Doctoral Degrees (Microbiology)

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Characterization of atypical class I lanthipeptides from the marine bacterium thalassomonas viridans XOM25ᵀ
(Stellenbosch : Stellenbosch University, 2024-03) Vermeulen, Ross; Dicks, Leon Milner Theodore ; Van Staden, Anton du Preez; Trindade, Marla; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The natural products (NPs) that are produced by living organisms have historically served as a pivotal starting point for numerous highly effective therapeutic agents. These compounds often possess distinct chemical structures that enable valuable mechanisms and modes of biological activity. Natural products are thus a promising reservoir that potentially contains the key to the treatment of diseases like cancer, infections, resistance, neurological disorders, and even a range of agricultural and environmental challenges. The rate of novel drug discovery is exhibiting a gradual decline. However, recent advancements in biotechnology, such as next-generation sequencing (NGS) and heterologous gene expression, are empowering researchers to access unexplored realms of biological and chemical diversity. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of NPs that have experienced significant growth by combining NGS and heterologous expression. Within the RiPP group, lanthipeptides are characterized by the post-translational installation of lanthionine rings or thioether cross-linkages. The incorporation of lanthionine rings offers several advantages such as enhanced stability and expanded structural diversity at low genetic cost. However, the process of post-translational modification poses distinct challenges when attempting to heterologously produce lanthipeptides exhibiting previously unobserved and markedly distinct characteristics. To date, the majority of described lanthipeptides play an antimicrobial role which provides a competitive advantage to the native producer by inhibiting the growth of similar or closely related species. Recently, a significant number of cryptic lanthipeptides have been heterologously produced which do not exhibit antimicrobial activity against species related to the native host. Many of these seemingly inactive and atypical lanthipeptides originate from Gram-negative bacteria associated with the marine environment. In this work, a significantly dissimilar lanthipeptide operon was identified in the genome of the marine Gram-negative marine bacteria Thalassomonas viridans XOM25T. The operon was used to construct an Escherichia coli BL21 expression system capable of co-producing the peptide substrates and post-translational modification machinery. The target peptides were subsequently isolated, confirmed to contain post-translationally installed lanthionine rings, and designated as the viridisin operon. Finally, the antimicrobial, stereochemical and pharmaceutical potential of the viridisins were assessed. The viridisins have similar stereochemistries to other lanthipeptides from Gram- negative bacteria, and likewise, they do not have antimicrobial properties. However, this study proved that the viridisin were able to modulate zebrafish larvae behavior. While the results of the zebrafish behavioral screening do not provide definitive proof of the mechanisms at play, such findings allude to the remarkable diversity of biological roles that RiPPs fulfil – intended or otherwise.
Characterization of bacteriocin 423 produced by Lactobacillus pentosus
(Stellenbosch : Stellenbosch University, 2000-12) Van Reenen, Carol A. (Carol Ann); Dicks, Leon Milner Theodore; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Worldwide, bacteriocins, particularly those produced by food-related lactic acid bacteria, are receiving attention due to the possible use of these peptides as natural preservatives in food, replacing potentially harmful chemical preservatives. Bacteriocins are ribosomally synthesized proteins or peptides that inhibit closely related microorganisms. Most bacteriocins produced by lactic acid bacteria are small, heat resistant peptides that inhibit other Gram-positive bacteria, including food-borne pathogens such as Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Staphylococcus aureus, but do not inhibit Gram-negative bacteria, molds or fungi. Bacteriocins are produced as inactive prepeptides that become active after the N-terminal leader peptide is cleaved off. Small heat resistant bacteriocins are either lantibiotics (Class I), containing unusual posttranslationally modified amino acids, or peptides that are non-Ianthionines (Class II). The Class II bacteriocins are further divided into four different groups: Class lIa, the anti-listerial bacteriocins containing the YGNGV consensus sequence in the N-terminal of the protein, Class lib, bacteriocins consisting of two peptides, Class IIc, bacteriocins that are secreted via the sec pathway, and Class lid, bacteriocins that do not belong in the previous three subgroups. A bacteriocin producing lactic acid bacterium was isolated in our laboratory from traditionally home fermented South African sorghum beer. The producing bacterium was found to be a facultative heterofermentative Lactobacillus sp. and was identified as Lactobacillus plantarum or Lactobacillus pentosus by using the API 50 CHL carbohydrate fermentation system and numerical analysis of total soluble cell protein patterns. RAPD-PCR analysis identified the strain as L. plantarum, but 16S rRNA sequencing confirmed its identification as L. pentosus. The bacteriocin, first designated plantaricin 423 and later bacteriocin 423, was identified as a Class lIa small heat resistant anti-listerial bacteriocin containing the YGNGV consensus motif. Bacteriocin 423 inhibited a variety of Gram-positive bacteria, including Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp., Staphylococcus spp., Bacillus spp., Clostridium spp. and Listeria spp. The bacteriocin was inactivated by proteolytic enzymes and active over a wide pH range (pH 1-10). Bacteriocin 423 lost 50 % of its activity after autoclaving for 15 min at 121°C, but was not affected by lesser heat treatments. Bacteriocin production was increased by optimizing the growth medium, which consisted of glucose, tryptone, yeast extract, potassium phosphate, sodium acetate, ammonium citrate, manganese sulphate, Tween 80 and casamino acids. The bacteriocin was found to be plasmid-encoded. Genetic analysis of the bacteriocin operon indicated a high percentage of homology to the operon of another Class lIa bacteriocin, pediocin PA-1, although the structural genes of the two bacteriocins were markedly different. The structural gene of bacteriocin 423 was amplified by PCR and cloned into a yeastJE. coli vector between the ADH1 promoter and terminator sequences and fused in-frame to the MFa1 secretion signal sequence. Saccharomyces cerevisiae transformed with this plasmid expressed the bacteriocin. The sequence of prebacteriocin 423 (MMKKIEKL TEKEMANIIGGKYYGNGVTCGKHSCSVN WGOAFSCSVSHLANFGHGKC) is similar, but not identical to any other reported Class lIa anti-listeria I peptide.
