OpenUCT :: Browsing by Author "Abratt, Valerie Rose"

Browsing by Author "Abratt, Valerie Rose"

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Open Access
Bacteriodes fragilis interactions with the collagen type I component of the host extracellular matrix
(2010) Galvao, Bruna Patricia Gomes de Vasconcelos; Abratt, Valerie Rose
Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The increasing incidence of antibiotic resistance by the bacterium has prompted the search for novel therapeutic targets. The idea of antivirulence over antimicrobial drug development is becoming increasingly popular and invites detailed research into the virulence factors of target pathogens. The ability to adhere to, and degrade components of the extracellular matrix, such as collagen, is critical in bacterial colonisation and host cell invasion. The aim of this dissertation was to specifically identify the collagen type I adhesins and proteases in the common research strain, B. fragilis 638R, as well as in 23 clinical isolates from Groote Schuur Hospital (GSH), Cape Town.
Open Access
The classroom transferability of a university-based inset programme of workshops in practical work for senior high school Biology educators
(2006) Joubert, Nicola; Rochford, Kevin; Abratt, Valerie Rose
Professional criteria for assessing (a) the success and transferability of the programme and (b) the quality of the research evidence gathered from the Biology teachers and their learners in Cape Town, were adopted from a combination of the theoretical frameworks for INSET evaluation recommended by several authors, including the American National Science Standards (1996), Tamir (1997) and Oyasi & Oyasi (2000). The post-workshop data indicated that educators enjoyed the practical activities, and were active in implementing a number of them with their classes in subsequent years. It further revealed that their confidence in engaging in practicalwork improved significantly. To verify or corroborate these findings, ten educators observed at from the 1999 course were interviewed from 2002 to 2005. One educator was observed at two different schools, with different socio-economic backgrounds. The interviews were transcribed and five of the educators were observed while they dealt with the practical activities learnt during this series of workshops. The visual data from the classroom observations, and the interviewswere further processed and compared to the quantitative statistical data. It was It was found that, of the eight schools, four well-resourced schools implemented the programme successfully. This was in terms of the number of practical activities from the course that had been transferred to the classroom. Three of the under-resourced schools, with larger classes, also implemented the course successfully. This was due to the skills and motivation which the educators gained whilst participating in this series of workshops. In two of the schools the high rate of vandalism and the heavy workload of the educators was excessive to the point that they could not implement the programme successfully.
Open Access
A clinical and molecular analysis of Clostridium difficile strains isolated from Groote Schuur Hospital
(2015) Brock, Tunehafo Elisabeth; Abratt, Valerie Rose; Reid, Sharon J
A clinical and molecular analysis of Clostridium difficile isolated from symptomatic patients at Groote Schuur Hospital was conducted in order to gain insight into the identity, epidemiology and pathogenesis of the various strains. C. difficile was detected and isolated by selective culture from stool specimens from 34 of the 162 symptomatic patients (20%). Three toxigenic-types were distinguished by PCR: A+B+ (47%), A-B+ (47%) and A-B- (6%), none of which harboured the binary toxin genes. Compared to the direct culture method, enzyme immunoassay-based detection tests (Meridian ImmunoCard and bioMérieux MiniVidas) were found to be lacking clinical sensitivity, while nucleic acid amplification tests (Hain Lifescience CDiff and Cepheid GeneXpert) were far more sensitive in the local clinical setting. PCR ribotyping identified all the A-B+ strains as PCR ribotype 017, which was the prevalent strain type (47%). Genotyping based on the tcdC gene and MLVA both grouped the ribotype 017 strains in a single clade. The antimicrobial susceptibility of all the isolates to metronidazole (MET), vancomycin (VAN), moxifloxacin (MOX) and erythromycin (ERY) were determined by the Etest method. All were sensitive to MET and VAN; however, four ribotype 017 strains displayed reduced susceptibility to MET. With regard to MOX, reduced susceptibly and full resistance were observed in 32% and 12% of the isolates, respectively, with all of these belonging to ribotype 017. MOX-resistant strains had a Thr82->Ile amino acid substitution in the GyrA enzyme and strains displaying reduced susceptibility to MOX had an Asp426->Asn amino acid substitution in GyrB. High-level resistance to ERY was observed in 47% of the isolates, which were primarily ribotype 017. ERY-resistant strains all harboured the ermB gene, suggesting that this was the genetic basis of the observed phenotype. Auto-aggregation analysis revealed that the ribotype 017 strains were significantly stronger auto-aggregators than the other ribotypes examined. Results of semi-quantitative RT-PCR analysis suggest that the expression level of the cwpV gene, encoding the CwpV protein, may play a role in auto-aggregation. In conclusion, this pilot study revealed that the GeneXpert method was the most accurate and sensitive technique for diagnosing CDI in the clinical setting at Groote Schuur Hospital. Ribotype 017 was the most prevalent strain type, and the antimicrobial resistance profile and increased auto-aggregation capacity of this ribotype may contribute to its high prevalence.
