Samantha Mc Carlie

PhD Student

Genetic analysis of disinfectant resistance in bacterial species

Serratia sp. HRI is a bacterium with high levels of resistance to various disinfectants. The bacterium's high levels of resistance provide a unique opportunity to study resistance to disinfectants. This work aims to understand why the bacterium is so highly resistant to disinfectants, specifically resistance to quaternary ammonium compound (QAC) disinfectants.

To understand the high level of antimicrobial resistance, we will analyse the bacterial response to disinfectants through differential gene expression. This is done using mRNA sequencing and transcriptome analysis before and after disinfectant exposure. The differential expression will be compared over time to examine how the bacterial response changes in reaction to disinfectants. The bacterial response can identify known and novel disinfectant resistance mechanisms.

Investigation into the role of efflux pumps is resistance to disinfectants

Increased antibiotic resistance in bacteria, particularly multidrug-resistant bacteria in medical, agricultural, and food production industries, requires disinfection protocols to control resistant bacterial populations. However, resistance to disinfectants has also developed. Understanding how bacteria become resistant to disinfectants is required to safeguard their efficacy. Efflux pumps are a common resistance mechanism used by bacteria to withstand antibiotics. However, their role in disinfectant resistance is less evident. The role of efflux pumps in the disinfectant tolerance of a resistant Serratia strain was tested using efflux inhibitors. The effect of short-term and long-term disinfection was investigated to better understand which resistance mechanisms are recruited. Results showed that efflux pumps are indeed involved in the resistance capabilities of the Serratia strain; however, it is unclear whether efflux pumps are the sole mechanism responsible for disinfectant resistance.

Gunter Staats

MSc Student

Boudine van der Walt

MSc Student

Investigation into the role of plasmids in disinfectant resistance

Resistance to disinfectants is a growing concern. The isolation of a highly resistant isolate (HRI) of Serratia sp. allows us to study various resistance mechanisms to provide more insight into developing such resistance phenotypes. Complete genome sequencing data of the HRI has revealed numerous unique genes in our HRI that produce multiple resistance mechanisms. These resistance genes need to be studied to establish their role in resistance. Plasmids have also been isolated from the HRI and a Serratia marcescens type strain. Both plasmids have mobile genetic elements, meaning that the transformation of the HRI plasmids into the type strain may increase its resistance. The project's main objective is to study the genes on the plasmids and establish whether the transfer of the plasmid to susceptible strains results in the transfer of resistance. And to further establish whether an increase in resistance could be due to the transposition of resistance genes from one plasmid (HRI) to the other (type) or from a new co-resident HRI plasmid. After completing this, the study will look at the behaviour of other susceptible bacterial species in response to transformation with the Serratia sp. HRI plasmids.

Development of a qPCR system to evaluate the removal of IBDV in poultry houses during pre-placement disinfection

The infectious bursal disease virus (IBDV) is non-enveloped, making it extremely difficult to remove from the environment. The focus remains on disinfection and hygiene to control and prevent future outbreaks of IBDV in broiler houses. Even so, IBDV may not be entirely eradicated or decreased by using certain disinfections. The need developed for a system to detect and evaluate the efficacy of disinfection protocols performed in chicken houses. GD Animal Health in the Netherlands has developed a system known as VIR-Check, which checks the viral load of five contagious intestinal viruses – rotavirus A and D, chicken astrovirus, avian nephritis virus three, and reovirus – pre and post disinfection. This research project aims to establish an RT-qPCR (reverse-transcription quantitative polymerase chain reaction) system that will be used to test the efficacy of pre-placement disinfection in poultry pens. The system will be optimised for the specific detection of IBDV from field samples obtained from a farm in Stutterheim in the Eastern Cape (South Africa).

Mart-Louise van Zyl

MSc Student

Deon Beauzec

MSc Student

Evaluation of a continual disinfection program in poultry production

With antibiotic resistance expanding globally, the use of antibiotics in animal production is being re-evaluated, and alternatives must be found. I am looking into the possible application of continuous disinfection during the growth cycle of broiler chickens, involving the application of disinfectants in drinking water and as a spray to clean and disinfect chicken houses when broilers are grown on a commercial scale. The study also looks into the effectiveness of different cleaning and disinfection programmes against bacteria and viruses. Finally, I am also investigating which organisms survive these cleaning methods and whether there is a possibility that the use of disinfectants instead of antibiotics can lead to the development of disinfectant resistance.

Interaction between antibiotic and disinfectant resistance

With antibiotic and disinfectant resistance on the rise within opportunistic pathogens, the need for a more in-depth understanding of the acquisition, transfer, and potential for cross-resistance has arrived. This project further investigates the relationship between antibiotic and disinfectant resistance in opportunistic pathogens using Serratia marcescens as a model. The main objectives are to determine accurate minimum inhibitory concentrations, investigate the presence of mobile genetic elements, isolate and analyse plasmids, take a closer look at the genetic composition of resistant and susceptible strains, and establish cross-resistance between selected isolates. This research will provide an in-depth understanding of resistance in a well-known nosocomial pathogen and open new avenues for the possible prevention of resistance development within the hospital setting.