Characterization of nisin F and its role in the control of respiratory tract and skin infections
(Stellenbosch : University of Stellenbosch, 2009-03) De Kwaadsteniet, Michele; Dicks, Leon Milner Theodore; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
Multidrug resistant strains of Staphylococcus aureus is presenting an increasing threat, especially immune compromised individuals. Many of these strains have developed resistance to newly approved drugs such as quinupristin-dalfopristin, linezolid and daptomycin. The search for alternative treatment, including bacteriocins (ribosomally synthesized antimicrobial peptides) of lactic acid bacteria is increasing . Lactococcus lactis subsp. lactis F10, isolated from freshwater catfish, produced a new nisin variant active against clinical strains of S. aureus. The operon encoding nisin F is located on a plasmid and the structural gene has been sequenced. The lantibiotic is closely related to nisin Z, except at position 30 where valine replaced isoleucine. The antimicrobial activity of nisin F against S. aureus was tested in the respiratory tract of Wistar rats. Non-immunosuppressed and immunosuppressed rats were intranasally infected with S. aureus K and then treated with either nisin F or sterile physiological saline. Nisin F protected immunosuppressed rats against S. aureus, as symptoms of an infection were only detected in the trachea and lungs of immunosuppressed rats treated with saline. The safety of intranasally administered nisin F was also evaluated and proved to have no adverse side effects. The potential of nisin F as an antimicrobial agent to treat subcutaneous skin infections was evaluated by infecting C57BL/6 mice with a bioluminescent strain of S. aureus (Xen 36). Immunosuppressed mice were treated with either nisin F or sterile physiological saline 24 h and 48 h after infection with subcutaneously injected S. aureus Xen 36. Histology and bioluminescence flux measurements revealed that nisin F was ineffective in the treatment of deep dermal staphylococcal infections. Non-infected and infected mice treated with nisin F had an influx of polymorphonuclear cells in the deep stroma of the skin tissue. This suggested that nisin F, when injected subcutaneously, may have modulated the immune system. Nisin F proved an effective antimicrobial agent against S. aureus-related infections in the respiratory tract, but not against subcutaneous infections. The outcome of nisin F treatment thus depends on the route of administration and site of infection.
Characterization of the adhesion genes of probiotic lactic acid bacteria
(Stellenbosch : Stellenbosch University, 2008-03) Ramiah, Kamini; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
One of the key selection criteria for potential probiotics is the ability to adhere and colonise the host gastrointestinal tract (GIT). Probiotics compete for receptor sites at the host intestinal surface, preventing the colonisation of pathogens, thereby protecting the host from infection. In addition, several important intestinal functions are mediated by the binding of probiotics to host tissue. However, the molecular mechanisms and genotypic characterization of adhesive elements have not received as much attention as other aspects of probiotic research. The present study aims to contribute to this area of research. The first part of the study focused on monitoring the expression of mucus adhesion genes mub, mapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of Lactobacillus plantarum 423, as well as mub, surface layer protein (slp) and EF-Tu of Lactobacillus acidophilus ATCC 4356 when grown in the presence of mucin, bile, pancreatin and at low pH. Real time PCR was used. mub, mapA and EF-Tu of strain 423 were up-regulated in the presence of mucus and expression increased under increasing concentrations of mucus. Expression of mapA was up-regulated under normal gut conditions (0.3%, w/v, bile; 0.3%, w/v, pancreatin; pH 6.5) and at higher levels of bile (1.0%, w/v) and pancreatin (1.0%, w/v). Expression of mub was downregulated in the presence of bile and pancreatin at pH 6.5, whilst the expression of EFTu and plaA remained unchanged. At pH 4.0, the expression of mub and mapA remained unchanged, whilst EF-Tu and plaA were up-regulated. Expression of mapA was down-regulated in the presence of 0.1% (w/v) cysteine, suggesting that the gene is regulated by a mechanism of transcription attenuation that involves cysteine. In the case of L. acidophilus ATCC 4356, none of the genes were up-regulated under increasing concentrations of mucin, whilst only slp and EF-Tu were up-regulated under normal and stressful gut conditions in vitro. In the second part of the study, male Wistar rats were used to evaluate which section of the gastrointestinal tract are colonised by L. plantarum 423 and Enterococcus mundtii ST4SA and determine the effect of adhesion. Fluorescent in situ hybridization (FISH) incorporating strain specific oilgonucleotide probes indicated strong fluorescent signals for L. plantarum 423 along the intestinal lining of the ileum and the cecum. L. plantarum 423 did not colonise the colon as indicated by real timePCR. Fluorescent signals were recorded for E. mundtii ST4SA across the epithelial barrier of cecum and colonic tissue, suggesting that translocation took place. Real time PCR revealed highest cell numbers of strain ST4SA in the cecum and the colon. Haemotoxylin eosin staining of rat tissue revealed no change in morphology or any toxic effects induced upon adhesion of the strains. 16S rDNA PCR and denaturing gradient gel electrophoresis (DGGE) revealed a decrease in enterobacterial species whilst the lactic acid bacterial content remained unchanged. Strains 423 and ST4SA agglutinated yeast cells in vitro, indicating the possible presence of mannose receptors. It is well known that these receptors play a crucial role in the elimination of type 1 fimbriated strains of E. coli. It is thus safe to speculate that mannose receptors may have played a role in diminishing the enterobacterial content in the gut. The third part of the study encompassed characterization of cell surface proteins of L. plantarum 423 and their role in adhesion to Caco-2 cell lines. The strain lacks the typical surface layer protein whilst a multifunctional “intracellular” protein, elongation factor Tu (EF-Tu) and glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) were detected. Removal of surface proteins reduced adherence of strain 423 to Caco-2 cell lines by 40%, suggesting that these proteins play a role in adhesion. The ability of strain 423 to competitively adhere, exclude and displace Clostridium sporogenes LMG 13570 and Enterococcus faecalis LMG 13566 from Caco-2 cell lines, was studied. Adhesion of C. sporogenes LMG 13570 and E. faecalis LMG 13566 was inhibited by 70% and 90%, respectively. Strain 423 excluded C. sporogenes LMG 13570 from Caco-2 cells by 73% and displaced the pathogen by 80%. E. faecalis LMG 13566 was excluded by 60% and displaced from Caco-2 cells by 90%. Despite removal of the surface proteins, L. plantarum 423 was still capable of competitively adhering to Caco-2 cells and reduced adherence of C. sporogenes LMG 13570 by 50% and E. faecalis LMG 13566 by 70%.