Open Access
The cloning and characterization of a Butyrivibrio fibrisolvens H17c glnA regulatory element in E. coli
(1997) Samsodien, Anwar; Abratt, Valerie Rose
Butyrivibrio fibrisolvens HI 7C is an important anaerobic bacterium which occurs in the rumen of most ruminants. A key factor affecting the growth of this bacterium is the availability of nitrogen sources, particularly in the form of ammonia. The aims of this study were to attempt the isolation of a gene/genes involved in the regulation of the B.fibrisolvens type III glutamine synthetase (GS), which is a key enzyme involved in ammonia assimilation in rumen bacteria. An existing B. fibrisolvens gene bank, as well as the B. fibrisolvens glnA gene cloned onto a low 11 copy number plasmid, were used to generate a heterologous, Escherichia coli- based, two plasmid, in trans system. This system was used to isolate an E. coli clone which showed increased GS activity levels (2.5-fold) and a retarded growth rate phenotype in complete media.
Open Access
Crystal structure of the Large Type III Glutamine Synthetase from Bacteroides Fragilis
(2010) van Rooyen, Jason M; Sewell, Bryan Trevor; Abratt, Valerie Rose
Glutamine synthetases are one of the most ancient functioning enzymes in existence and these large oligomeric complexes are found in all extant forms of life where they play a critical role in nitrogen metabolism. Over the past five decades, extensive biochemical studies together with structural investigations have helped build a picture of the mechanism of functioning and regulation in the GSI and GSII families. The most divergent GSIII family, however, is poorly characterized and has only recently been recognized. Structural studies, using both cryo-EM and X-ray crystallography, were undertaken on the type III GS, GlnN, from the opportunistic human pathogen, Bacteroides fragilis, with a view to better understanding the GSIII family in the light of the known structure functionrelationships of the other GS enzymes, and to investigate the potential for the design of selective inhibitors against the divergent family. A low-resolution (16 Ã) reconstruction of GlnN was first determined by single particle cryo-EM and image processing. This structure revealed that GlnN was a double-ringed dodecamer with D6 symmetry and the arrangement of active sites within the hexameric rings closely matched the GSI structure. Following the design of a rapid purification protocol and improvements to the stability and solubility of GlnN, conditions were discovered for the production of diffraction quality inhibitor-bound crystals. A second better diffracting crystal form was also produced following proteolytic processing. The crystal structure of GlnN was solved to near atomic resolution (3.0 Ã) following phase extension of low-resolution SAD phases, taking into account the cryo-EM structure. The higher resolution of the crystal structure revealed that, surprisingly, the orientation of the hexameric rings in GlnN is inverted in comparison to other families. These results have raised interesting questions surrounding the mechanism and driving forces responsible for the evolution of quaternary structure in the GS enzymes and have suggested that the GSI and GSII structure arose following truncation of a large GSIII-like ancestor. Despite the differences in higher order assembly, the GlnN monomer displayed a high degree of similarity with the GSI and GSII structures in the core active site region, thus, suggesting a conservation of reaction mechanism. Structure-based multiple sequence alignment showed that the residues forming the nucleotide binding pocket are the least conserved in the GS superfamily, and several residue positions, which represent altered modes of ligand binding, were suggested as potential avenues for the design of selective inhibitors against GlnN.
Open Access
DNA repair in bacteroides fragilis
(2008) Steffens, Laura Sione; Abratt, Valerie Rose
Bacteroides fragilis is a gut commensal in both humans and animals where it benefits the host through metabolizing indigestible compounds, stimulating the immune system and protecting against pathogen colonization. However, it is also an opportunistic pathogen, responsible for approximately half of anaerobic bacteraemias. Metronidazole is used to treat anaerobic infections. It diffuses into the celI as an inactive prodrug where it is reduced to form nitro anion and nitroso and hydroxylamine radicals. These chemically reactive compounds interact with DNA causing strand breaks and base mutations; the damage accumulates and leads to cell death. Mechanisms of metronidazole resistance in B. fragilis include decreased activity of oxidation/reduction enzymes, over-expression of multidrug efflux pumps and the conversion of metronidazole to non-toxic derivatives by nitroimidazole nitroreductases (encoded by nim genes). However, metronidazole resistance could also potentialIy be mediated by the over-expression or enhanced activity of DNA repair proteins. Thus, DNA repair in B. fragilis should be thoroughly investigated.
