSA) infections. All the lipids were synthesized, and its structures were confirmed by FTIR, 1H NMR, 13C NMR and HR-MS. The nontoxic nature of the synthesized lipids was demonstrated by cell viability results above 75% on all tested mammalian cell lines using the MTT assay. After the synthesis and characterization, the novel fatty acid-based lipids were employed to formulate three pH-responsive lipid-based nanodrug delivery systems (liposomes, micelles and lipid polymer hybrid nanoparticles) for efficient and targeted delivery of VCM for the treatment S. aureus and MRSA infections. These systems were characterised for their physicochemical properties (Zetasizer), in vitro drug release (dialysis bag), morphology (HR-TEM), in vitro cell viability studies (flow cytometry), in vitro cytotoxicity (MTT assay), in vitro antibacterial activity (broth dilution method) and in vivo antibacterial activity (mice skin infection model). The four formulated pH-responsive liposomes had a mean size ranging from 86.28 ± 11.76 to 282 ± 31.58 nm, with their respective PDI’s ranging from 0.151 ± 0.016 to 0.204 ± 0.014 at pH 7.4 and 6.0 respectively. The ZP values were negative at physiological pH (7.4) and shifted towards positivity with a decrease in pH (6.0). The encapsulation efficiency (%EE) and loading capacity were in the range of 29.86 ± 4.5% and 44.27 ± 9.2%, The drug release profiles of all formulations at both pH 7.4 and 6.0 were sustained throughout the studied period of 72 h. Enhanced in vitro antibacterial activity at pH 6.0 was observed for the DOAPA-VAN-Liposome and DLAPA-VANLiposome formulations. Flow cytometry studies indicated a high killing rate of MRSA cells using DOAPA-VAN-Lipo (71.98%) and DLAPA-VAN-Lipo (73.32%) using the MIC of 1.59 µg/ml. In vivo studies showed reduced MRSA recovery from mice treated with liposome formulations (DOAPA-VAN-Lipo and DLAPA-VAN-Lipo) by 4- and 2-folds compared to bare VCM-treated mice respectively. The pH-responsive oleic acid-based dendritic lipid amphiphile self-assembled into stable micelles with particle size, PDI, ZP and %EE of 84.16 ± 0.184 nm, 0.199 ± 0.011 and -42.6 ± 1.98 mV and 78.80 ± 3.26%, respectively. The micelles demonstrated pH-responsiveness with an increase in particle size to 141.1 ± 0.070 nm at pH 6.0. The drug release profiles of formulations at both pH 7.4 and 6.0 were sustained throughout the studied period of 72 h. The in vitro antibacterial efficacy of VCM-OLA-SPDA-micelle against MRSA was 8-fold better when compared to bare VCM, and the formulation was 4-fold better at pH 6.0 when compared to the formulation’s MIC at pH 7.4. The MRSA viability assay showed that the micelles had a high percentage killing of 93.39% when compared to bare VCM (58.21%) at the same MIC (0.98 µg/ml). The in vivo mice skin infection model also demonstrated an enhanced antibacterial effect, showing 8-fold reduction in MRSA burden on skin treated with VCM-OLA-SPDA-micelles when compared with the skin sample treated with bare VCM. The optimized pH responsive lipid polymer hybrid nanoparticles (LPHNPs) formulations, RH40_VCM_LPHNPs had a particle size, PDI and ZP of 64.05 ± 0.64 nm, 0.277 ± 0.057 and 0.55 ± 0.14Vm, respectively, whereas SH15_VCM_LPHNPs displayed a size of 73.41 ± 0.468 nm, PDI of 0.487 ± 0.001 and ZP of -1.55 ± 0.184 Vm at pH 7.4. There was a significant change in particle size and ZP to 113.6 ± 0.20 nm and 9.44 ± 0.33 Vm for RH40_VCM_LPHNPs, respectively, whereas for SH15_VCM_LPHNPs, there was no change in is size but a significant change in surface charge switch to 9.83 ± 0.52 Vm at pH 6.0. The drug release profiles of formulations at both pH 7.4 and 6.0 were sustained throughout the studied period of 72 h. The VCM release profile, together with release kinetic study on LPHNPs, demonstrated the influence of pH on the high rate of VCM release at pH 6.0 as compared to pH 7.4. The LPHNPs a had better antibacterial activity against S. aureus and MRSA at both pH conditions when compared to bare VCM. Furthermore, the MIC of LPHNPs against MRSA was better by 8-fold at pH 6.0 than at 7.4. In summary, synthesized novel lipid materials showed superior biosafety profiles and potential in the development of lipid-based pH-responsive nanoantibiotic delivery systems against bacterial infections and other disease types characterized by low pH. The data from this study has resulted in three first-authored research publications, one co-authored research publication and one coauthored review article.