Development of an antimicrobial wound dressing by co-electrospinning bacteriocins of lactic acid bacteria into polymeric nanofibers
(Stellenbosch : Stellenbosch University, 2012-12) Heunis, Tiaan de Jager; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Skin is the largest organ in the human body and serves as a barrier that protects the underlying tissue of the host from infection. Injury, however, destroys this protective barrier and provides a perfect opportunity for microorganisms to invade the host and cause infection, thereby affecting the normal wound healing processes. Furthermore, the ability of microbial pathogens to rapidly develop resistance towards a variety of antimicrobial compounds hampers the effective treatment and control of infections. Antimicrobial-resistant pathogens are increasingly being isolated from patients, placing a huge burden on the health care sector. The search for new and novel antimicrobial agents and treatments is thus of utmost importance and will continue to play an integral role in medical research. Antimicrobial peptides (AMPs) may serve as possible alternatives to antibiotics, or may be used in combination with antibiotics to reduce the risk of antimicrobial resistance. AMPs play a role in innate defence and are produced by a variety of mammals, plants, reptiles, amphibians, birds, fish and insects. The AMPs of bacteria (bacteriocins), especially those of lactic acid bacteria (LAB), are receiving increased attention as antimicrobial agents to treat bacterial infections. Electrospun nanofibers have characteristics that make them suitable as wound dressings, i.e. high oxygen permeability, variable pore size, high surface area to volume ratio and nanofibers are morphologically similar to the extracellular matrix. The ability to incorporate of a variety of biologically active compounds into nanofibers increases their potential as wound dressings. A novel approach would be to incorporate bacteriocins from LAB into nanofiber scaffolds to generate antimicrobial wound dressings. In this study, the feasibility of co-electrospinning bacteriocins from LAB into nanofibers was investigated. Plantaricin 423, produced by Lactobacillus plantarum 423, was successfully co-electrospun into poly(ethylene oxide) (PEO) nanofibers. Plantaricin 423 retained activity after the electrospinning process and continued to inhibit the growth of Lactobacillus sakei DSM 20017T and Enterococcus faecium HKLHS. Viable cells of L. plantarum 423 were also successfully co-electrospun into PEO nanofibers, albeit with a slight reduction in viability. A nanofiber drug delivery system was developed for plantaricin 423 and bacteriocin ST4SA, produced by Enterococcus mundtii ST4SA, by blending PEO and poly(D,L-lactide) (PDLLA) in a suitable solvent before electrospinning. Nanofibers were produced that released the bacteriocins over an extended time period. The PEO:PDLLA (50:50) nanofiber scaffold retained its structure the best upon incubation at 37 °C and released active plantaricin 423 and bacteriocin ST4SA. Nisin A was also successfully co-electrospun into a PEO:PDLLA (50:50) nanofiber scaffold and nisin A, released from the nanofibers, inhibited the growth of Staphylococcus aureus in vitro. Nisin A-containing nanofiber scaffolds significantly reduced viable S. aureus cells in infected skin wounds and promoted wound healing in non-infected wounds. As far as we could determine we are the first to show that bacteriocin-eluting nanofiber scaffolds can be used to treat skin infections and influence wound healing.
Expression of genes encoding bacteriocin ST4SA as well as stress proteins by Enterococcus mundtii ST4SA exposed to gastro-intestinal conditions, as recorded by real-time polymerase chain reaction (PCR)
(Stellenbosch : Stellenbosch University, 2007-03) Granger, Monique; Dicks, Leon Milner Theodore; Van Reenen, C.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The tolerance of Enterococcus mundtii ST4SA to stressful gastro-intestinal conditions in humans and animals is vital to its success as a probiotic. The need for new effective probiotics with stronger inhibitory (bacteriocin) activity has arisen due to the increasing number of antibiotic resistant pathogens. Enterococci are used in the fermentation of sausages and olives, cheese making and as probiotics. Their role as opportunistic pathogens in humans makes them a controversial probiotic (Moreno et al., 2005). Enterococci occur naturally in the gastro-intestinal tract which renders them intrinsic acid and bile resistance characteristics. E. mundtii ST4SA produces a 3950 Da broad-spectrum antibacterial peptide active against Gram-positive and Gram-negative bacteria, and viruses. The bacteria include Enterococcus faecalis, Streptococcus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae and Staphylococcus aureus. E. mundtii ST4SA inactivates the herpes simplex viruses HSV-1 (strain F) and HSV-2 (strain G), a measles virus (strain MV/BRAZIL/001/91, an attenuated strain of MV), and a polio virus (PV3, strain Sabin). This study focuses on the genetic stability of E. mundtii ST4SA genes when exposed to stress factors in the human and animal gastrointestinal tract. Based on results obtained by real-time PCR, the expression of genes encoding bacST4SA, RecA, GroES and 23S rRNA by E. mundtii ST4SA were not affected when the cells were exposed to acid, bile and pancreatic juice. This suggests that these genes of E. mundtii ST4SA will remain stable in the intestine. This could indicate that other genes of E. mundtii ST4SA could remain stable in the host. Further studies on the stability of genes encoding antibiotic resistance and virulence factors should be conducted to determine their stability and expression in the host in stress conditions. Concluded from this study, E. mundtii ST4SA is an excellent probiotic strain.