Open Access
DNA repair in Bacteroides fragilis Bf-2
(1987) Abratt, Valerie Rose; Woods, David R; Jones, David T
Repair deficient mutants of Bacteroides fragilis have been isolated in order to study the responses of this organism to various DNA damaging agents at the physiological and molecular levels. Two types of mutants were isolated by ethyl methane sulphonate mutagenesis of B.fragilis followed by selection for sensitivity to mitomycin C. One mutant (UVS9) showed sensitivity to both mitomycin C and far-UV irradiation. The other (MTC25) was more sensitive to mitomycin C than UVS9, but showed wild-type resistance to UV radiation. Both mutant strains had wild-type resistance to methyl methane sulphonate.
Open Access
The effect of metronidazole on Bacteroides fragilis and Escherichia coli
(1992) Dachs, Gabriele Ursula; Abratt, Valerie Rose; Woods, David R
The antibiotic metronidazole is used extensively in the clinical treatment of anaerobic infections, including those caused by the anaerobic pathogen Bacteroides fragilis. Metronidazole is an inert substance that requires reductive activation to become cytotoxic. In its activated form metronidazole induces DNA damage. Relatively little is known about the cytotoxic effects of this drug in vivo. The aim of the work reported in this thesis was to analyze the mode of action of metronidazole in living systems. Furthermore, the potential for bacterial cells to develop resistance mechanisms to metronidazole is largely unknown, and therefore the role played by B. fragilis genes in influencing the potency of metronidazole was investigated. Bibliography: pages 172-201.
Open Access
Evaluating oxalate-degrading Lactobacillus spp. for their ability to be used as probiotics in the treatment of kidney stone disease
(2010) Kabanda, Siti M; Abratt, Valerie Rose; Reid, Sharon J
Although the direct cause of kidney stone formation is not known, reports have suggested it is probably a multifactorial disease. Lactobacillus strains which potentially had increased ability to degrade oxalate were previously isolated from a healthy low kidney stone risk group. The aim of this study was to identify these natural Lactobacillus strains and evaluate their potential for use as probiotics in reducing the risk of kidney stone disease. Identification was achieved by PCR amplification and sequencing of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer (ITS) region. The strains were identified as follows; Lactobacillus gasseri 7(3), L. gasseri 17(4), Lactobacillus reuteri 17(7) and L. reuteri 16(9). Their probiotic characteristics were also evaluated.
Open Access
Evaluation of five bifidobacterium isolates as potential probiotics and genetic analysis of their ability to withstand oxidative stress
(2010) Nonyane, Molati Albert; Reid, Sharon J; Abratt, Valerie Rose
The diverse microbiota of the human gastrointestinal tract plays a major role in the general health of humans. A number of bacterial strains with distinctive properties have been isolated and used commercially as probiotics in order to harness these health benefits and offer them to unhealthy hosts. There are set criteria that have to be followed before new probiotics can be introduced into the market. Five Bifidobacterium isolates with extremely high sucrase activity were randomly selected from a faecal sample from a healthy donor for further characterisation as potential probiotics with the ability to utilize fructo- oligosaccharide substrates in the gut. Phylogenic identification of the isolates to the species level was carried out using sequences of the 16S rRNA gene, 16S-23S rRNA gene spacer region, the heat shock protein (hsp60) and Elongation factor Tu (tuf).
Open Access
Genetic effects of prolonged UV-B exposure in a Namaqualand daisy - Dimorphotheca sinuata
(2001) Mpoloka, Sununguko Wata; Thomson, Jennifer Ann; Abratt, Valerie Rose; Mundree, Sagadevan G; Riddoch, Bruce
This thesis describes investigations into the genetic effects of long term UV-B exposure in Namaqualand daisies (Dimorphotheea sinuata) grown for several generations under ambient and enhanced UV-B levels. Enhanced UV-B radiation was found to have a major effect on the biochemical composition of the chloroplast accompanied by impairment of photosynthetic function, involving a down-regulation of photosynthetic genes and an up-regulation of flavonoid biosynthesis.