Wanja Swart

MSc Student

Twyne Skein

MSc Student

Development of a universal vaccine for infectious coryza

Avibacterium paragallinarum is the causative agent of infectious coryza, a disease in chickens. The disease results in a reduction in the growth and feed conversion rates and egg production. One way to combat infectious coryza is by vaccination. However, vaccines need to contain the exact serovars of A. paragallinarum prevalent in an area, or else vaccination failure occurs. This research tries to identify shared common antigenic proteins among the different serovars of A. paragallinarum occurring in South Africa and to express them in the yeast Yarrowia lipolytica. If this works, it will circumvent the need to undertake complicated and often unreliable serotyping or molecular typing to ascertain which serovars infect chickens in an area.

Screening monoclonal antibodies for the serotyping of Avibacterium paraganllinarum

Avibacterium paragallinarum causes infectious coryza in chickens, which can decrease egg production and even lead to the chicken's death if untreated. Although commercial vaccines are available on the market, chickens are still infected, as A. paragallinarum is divided into three serovars (A, B, and C), and the vaccines provide no cross-protection. Hybridomas stored in the -80°C freezer for many years were isolated from field and vaccine strains. My experiment aims to find which serovars are present in these hybridomas using ELIZA as the serotyping method. The project aims to find reliable Mabs that differentiate between the serovars, especially the C group serovar.

Azil Coertzen

BSc Hons Student

Bernadette Belter

MSc Student

Expression of antigens of SARS-C0V-2 virus

The rise of SARS-CoV-2 variants with specific mutations has raised serious concerns, such as that these mutations will affect virus sensitivity to antibodies and the potential exists that the current vaccines might be ineffective. This has been proven in some cases where specific variants are not neutralised by antibodies produced against the vaccine strain, since they do not produce effective cross-neutralising antibodies. This again prompts the importance of SA institutions to play a part in developing vaccines and diagnostic assays that incorporate the new variants, including B.1.351, which has been shown to produce cross-neutralising antibodies with the potential to neutralise other variants and limit infections.

My project would aim to express SARS-CoV-2 and the South African B.1.351 variant proteins, such as the spike and nucleocapsid proteins in the yeast Yarrowia lipolytica. Variations of the spike protein will also be examined to optimise expression in this system. The expression will be carried out using expression vectors that allow for the surface display, internalisation, and secretion of these proteins using the patented Yarrowia lipolytica expression system. Upon expression, various protein purification strategies will have to be explored for downstream applications, such as potential subunit vaccine development and diagnostic assays.

Development of phage display-derived antiviral peptides against Newcastle disease virus

Newcastle disease virus is a pathogen that causes significant losses in the poultry industry. The first part of this project seeks to determine the neutralising efficacies of six antiviral peptides against the Newcastle disease virus and compare them with the peptides displayed on phage M13, their native expression system. The second part of the project aims to express the most effective of the six antiviral peptides in the plant Nicotiana benthamiana by displaying it on modified tobacco mosaic virus-like particles. Lastly, the peptide produced in the plant must be tested against the Newcastle disease virus again to determine whether the peptide retains its efficacy when produced heterologously. If this works, the process could potentially be used to produce other functional binding peptides displayed on virus-like particles in plants.

Carlo Visser

MSc Student

Jacky Huang

MSc Student

Evaluation of bacteriophage enzymes as an alternative to antibiotics

Antibiotic resistance is a rising global crisis, with the WHO predicting that by the year 2050, an estimated 10 million people will die each year as a result of antibiotic resistance. This project aims to combat rising antibiotic resistance by utilising antibiotic alternatives, which in this case is the use of the phage-derived proteins endolysin and holin. Therefore, this project aims to assess the efficacy of both endolysin and holin anti-bactericidal activity and to further assess their potential to act as an antibiotic alternative.

Metagenomics evaluation of the bacterial diversity in diseased Pecan nut trees

The project will focus on pecan-nut trees' bacterial composition, which displays overall decline. These diseased trees displayed an infection within the tree stem, which has led to the trees producing minimal to no harvest of leaves or pecan nuts. The tree stem, roots, and soil samples will be taken and further analysed in the lab. DNA will be extracted from these samples and further sequenced and analysed using metagenomics technology. Bacteria will also be cultured from these samples, grown, and further sequenced for identification. Results from the culturing of bacteria and identification via metagenomics will be compared. This research will lead us closer to establishing the cause of the decline in pecan-nut trees. The research will also determine whether the origin of the disease is bacterial.

Bianca Delport

MSc Student