Gastrointestinal persistence of the probiotic bacteria Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, and their anti-listerial activity
(Stellenbosch : Stellenbosch University, 2018-12) Van Zyl, Winschau Fayghan; Deane, S. M.; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Gastrointestinal diseases, and in particular those caused by bacterial infections, are a major cause of morbidity and mortality worldwide. Treatment is becoming increasingly difficult due to the increase in number of species developing resistance to antibiotics. Different treatment strategies need to be developed. Probiotic lactic acid bacteria (LAB) have considerable potential as alternatives to antibiotics, both in prophylactic and therapeutic applications. Lactic acid bacteria have a long history of safe use in food and therapeutic products and is increasingly recognised for their beneficial effects. However, the underlying mechanisms by which probiotic LAB enhance the health of the consumer have not been fully elucidated. Demonstrating key antimicrobial and protective probiotic mechanisms in vivo will allow for industry and consumers to choose scientifically validated probiotics for the prevention or treatment of targeted gastrointestinal diseases. The present study aimed to contribute to this area of probiotic research. The first part of this study focus on monitoring the survival, metabolic activities and persistence of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA in the gastrointestinal tract (GIT) of mice using a bioluminescence imaging (BLI) system. The route and destination of both probiotic strains in the GIT were determined after single and multiple doses. Both strains prominently colonized the cecum and colon. Enterococcus mundtii ST4SA persisted in the GIT and faeces the longest and in higher numbers while also actively colonizing the small intestine. This is the first report of in vivo and ex vivo BLI of E. mundtii ST4SA in a murine model. The second part of the study encompassed the development of a novel system that facilitates the rapid and efficient isolation of double-crossover integration or deletion mutants of L. plantarum 423 and E. mundtii ST4SA. The system was useful in the construction of L. plantarum 423 and E. mundtii ST4SA bacteriocin and adhesion gene mutants. The newly described method expands the LAB molecular research genetic toolkit and has significant potential to allow genetic modification of most, if not all LAB species. This provides the unique opportunity to study the role of specific probiotic LAB genes in complex environments using reverse genetics analysis. In the final part of the study, the ability of L. plantarum 423 and E. mundtii ST4SA to competitively exclude L. monocytogenes EGDe, an intestinal pathogen, from the GIT of mice was proven. Valuable insight was gained on the molecular modes of action of the two probiotic strains. Plantaricin 423 and mundticin ST are bacteriocins produced by L. plantarum 423 and E. mundtii ST4SA, respectively. Bacteriocin-negative mutants of L. plantarum 423 and E. mundtii ST4SA failed to exclude L. monocytogenes EGDe from the GIT, confirming in situ bacteriocin production as an anti-infective mediator. Additional confirmation of in situ bacteriocin production as a mechanism of action was provided by using variant strains of L. monocytogenes EGDe expressing the immunity genes of plantaricin 423 and mundticin ST, respectively, which provided resistance to the respective bacteriocins. Furthermore, the exclusion of L. monocytogenes EGDe from the GIT was reduced when mice were administered with L. plantarum 423 and E. mundtii ST4SA adhesion gene knockout strains. These results substantiate our understanding of the functional attributes of probiotics currently available to consumers and the improvement of future probiotic products.
In vitro and In vivo characterization of Amyloliquecidin, a novel two-component lantibiotic produced by Bacillus amyloliquefaciens
(Stellenbosch : Stellenbosch University, 2015-04) Van Staden, Anton Du Preez; Dicks, Leon Milner Theodore; Deane, Shelly May; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Antimicrobial resistance is one of the major problems faced by the medical industry today. The ability of bacteria to rapidly acquire resistance against antibiotics and the over prescription and inappropriate use of antibiotics further exacerbate this crisis. Few new antimicrobials are, however, making it through the drug discovery pipeline. The search and development of novel and effective antimicrobials is therefore of the utmost importance. Lantibiotics are ribosomally synthesized cationic antimicrobial peptides with extensive post-translational modifications. They are active against a wide range of Gram-positive bacteria, including antibiotic-resistant strains. They are characterized by the presence of lanthionine and methyllanthionine rings and have been suggested as alternatives or for use in conjunction with antibiotics against resistant pathogens. Staphylococcus aureus is the most common bacteria isolated from skin and soft tissue infections (SSTIs). Strains of S. aureus have emerged with resistance against antibiotics with the most common being methicillin-resistant S. aureus (MRSA). Several lantibiotics are active against MRSA in vivo and have even shown superior activity to traditional antibiotics. Lantibiotics therefore show much promise for the treatment of SSTIs caused by resistant- and non-resistant S. aureus. In this study the bacterially diverse soil of the Fynbos in the Western Cape was screened for novel antimicrobials. Two antimicrobial producing Bacillus strains were isolated, Bacillus clausii AD1 and Bacillus amyloliquefaciens AD2. Both of these strains produce lantibiotics with B. clausii AD1 producing a known lantibiotic, clausin. B. amyloliquefaciens AD2 produces a novel two-component lantibiotic which was designated amyloliquecidin. The lantibiotic operon of amyloliquecidin was sequenced and annotated. All the genes required for successful production of amyloliquecidin are present in the operon. Amyloliquecidin was characterized in vitro and along with clausin is active against clinical strains of S. aureus (including MRSA), Enterococcus spp., Listeria spp. and beta-haemolytic streptococci. Amyloliquecidin has remarkable stability at physiological pH compared to nisin and clausin. A comparative in vivo murine infection model was used to evaluate the effectiveness of amyloliquecidin, nisin, clausin and Bactroban (commercial S. aureus topical treatment) in treating wound infections caused by S. aureus. All the lantibiotics proved to be just as effective as the Bactroban treatment. Furthermore, the tested lantibiotics did not have a negative influence on the wound closure rates of infected and non-infected wounds. Bactroban had a negative effect on wound healing compared to the lantibiotics. To our knowledge amyloliquecidin is the third two-component lantibiotic isolated from Bacillus. This study represents the first to test the effectiveness of amyloliquecidin in vivo and is one of a handful to test lantibiotics as topical treatments.