Open Access
Glutamine synthetase in Bacteroides fragilis
(2007) Tumba, Nancy; Abratt, Valerie Rose
Bactereroides fragilis is a gram-negative, non spore-forming, obligate anaerobe of the human intestinal microbiota. It is, however, an opportunistic pathogen and has been ranked as the most prevalent isolate in cases of anaerobic septicaemia. Similar to most bacteria, ammonium is assimilated in B. fragilis through the action of glutamine synthetase (GS). Glutamine is vital to nitrogen metabolism as it serves as a precursor to many secondary metabolites. GS enzymes are, therefore, vital to the growth of the organism and many prokaryotes are known to possess two or more isoforms of the enzyme. In addition, GS expression and regulation is usually tightly regulated in concert with the availability of nitrogen. Previous studies have identified a single GSIII encoding gene (glnN) in B. fragilis. In this dissertation, an additional ORF coding for a putative GSI enzyme in B. fragilis was identified, isolated and functionally characterized. A putative regulatory protein was also identified and its functional contribution to nitrogen metabolism was determined, in order to extend our understanding of nitrogen assimilation in B. fragilis.
Open Access
Identification of Bacteroides genes involved in Metronidazole resistance
(2004) Casanueva, Ana; Abratt, Valerie Rose
Bacteroides species are Gram-negative obligate anacrobes that live in the gastrointestinal tract of mammals and are thought to account for approximately 30% of the colonic microbiota. Certain Bacteroides species, such as B. fragilis and to a lesser extent B. thetaiotaomicron, can become opportunistic pathogens and cause severe infection. The antibiotic of choice for treating such infections is metronidazole, a DNA damaging agent. Metronidazole enters the bacterial cell as an inert prodrug, and is activated by cellular reduction into a cytotoxic compound which is thought to cause DNA strand breaks. Certain metronidazole resistant B. fragilis strains have been described, where the drug was not reduced inside the cell due to decreased activity of the metabolic enzymes which are involved in this process. Little is known about the mechanisms involved in repair of metronidazole damage and the potential for resistance. In this study, two difIerent approaches were used to isolate and analyse Bacteroides genes involved in metronidazole resistance, with emphasis on DNA repair genes. These methods were transposon mutagenesis of Bacteroides, and functional complementation of E. coli metronidazole sensitive mutants with genes from B. fragilis.
Open Access
Metronidazole resistance in clinical Bacteroides fragilis isolates from Groote Schuur Hospital, Cape Town, South Africa
(2014) Meggersee, Rosemary Lauren; Abratt, Valerie Rose
Bacteroides fragilis, an anaerobic gut commensal and opportunistic pathogen, is a leading cause of anaerobic abscesses and bacteraemias. Treatment of infections is complicated by the emergence of resistance to several of the antibiotics, specifically metronidazole, used in the clinical setting. The aim of this thesis was to examine the levels of antibiotic resistance of 23 B. fragilis strains isolated at Groote Schuur Hospital, Cape Town, and determine the metronidazole (Mtz) resistance mechanisms present in order to evaluate the clinical risk of the spread of drug resistance. It also reports the identification and functional characterisation of a putative, novel nim gene in the B. fragilis 638R genome. Measurement of the minimum inhibitory concentration of the strains to antibiotics showed that 8% were highly resistant to imipenem and cefoxitin and 65% to tetracycline. All strains were sensitive to clindamycin. Two strains, B. fragilis GSH8 and GSH15 were Mtz resistant and strain GSH15 showed multidrug resistance to metronidazole, imipenem, cefoxitin and tetracycline. The genetic basis of the resistance to the various antibiotics was examined and could largely be attributed to the presence of previously published resistance genes. There were several exceptions to this which were investigated further at the genetic level with particular focus on imipenem and Mtz. In the cases of Mtz resistance, PCR screening did not detect any of the known nim genes but both strains showed increased lactate dehydrogenase (LDH) activity suggesting the involvement of the LDH/ pyruvate: ferredoxin oxidoreductase (PFOR) pathway. However, there was no reciprocal decrease in PFOR activity so the LDH/ PFOR pathway was not involved in the observed Mtz resistance. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that induction by exposure to sub-lethal doses of Mtz caused a slight increase in transcription of the efflux gene bmeB5. However, this was not statistically significant and still did not account for the observed Mtz resistance. A putative nim-like gene was identified on the B. fragilis 638R genome and was present in most of the strains examined. Phylogenetic and three dimensional analysis of the derived amino acid sequence revealed that the 638Rnim was more closely related to NimA from Deinococcus radiodurans (drNimA) than to any of the other nim genes. Heterologous overexpression of 638Rnim in an Escherichia coli Mtz sensitive mutant resulted in a 3-fold increase in Mtz resistance as compared to the control (6 vs 2 mg/L respectively). However, its possible role in Mtz resistance in B. fragilis could not be confirmed by overexpression or interruption of the gene in the homologous host. qRT-PCR showed that increased transcription of the gene approached statistically significant levels upon Mtz induction. This nim-like gene, therefore, warrants further investigation under a range of different induction conditions. The findings of this research provide useful information on the state of B. fragilis antibiotic resistance present in Groote Schuur Hospital and the incidence of the associated resistance genes. It also identifies and presents a preliminary characterisation of a putative novel nim gene as a basis for further functional studies on Mtz resistance mechanisms.