Design of advanced materials and nano delivery approaches for enhancing activity against Methicillin resistant Staphylococcus aureus.
(2018) Omolo, Calvin Andeve.; Govender, Thirumala.; Mocktar, Chunderika.
Infectious diseases, including bacterial infections, continue to be a significant cause of morbidity and mortality globally, antimicrobial resistance has further made them fatal. Limitations of conventional dosage forms have been found to be one of the contributing factors to antimicrobial resistance. Novel nano delivery systems are showing potential to combat antimicrobial resistance. The search for novel materials for efficient delivery of antibiotics is an active research area. The aim of the study was to design and synthesize advanced materials and explore nano-based strategies for preparations of novel drug delivery systems to treat SA and MRSA infections. In this study two novel materials; a linear polymer dendrimer hybrid star polymer (3-mPEA) comprising of a generation one poly (ester-amine) dendrimer (G1-PEA) and copolymer of methoxy poly (ethylene glycol)-b-poly(ε-caprolactone) (mPEG-b-PCL) and oleic acid based quaternary lipid (QL) were synthesized and characterized and Poloxamer 188 (P188) material available in the market were employed to formulate three nano drug delivery systems for efficient and targeted delivery of antibiotics. The synthesized materials and the drug delivery system were found to be biosafe after exhibiting cell viability above 75% in all the cell lines tested on using MTT assay. The formulated nano based systems were evaluated for sizes, polydispersity indices (PDI), zeta potential (ZP), surface morphology, drug release, in vitro and in vivo antibacterial activity. Nanovesicles were formulated from 3-mPEA and they had sizes, PDI, ZP and entrapment efficiency of 52.48 ± 2.6 nm, 0.103 ± 0.047, -7.3 ± 1.3 mV and 76.49 ± 2.4%. respectively. QL lipid was employed to formulate vancomycin (VCM) loaded liposomes with Oleic acid based ‘On’ and ‘Off’” pH responsive switches for infection site and intracellular bacteria targeting. They were found to have the size of 98.88 ± 01.92 at pH 7.4. and exhibited surface charge switching from negative at pH 7.4 to positive charge accompanied by faster drug release at pH 6.0. Fusidic acid nanosuspension (FA-NS) with size, PDI and ZP of 265 ± 2.25 nm, 0.158 ± 0.026 and -16.9 ± 0.794 mV respectively was formulated from P188. The drug release profile from both the nanovesicles and liposomes was found to have sustained release. In vitro antibacterial activity for the nanovesicles, FA-NS and liposomes showed 8, 6 and 4-fold better activity at pH 7.4, while the liposome being a pH responsive antibacterial system at pH 6 showed 8- and 16- fold better activity against both Methicillin susceptible (MSSA) and resistant Staphylococcus aureus (MRSA) respectively when compared with the bare drugs. An in vivo BALB/c mice, skin infection model revealed that treatment with VCM-loaded nanovesicles, liposomes and FA-Ns significantly reduced the MRSA burden compared to bare drugs and untreated groups. There was a 20, 6.33 and 76-fold reduction in the MRSA load in mice skin treated with nanovesicles, liposomes and FA-NS respectively compared to those treated with bare VCM and fusidic acid. In summary, synthesized material showed to be biosafe and potential for the development of nano-based drug delivery systems of antibiotics against bacterial infections. The data from this study has resulted in one book chapter and 3 first authored and 3 co-authored research publications.
Design of advanced multifunctional biomaterial-based biomimetic and pH-responsive hybrid nanocarriers for antibiotic delivery against bacterial infections and sepsis.
(2023) Elhassan, Eman Hussain Elmubarak.; Govender, Thirumala.; Omolo, Calvin Andeve.