Isolation and identification of polysaccharide (gum)-producing bacteria from a sugarcane factory and strategies to prevent their growth
(Stellenbosch : Stellenbosch University, 2020-03) Nel, Sanet; Dicks, Leon Milner Theodore; Endo, A.; Davis, S. B.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Contrary to expectations, Leuconostoc mesenteroides was not the major gum-producing bacterium isolated from sugarcane. Instead, high cell numbers of W. confusa and W. cibaria were recorded. The dominance of Weissella spp. on the prepared cane is significant because these bacteria are not usually associated with deteriorated sugarcane and have not previously been isolated from a sugarcane processing factory. This study also report, for the first time, on the isolation of B. amyloliquefaciens from a sugarcane processing factory. The second aim of the study was to evaluate the efficacy of biocides to prevent/limit microbial growth in the factory. Two dithiocarbamate biocides, Busan®1021 and Preventol®Z, each at a 20 ppm dose, were tested against seven gum-producing bacteria. Preventol®Z demonstrated a bactericidal (killing) effect against Le. mesenteroides A16-9, Le. lactis B9-3, B. subtilis B7-19 and B. amyloliquefaciens B7-51 after 6 h of contact, but had only a bacteriostatic (growth inhibiting) effect on W. cibaria A1-17, W. confusa B1-24 and Lb. fermentum B19-18 when tested under the same conditions. Busan®1021 had a bactericidal effect on all seven species. Bacillus subtilis B7-19 and B. amyloliquefaciens B7-51 were susceptible to both biocides, but only for the first 2 h of exposure, after which the killing effect remained constant. Based on results obtained in this study, the concentrations of Preventol®Z and Busan®1021 may need to be increased, or dosage intervals altered, to kill all gum-producing bacteria. Although sugarcane processing factories have little to no control over the quality of cane entering the factory, poor sanitation in the factory and incorrect process control can also contribute to sucrose loss due to microbial activities and subsequent gum formation. Factories should therefore be mindful of correctly controlling high-temperature processes and reduce the recirculation of sump contents which are not treated with biocides. This study provided valuable knowledge on the identities of gum-producing bacteria in sugarcane processing factories, and their susceptibility to two commercial biocides.
Probiotic properties of lactic acid bacteria evaluated in a gastro-intestinal model and in in vivo pig trials
(Stellenbosch : Stellenbosch University, 2005-03) Mare, Louise; Dicks, Leon Milner Theodore; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: This study describes the use of a gastro-intestinal model to screen lactic acid bacteria isolated from the gastro-intestinal tract of post-weaned piglets (raised on six different diets) for probiotic properties. Intestinal bacteria were isolated from ,the stomach, duodenum, jejunum, caecum, ileum and colon. The highest cell numbers (6 x 107 cfulg) were isolated from the ileum. No significant differences in viable cell counts were recorded for piglets raised on the six diets. Isolates with the best overall probiotic properties were identified as members of Lactobacillus salivarius and Lactobacillus fermentum. The two strains selected for further studies were Lactobacillus plantarum 423 (originally isolated from sorghum beer) and Lactobacillus salivarius 241 (isolated from pig intestine). Enterococcus faecalis FAIR E 92 was originally isolated from pig intestine and was included in this study as a non-pathogenic challenge strain. L. plantarum 423 produces a bacteriocin plantaricin 423, active against E. faecalis FAIRE 92. L. plan/arum 423 and L. salivarius 241 were included in the gastro-intestinal model and their adhesion to the mucus of porcine ileum studied with fluorescent-in-si/u-hybridization (FISH). A decrease in viable cell numbers of L. plan/arum 423 was recorded in the duodenum, jejunum and ileum in the presence of bile and pancreatic juice. However, higher cell numbers were recorded in the caecum and anterior colon, which suggested that strain 423 recovered from these stress factors. Plantaricin 423 was detected for up to 28 hours in the duodenum, jejunum, ileum and middle colon. Lower cell numbers (one log unit) of L. salivarius 241 were recorded in the gastro-intestinal model over seven days, compared to strain 423. Piglets of one, 14 and 28-days-old were dosed with L. plan/arum 423 and L. salivarius 241, separately and in combination (1: 1). In a separate experiment, 14-day-old piglets were challenged twice with E. faecalis FAIRE 92, followed by dosage with strains 423 and 241. New-borne piglets dosed with L. plantarum 423 gained more weight (4 kg over 19 days) compared to piglets dosed with L. salivarius 241 (2.2 kg over 19 days), or a combination of the two strains (2 kg over 19 days). Piglets of 14 and 28-days-old, on the other hand, gained more weight when dosed with a combination of strains 423 and 241. The cell numbers of E. faecalis FAIR E 92 and other enterococci decreased drastically (two log units) when the piglets were dosed with the latter two strains. Overall, piglets of various ages reacted differently when administered L. plantarum 423 and L. salivarius 241, separately or in combination. Fluorescent-in-situ-hybridization (FISH) was used to study the in vivo adhesion of L. plantarum and L. salivarius to mucus in the stomach, duodenum, jejunum, ileum, caecum and colon. The highest number of L. plantarum cells was recorded in the ileum, whereas L. salivarius favoured adhesion to the duodenum. A decrease in cell numbers of E. faecalis in the ileum mucus was recorded when a combination of the probiotic strains 423 and 241 was administered. This study provided a reliable estimation of the presence and/or adhesion of L. plantarum and L. salivarius to various parts of the porcine gastro-intestinal tract, without the use of expensive cultivation techniques. Insight was gained into the co-evolution existing between probiotic bacteria and the porcine gastro-intestinal tract, emphasizing the use of gastro-intestinal models to study the dynamics of the gastro-intestinal tract.
Profiling of the secondary metabolites and the characterization two novel antilisterial peptides, xenopep and rhabdin, produced by xenorhabdus khoisanae
(Stellenbosch : Stellenbosch University, 2022-04) Booysen, Elzaan; Dicks, Leon Milner Theodore; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: In the early 1900’s the discovery of sulfonamides and penicillin paved the way for antibiotics and led to a boom in the discovery of other antibiotics. Unfortunately, this boom was short lived and soon the discovery and approval of novel antibiotics by the food and drug association and other similar associations dwindled. With the ever-increasing prevalence of antibiotic resistant pathogens this soon became a problem that was not foreseen. Most antibiotics currently on the market have been isolated from a select few genera. With nearly all the antibiotics from such few sources, bacteria were able to acquire resistance at an enhanced pace. This study focused on a relatively unexplored niche for novel antibiotics, from the genus Xenorhabdus. Species of this genus is mutually associated with Steinernema nematodes and have a unique life cycle. Xenorhabdus spp. are known to produce various secondary metabolites (SMs) that have antimicrobial, insecticidal, antiviral, immunosuppressant and proteolytic properties. Species from this genus use different synthesis machineries to produce these compounds, although the majority are produced via the non-ribosomal peptide synthesis. The ability of non-ribosomal peptides to incorporate non-proteogenic amino acids, D-amino acids, fatty chains, or polyketide chains result in unique resistance to proteinases and environmental stressors. Xenorhabdus khoisanae J194 is mutually associated with Steinernema jeffreyense J194, a nematode that was isolated from soil in the Eastern Cape. Culture conditions, especially oxygen, greatly affected SM production of X. khoisanae J194. PAX peptides, xenocoumacins and xenoamicins were identified in the cell-free crude extract of X. khoisanae J194 cultures. Two novel antilisterial peptides, xenopep and rhabdin, were also detected in the cell-free crude extract of. Xenopep has a narrow spectrum of activity and inhibited the growth of only, Listeria monocytogenes and Staphylococcus epidermidis, while rhabdin is active against both Gram-positive and Gram-negative bacteria. Xenopep and rhabdin share numerous characteristics and both contain a tetra-peptide in their structure including a tetra-peptide in their structure. Both peptides share the same amphipathic characteristic and behave similar suspension. Membrane potential and ATP release assays have shown that xenopep formed pores/lesions in the cell membrane of L. monocytogenes within minutes, followed by a rapid decrease in cell numbers over 3 hours. Scanning electron microscopy (SEM) images of L. monocytogenes treated with xenopep became elongated and formed filaments. This suggests that xenopep may inhibit penicillin binding protein three. This is the first study reporting on SMs produced by X. khoisanae when cultured under different conditions and is the first detailed description of antilisterial peptides produced by the species.
Safety of antibiotic and probiotic feed additives for Gallus gallus domesticus
(Stellenbosch : Stellenbosch University, 2018-09-05) Neveling, Deon Pieter; Dicks, Leon Milner Theodore; Smith, Carine; Pieterse, Elsje; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The inclusion of antibiotics in broiler feed is of great concern, as many resistant pathogenic bacteria may spread to other farming animals and humans. Alternative methods are thus required to improve broiler health and performance without detrimental consequences. The objective of this study was to evaluate the effect of a multi-species probiotic on the health and growth performance of Gallus gallus domesticus. Bacteria from different segments of the gastrointestinal tract (GIT) of healthy free-range broilers were isolated, identified to species level by amplifying the genes encoding 16S rDNA, recA and gyrB, and comparing the sequences with those listed in GenBank. A select few isolates were screened for probiotic characteristics. Among the 609 isolates sampled from the GIT, Lactobacillus johnsonii DPN184, Lactobacillus salivarius DPN164, Lactobacillus crispatus DPN167, Lactobacillus gallinarum DPN164, Enterococcus faecalis DPN94 and Bacillus amyloliquefaciens DPN123 tolerated acidic conditions (pH 2 to 3), were resistant to bile salts (0.2 to 2.0 % w/v) and produced exopolysaccharides. Bacillus amyloliquefaciens DPN123, isolated from the duodenum, produced extracellular amylase, phytase and antimicrobial lipopeptides (surfactin and iturinA1). Enterococcus faecalis DPN94, isolated from the jejunum and ileum, produced phytase and bile salt hydrolase. The genome of E. faecalis DPN94 contained several genes that may encode virulence, but not the production of cytolysin. Differences in opinion exist regarding the role virulence genes may play in the colonisation of epithelial cells. Lactobacillus johnsonii DPN184, isolated from the cecum, produced hydrogen peroxide. Lactobacillus salivarius DPN181, isolated from the colon, produced hydrogen peroxide and high levels of lactic acid. Lactobacillus crispatus DPN167 was isolated from the crop, proventriculus and ventriculus, and produced hydrogen peroxide and bile salt hydrolase. Lactobacillus gallinarum DPN164 was isolated from the jejunum and ileum. A multi-species probiotic, consisting of L. johnsonii DPN184, L. salivarius DPN164, L. crispatus DPN167, L. gallinarum DPN164, E. faecalis DPN94 