Despite the notable improvements in the management of bacterial infections and sepsis, the mounting threat of antibiotic resistance on a global scale is leading towards a post-antibiotic era. Nano-drug delivery systems have improved the delivery and efficacy of various antibiotics. Biomimicry and stimuli-responsiveness have recently been used to improve the targetability of these nanocarriers, and enhance their localization at infected sites, thus improving overall therapeutic outcomes and reducing toxicity. Strategies such as targeting bacterial biofilms and efflux pumps can further enhance the delivery and effectiveness of antibiotics. Developing smart biomaterials with multifunctional properties to confer biomimetic, stimuli-responsive and antivirulence properties to antibiotic nanocarriers is the focus of ongoing research. Therefore, the general aim of this study was to investigate the potential of various novel multifunctional biomaterial-based hybrid nanocarriers (HNs), including biomimetic and/or pH-responsive HNs in enhancing the targeted delivery of antibiotics and modulating the proinflammatory response against bacterial infections and sepsis. In this study, two biomaterials with multifunctional activities, hyaluronic acid-lysine conjugate (HA-Lys) and tannic acid (TA), were employed to design, formulate, and extensively characterize innovative biomimetic and pH-responsive HNs for efficient and targeted delivery of antibiotics. The novel HA-Lys was synthesized and fully characterized using proton nuclear magnetic resonance (1H NMR) spectroscopy and Fourier-transform infrared spectroscopy (FT-IR). Then it was successfully employed with tocopherol succinate (TS) and Oleylamine (OLA) to fabricate biomimetic pH-responsive vancomycin-loaded hybrid nanostructured lipid carriers (VCM-HNLCs). The prepared VCM-HNLCs were spherical and had average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 110.77  1.69 nm, 0.11  0.02, -2.92  0.21 mV, 76.27  1.20 % and 8.36  0.25 %, respectively. Both HA-Lys conjugate and its respective nanoformulations had excellent biosafety profiles (>70 % cell viability and ˂ 1 % hemolytic effect). Possible VCM-HNLCs competitive inhibition activity to toll-like receptors 2 and 4 (TLR2 and TLR4) was demonstrated via microscale thermophoresis (MST) analysis, which showed a 5-times and 16-times lower Kd values than their natural substrates peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively. VCM-HNLCs exhibited a pH-responsive drug release profile under acidic conditions, higher bacterial killing kinetics, enhanced antibacterial, anti-biofilm, and efflux pump inhibition activities over bare VCM. Also, they showed an improved activity in neutralizing reactive oxygen species (ROS) and modulating the inflammatory response induced by LPS. On the other hand, tannic acid (TA) and Oleylamine (OLA) were successfully employed to formulate biomimetic ciprofloxacin-loaded tannic acid hybrid nanoparticles (CIP-loaded TAH-NPs) to enhance the efficacy of CIP against bacterial infections and sepsis. The prepared HNs had onion-shaped morphology, with average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 85.65 ± 0.89 nm, 0.126 ± 0.01, +16.3 ± 0.23 mV, 68.73 ± 0.54 % and 6.86 ± 0.09 %, respectively. The hemolysis and MTT assays confirmed the biosafety and non-hemolytic activity of CIP-loaded TAH-NPs formulations (>70 % cell viability and ˂ 1 % hemolytic effect). The results of MST investigations and in-silico simulations demonstrated that TA and its nanoformulation (CIP-loaded TAH-NPs) competitively inhibited TLR4 compared to its natural substrate LPS. CIP-loaded TAH-NPs showed a diffusion-based sustained release profile at physiological pH 7.4. Also, in comparison to bare CIP, the hybrid nanovesicles demonstrated improved antibacterial, anti-biofilm and efflux pump inhibition properties, as well as faster bacterial killing kinetics. Moreover, they showed a significant neutralization of ROS and the ability to control the inflammatory responses brought on by LPS. In summary, VCM-HNLCs and CIP-loaded TAH-NPs were successfully formulated and showed significant improvement in antibiotics efficacy and overall therapeutic outcomes. This study confirmed the potential of biomimetic stimuli-responsive antibiotic hybrid nanocarriers for enhancing antibiotic efficacy against bacterial sepsis and addressing the antimicrobial resistance crisis. The data from this study has resulted in one first-authored review article, two first-authored research publications and one co-authored review article.
Molecular characterization of multi-drug resistant (MDR) gram-negative bacterial pathogens from environments, patients and staff in a teaching hospital in Ghana.
(2023) Yeboah, Esther Eyram Asare.; Essack, Sabiha Yusuf.; Owusu-Ofori, Alexander.; Agyepong, Nicholas.; Abia, Akebe Luther King.; Amoako, Daniel Gyamfi.; Mbanga, Joshua.