and B. amyloliquefaciens DPN123, was added to the feed of broilers and its effect on growth performance, size of the lymphoid organs, gizzard mass, mineral content of the tibia bones and red blood cell parameters determined. A separate group of broilers was administered a combination of sulphadiazine, colistin and trimethoprim through their feed and subjected to the same tests. A third group of birds received standard feed without additives and served as control. On day 19, the villi of broilers on antibiotics had larger surface areas, and higher lymphocyte and basophil counts compared to broilers from probiotic and control treatment groups. On day 29, the cecal microbiome of broilers from the control and probiotic treatment groups were similar but differed significantly from broilers that received antibiotics. Probiotic administration did not alter homeostasis of the normal GIT microbiome, suggesting that probiotics rather modulate the microbiome by preventing dysbiosis induced by pathogenic microorganisms. Birds on antibiotics had lower levels of Enterobacteriaceae and higher levels of unidentified Clostridiales, Brucellaceae, Synergistaceae, Erysipelotrichaceae and Coriobacteriaceae in their ceca. The multi-species probiotic repressed the growth of Listeria monocytogenes EDGE in vivo, most probably by lowering the cell’s metabolic activity, by competing with Listeria for receptor sites on the gut wall or mucosa, or by production of antimicrobial compounds such as short-chain fatty acids, hydrogen peroxide and lipopeptides. Salmonella Enteritidis 147 invaded Caco-2 cells and altered claudin-3 tight junctions between the cells, leading to monolayer disruption. Salmonella decreased tight junctions by invading eukaryotic cells which led to cell death. Interaction of S. Enteritidis with broiler epithelial cells led to the up-regulation of lysozyme C and G, cathelicidin 2 and 3, myeloid protein 1, trypsin inhibitor CITI-1, gallinacin-2 and ubiquitin-fold modifier 1, and the down-regulation of glutaredoxin-1, gallicin-7 and vigilin. Up-regulated proteins acted as chemotactic compounds, inhibitors of microbial enzymes, and played critical roles during stress. Down-regulated proteins activated natural killer cells, and regulated apoptosis and antimicrobial defence systems. The multi-species probiotic was not cytotoxic, but the metabolic end products were. The probiotic bacteria adhered to Caco-2 cells but did not invade them, and decreased claudin-3 tight junctions but did not disrupt the monolayer. Probiotics decreased claudin-3 tight junctions by producing short-chain fatty acids, hydrogen peroxide and antimicrobial lipopeptides. In broilers administered with the multi-species probiotic, transgelin 2/3, elongation factor-1 beta and anterior gradient 2 were up-regulated, but carnitine O-acetyltransferase, adenylate kinase 2, superoxide dismutase Cu-Zn and protein SET down-regulated. Upregulated proteins were involved in the proliferation, migration and healing of cells and regulation of the cytoskeleton, whereas down-regulated proteins were important in fatty acid transport, energy homeostasis, nucleotide metabolisms, free-radical elimination and signal transduction. Concluded from these studies, the multi-species probiotic was non-toxic and interacted with epithelial cells in a symbiotic manner. Feeding of Salmonella enterica serovar Enteritidis A9 to broilers had no effect on body mass, and no significant differences were observed with respect to immune organ weights, haematological parameters and serum interferon gamma levels. Colonisation of Salmonella in the cecum of broilers that received oxytetracycline was, however, lower on days 11 (one day post infection, dpi 1) and 14 (dpi 4), but then increased to levels corresponding to those of birds in the control and probiotic groups. At first, the antibiotics decreased the cell numbers of Salmonella in the cecum, but higher levels were recorded with continuous administration. The increase in cell numbers may be due to antibiotics disturbing the microbiome in the GIT, indirectly favouring the colonisation of Salmonella. On day 29 (dpi 19), the cell numbers of Salmonella in the cecum of broilers administered with the multi-species probiotic were similar to those of infected and uninfected birds. Broilers that received oxytetracycline displayed higher serum bactericidal activity against Salmonella on day 11 (dpi 1) compared to birds from the probiotic and control groups. In addition, on day 29 (dpi 19) birds on probiotics had higher serum bactericidal activity against Salmonella than birds in the control group. Broilers receiving the multi-species probiotic had higher levels of lysozyme in their serum on day 11 (dpi 1) compared to uninfected broilers. Broilers receiving the antibiotic and probiotic had higher T lymphocyte responses compared to broilers from the control treatment groups on day 17 (dpi 7). These results suggested that antibiotic and probiotic feed additives stimulated the immune response of broilers infected with Salmonella. The designed multi-species probiotic possessed numerous beneficial characteristics and its daily use as a feed additive was deemed safe, as probiotic use did not negatively affect the performance of healthy birds. The probiotic strains adhered to intestinal epithelial cells and crosstalk between these cells did not induce negative proteomic changes. The multi-species probiotic also increased broiler immune responses during Salmonella infection, which suggests that the strains may be used as an alternative feed additive to improve broiler health and performance.
Survival of probiotic lactic acid bacteria in the intestinal tract, their adhesion to epithelial cells and their ability to compete with pathogenic microorganisms
(Stellenbosch : Stellenbosch University, 2008-03) Botes, Marelize; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Research on probiotics has increased over the past years, which led to commercialization of a number of probiotic supplements and functional foods. In vitro assays such as tolerance to acid and bile, adhesion to mucus and epithelial cells, antimicrobial activity and antibiotic resistance tests are performed to screen lactic acid bacteria for probiotic properties. Enterococcus mundtii ST4SA produces an antimicrobial peptide (peptide ST4SA) with activity against Gram-positive and Gram-negative bacteria. Lactobacillus plantarum 423 produces plantaricin 423, a typical class II bacteriocin, active against a number of Gram-positive bacteria. A gastro-intestinal model (GIM) simulating the gastro-intestinal tract (GIT) of infants, was developed to study the survival of E. mundtii ST4SA and L. plantarum 423 and evaluate them as possible probiotics. Growth of the two strains in the GIM was compared to the growth of commercially available probiotics. Infant milk formulations were used as growth medium. Changes in pH, the addition of bile salt and pancreatic juice, and intestinal flow rates were controlled by peristaltic pumps linked to a computer with specifically designed software. Strain ST4SA was sensitive to low pH and high concentrations of bile salts. Growth of strain ST4SA was repressed in the first part of the GIM, however, the cells recovered in the ileum. Strain 423 was also sensitive to acidic conditions. However, the cells withstood the presence of bile and pancreatin in the first part of the GIT. Neither of the two strains displayed bile salt hydrolase (BSH) activity. Both strains were resistant to amoxicillin, ampicillin, chloramphenicol, cefadroxil, roxithromycin, meloxicam, doxycycline, erythromycin, novobiocin, rifampicin, tetracyclin, bacitracin, oflaxacin and cephazolin, anti-inflammatory drugs Na+- diklofenak and ibuprofen, and painkillers codeine terprim hydrate aminobenzoic acid, metamizole aspirin and paracetamol. Strain 423 was resistant to ciprofloxacin. Genes encoding cytolysin, non-cytolysin β-hemolysin and cell aggregation substances were detected on the genome of strain ST4SA but they were not expressed. L. plantarum 423 does not contain genes encoding gelatinase, cell aggregation, enterococcus surface protein, hemolysin, non-cytolysin β- hemolysin and enterococcus endocarditis antigen. Both strains inhibited the growth of Listeria monocytogenes ScottA in the GIM. Survival of the strains improved when used in combination and compared well with the survival of commercially available probiotics. Adhesion to epithelial cells is an important prerequisite for bacterial colonization in the GIT. The adhesion of E. mundtii ST4SA and L. plantarum 423 was studied using Caco-2 (human colon carcinoma epithelial) cells. Both strains revealed good adhesion compared to other probiotic strains. No correlation was found between hydrophobicity, auto-aggregation and adhesion to Caco-2 cells. Antibiotics and anti-inflammatory medicaments had a negative effect on adhesion. Different combinations of proteins were involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. E. mundtii ST4SA, L. plantarum 423 and L. monocytogenes ScottA were stained with fluorescent dyes to visualize adhesion to Caco-2 cells. Adhesion of L. monocytogenes ScottA to Caco-2 cells was not reduced in the presence of strains ST4SA and 423. Cell-free culture supernatants of both strains inhibited the invasion of L. monocytogenes ScottA. The cell structure of Caco-2 cells changed in the presence of L. monocytogenes ScottA. Strains ST4SA and 423 protected Caco-2 cells from deforming.
Therapeutic properties of the lantibiotic nisin F
(Stellenbosch : Stellenbosch University, 2013-03) Brand, Anneke Mari; Dicks, Leon Milner Theodore; Smith, Carine; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Bacterial resistance against antibiotic treatments is a global concern and resistance to almost every known antibiotic has already been reported. There is thus a significant need for the development of novel antimicrobial drugs. In addition to probiotic traits, certain bacteria have the ability to produce antimicrobial peptides, referred to as bacteriocins. Lantibiotics, a group of small ribosomally synthesized bacteriocins, recently gained interest for their application in the medical field. Lantibiotics have a very specific structure, including lanthionine rings, that stabilise the peptides. Due to their small size and specific action, these peptides reach specific sites of infection without affecting the composition of the host’s natural microbiota. As with any therapeutic agent, antimicrobial peptides are also prone to in vivo degradation, binding, clearance via immune action and development of bacterial resistance. Nisin F, a class Ia lantibiotic produced by Lactococcus lactis subsp. lactis F10, has already shown activity against the well-known pathogens Stapylococcus aureus, Listeria monocytogenes and various antibiotic resistant strains. The aim of this study was to assess the antimicrobial activity of nisin F against systemic S. aureus infections in mice and possible immune responses elicited by the peptide. A single administration of nisin F to the peritoneal cavity protected mice from S. aureus infection for at least 15 min. After continuous administration, the peptide showed no significant antimicrobial activity against S. aureus. The peptide did, however, convey some degree of protection to infected mice by stimulating a pro-inflammatory action through lymphocyte protection. When administered to uninfected mice, nisin F had an immune boosting effect via interleukin (IL)-6 and IL-10 without being detrimental to the host. The ex vivo effects of nisin F was compared to nisin A, a natural nisin variant, and Nisaplin®, a commercially purified form of nisin A. None of the three peptides inhibited the functional capacity of leukocytes in terms of 1L-1β en IL-6 production, not even in the presence of an external stimulus (lipopolysaccharides from Escherichia coli). Cytotoxicity was detected in response to high dosages of nisin F. Serum inhibited the antimicrobial effect of nisin F and nisin A, but Nisaplin® remained unaffected. Nisin F was applied against systemic infection for the first time and the immunological effect of the peptide was investigated. Nisin F partially protected mice against S. aureus infections through immunomodulatory effects. This study provided valuable knowledge on the in vivo application of nisin F. With further optimization of nisin F preparation and application systems, the peptide might be more effective against in vivo infections.

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Browsing Doctoral Degrees (Microbiology) by browse.metadata.advisor "Dicks, Leon Milner Theodore"

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