Multidrug resistant Gram-negative bacteria (MDR GNB) are implicated in serious infections both of community and nosocomial origin and may be disseminated in the hospital in the absence of efficient infection prevention and control (IPC) practices. The prevalence and risk factors for rectal colonization of MDR GNB among patients, the carriage of MDR GNB on healthcare workers’ (HCWs’) hands and the contamination patients’ environments with MDR GNB were investigated in a teaching hospital in Ghana. In this prospective study, conducted between April 2021 to July 2021, the phenotypic profiles of the MDR GNB isolates were determined using the VITEK 2 system. Risk factors for colonization with MDR GNB were assessed using univariate and multivariate analysis of associated data. The resistome, virulome, mobilome and genetic relatedness of MDR extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and ESBL-producing or carbapenem resistant Klebsiella pneumoniae isolates from patients and their environment were also determined using whole genome sequencing performed on the Nextseq 550 (2 x 150 bp) and bioinformatics analysis. A total of 585 samples were collected from patients, HCWs’ hands and the hospital environment within the study period. The prevalence of MDR GNB rectal colonization among patients was 50.62% on admission and 44.44% after 48 hours. MDR GNB, frequently E. coli and K. pneumoniae were isolated from 6 (5.26%) and 24 (11.54%) of HCW’s hand swabs and environmental swabs, respectively. Previous hospitalization (p-value = 0.021, OR,95% CI= 7.170 (1.345-38.214) was significantly associated with colonization by MDR GNB after 48 hours of admission while age (21-30 years) (p-value =0.022, OR, 95% CI =0.103(0.015-0.716) was significantly identified as a protective factor associated with a reduced risk of rectal MDR GNB colonization. Rectal carriage and acquisition of ESBL-producing E. coli among patients was 13.65% and 11.32% respectively. blaTEM-1B and blaCTX-M-15 were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Multiple virulence genes, predominantly, terC were detected in the ESBL E. coli isolates. Sequence types (STs) were diverse and included one novel ST (ST13846) present in two isolates. Phylogenetic analysis grouped the ESBL E. coli isolates into four main clusters. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities. Of the ten selected MDR K. pneumoniae isolates, the β-lactamase gene, blaCTX-M-15 was observed in six isolates. Mutations were found in both ompK36 and ompK37 in all isolates (both carriage isolates and isolates from hospital environments). Genes encoding resistance to fluoroquinolone (qnrB), aminoglycosides (aadA1, aadA2, aac(3)-IIa, aac(6')-Ib-cr,aph(3'')-Ib , aph(6)-Id) sulphamethoxazole/trimethoprim (sul1, sul2, dfrA14, dfrA15) were also detected. The K. pneumoniae isolates belonged to seventeen different STs with ST39 most commonly observed and common to both carriage isolates and isolates from hospital environments. A myriad of virulence genes, including irp1, irp2, iutA, gndA, ompA, fes, fep, mrkD and fimH were detected in both carriage and isolates from the hospital environment. IncFIB was the most abundant plasmid replicon occurring in nine (four carriage isolates and five isolates from hospital environments). ESBL-producing K. pneumoniae isolates appeared to be introduced into the hospital from the community. The high colonization of MDR GNB in patients, the carriage of MDR GNB on HCW’s hands, the contamination of hospital environments and the circulation of ESBL-producing E. coli and K. pneumoniae isolates with diverse genomic characteristics, highlights the need for patient screening, and stringent infection prevention and control practices to prevent the spread of MDR GNB in hospitals. The observed clonal relatedness among isolates from patients and the hospital environment, as well as between different patients, suggests a possible transmission within and between sources, hence infection prevention and control practices need to be enhanced to prevent the dissemination and transmission of these resistant strains in the hospital. This study further highlights the usefulness of whole genome sequencing as an effective tool in AMR surveillance.
Molecular profile of gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana.
(2017) Agyepong, Nicholas.; Essack, Sabiha Yusuf.; Owusu-Ofori, Alex.; Govinden, Usha.
Gram-negative ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp) pathogens are a major healthcare concern globally due to their increasing multidrug resistance and ability to cause debilitating infections. Phenotypic and genotypic characteristics of multidrug resistant Gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana were investigated. Two hundred (200) clinical, non-duplicate Gram-negative bacterial pathogens were randomly selected from various human specimens routinely processed by the diagnostic microbiological laboratory in the hospital. Multidrug resistant (isolates resistant to at least one agent in three or more antibiotic class) isolates selected from each group of Gram-negative ESKAPE pathogens constituted the final sample. Identification and antibiotic susceptibility profiles were carried out using Vitek-2. Identity of isolates for whole genome sequencing was further confirmed by MALDI-TOF MS. Four P. aeruginosa and 10 K. pneumoniae were subjected to whole genome sequencing based on their extensively drug resistant profiles and resistance to third-generation cephalosporins respectively using Illumina MiSeq, after genomic DNA extraction using the NucliSens easyMAG®. Antibiotic resistance genes and plasmids were identified by mapping the sequence data to an online database using ResFinder and plasmidFinder respectively. MLST was also determined from the WGS data. The raw read sequences and assembled whole genome contigs have been deposited in GenBank under project number PRJNA411997. An average multidrug resistance of 89.5% was observed, ranging from 53.8% in Enterobacter spp to 100.0% in Acinetobacter spp and P. aeruginosa. Gram-negative ESKAPE bacteria constituted 48.5% (97) of the 200 isolates. P. aeruginosa (n=4) belonging to ST234 harboured blaDIM-1, blaIMP-34, blaOXA-10, blaOXA-129, blaOXA-50, blaPAO aadA1, aac4 aph(3’)-IIb, fosA, sul1, dfrB5, catB7, arr-2 conferring resistance to β-lactams, aminoglycosides, fosfomycin, sulphonamides, trimethoprim phenicols and rifampin respectively. qnrVC was detected in two of the four isolates . Both blaDIM-1 and blaIMP-34-like positive contigs showed identical DNA sequences and were linked to type 1 integron structures. BlaDIM-1 was 100% identical to the blaDIM-1 prototype gene, while blaIMP-34-like had two base pair (bp) differences T190C and C314G respectively compared to blaIMP-34, leading to one amino acid substitution in IMP-34-like indicating that, the gene may have independently evolved, perhaps due to selection pressure. Blast analysis did not reveal identical genetic structures deposited in NCBI, neither among the nucleotide collection, completed genomes nor among the completed plasmids. β-lactamases (blaCTX-M-15, blaSHV-11, blaTEM-1B) and resistance genes for aminoglycosides (aac(3)-IIa-like,aph(3')-Ia) quinolones/fluoroquinolones (oqxA-like,oqxB-like,qnrB10-like,qnrB2) and others including fosfomycin (fosA), trimethoprim (dfrA14), and sulphonamide (sul2) were found in the K. pneumoniae (n=10). Multiple and diverse mutations of the quinolone resistance-determining regions gyrA, gyrB and parC genes were detected in the K. pneumoniae (n=4), which were clonally distinct. The diversity of resistance genes expressed by Gram-negative ESKAPE pathogens conferring resistance to multiple antibiotics is problematic in a resource-constrained country like Ghana, necessitating urgent antibiotic stewardship and infection prevention and control interventions.
Synthesis and antimicrobial evaluation of novel 2,4-dihydro-3h-pyrazol-3-one hybrids: a new class of antibiotics.
(2019) Jain, Kavita.; Karpoormath, Rajshekhar.
The rise in multidrug resistance (MDR) pathogenic microbes has emerged as a critical global health burden. To address this problem, the scientific community and Pharmaceutical industries worldwide are focused in developing newer, safer and cost-effective antimicrobial agents. Design and development of potential antimicrobial agents has been one of the focus areas of the Synthetic and Medicinal Chemistry Research Group (SMCRG), UKZN. In continuation of the ongoing research in SMCRG and the need to discover newer antimicrobials, I envisaged to synthesize novel antimicrobial agents containing 2,4-Dihydro-3H-pyrazol-3-one (pyrazolone) as a core pharmacophoric moiety. Pyrazolones are versatile heterocyclic building blocks and is a core scaffold in several marketed drugs such as for analgesic and antipyretic (phenazone, metamizole, propyphenazon, and remifenazone); neuroprotective agent (edaravon); antispasmodic (dipyron); anti-inflammatory agents (famprofazone, phenylbutazone, and remifenazone) and more recently there have been several reports on the pyrazolone analogs as potential antimicrobial agents. In this research work I have synthesized a series of potential pyrazolone hybrids containing substituted 1,3,4-thiadiazole, thiazolidinone, triazole, oximes, and chalcones as antimicrobial agents (Figure 1). The work in this thesis is divided into 7 chapters:

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Browsing Doctoral Degrees (Pharmaceutical Sciences) by Subject "Antibiotics."