Open Access
Molecular characterisation of selected gastrointestinal microbiota in South African HIV-positive patients during HAART
(2012) Du Plessis, Sarah Jane; Abratt, Valerie Rose; Reid, Sharon J
Progression of the HIV disease is characterised by a massive depletion of CD4+ T cells and it has been shown that patients living with a more advanced HIV infection have a higher risk of developing diarrhoea due to the disruption of the gastrointestinal microbiota caused by either the HIV-infection or the use of antibiotics and drugs such as highly active antiretroviral therapy (HAART). An imbalance in the microbial composition, attributable to a disturbed mucosal barrier, as well as increased permeability and inflammation caused by HIV, can influence the metabolic (carbohydrate fermentation) and protective functions provided by the microbiota. The effect of HIV on the intestinal microbiota has not been widely examined and those studies that have focused on HIV and the gastrointestinal tract, have investigated it mainly from a virological perspective. Consequently, the aim of the study was to ascertain whether the diversity and/or abundance of the endogenous intestinal microbiota of South African HIV-positive patients was disrupted on account of HIV within the gastrointestinal tract. An additional aim was to determine whether the administration of HAART affected the microbiota during a 6 month longitudinal study. The diversity of the intestinal microbial composition was characterised with respect to the total bacteria, Bifidobacterium and Lactobacillus species using PCR-DGGE. qPCR was used to determine the abundance of total bacteria, Bifidobacterium, Lactobacillus, Escherichia coli, the Bacteroides/Prevotella, Clostridium coccoides and Clostridium leptum groups. ... In addition, three potential intestinal pathogens (Clostridium difficile, Campylobacter jejuni and Salmonella enterica) were monitored by qPCR during this period, to determine their prevalence in the HIV-positive patients.
Open Access
The molecular characterisation of the recA locus in the opportunistic pathogen Bacteroides fragilis
(2012) Nicholson, Samantha Anne; Abratt, Valerie Rose
Bacteroides fragilis is a human gut commensal and an opportunistic pathogen causing anaerobic abscesses and bacteraemias which are treated with the drug, metronidazole, a DNA damaging agent. The RecA protein is thought to be involved in the repair of metronidazole damage as well as damage caused by oxidative stress. The ability to survive oxygen stress is a strong indicator in an anaerobic bacterium of pathogenic potential and bacterial persistence in the oxygen rich peritoneal cavity. The aim of this thesis was to characterise the B. fragilis recA gene cluster with respect to its genomic context and the transcriptional regulation of the genes in response to metronidazole and oxygen stress. The possible functional roles of the proteins encoded by these genes in protection against these processes would also be evaluated. The functional characterisation of the RecA protein from B. fragilis showed that it was important for survival after exposure to nitrogen (metronidazole) and oxygen (hydrogen peroxide) radicals. RecA was shown to be important for the maintanence of genomic integrity even under normal growth conditions, and overexpression of this protein was shown to be important for increased survival after exposure to metronidazole. RT-PCR of B. fragilis cDNA showed that the recA gene was co-transcribed as an operon together with two upstream genes. Bioinformatic analysis revealed that the first ORF, BF638R1248, was a putative saccharopine dehydrogenase gene (sdh), encoding the SDH protein which may be involved in lysine degradation. The second ORF, BF638R1246/7 had homology to bcp genes, and encoded a putative Bactoferritin co-migratory protein (BCP) belonging to the thiol specific antioxidant superfamily. The functional roles of these proteins suggested that they might also be involved in survival after univalent electron stress. Quantitative RT-PCR showed that all three genes were transcriptionally regulated, but at different levels, after exposure to either metronidazole or H2O2. This suggests that in addition to being expressed as an operon, the genes may also possess independent regulatory elements. Functional characterisation of sdh and bcp was done using a gene mutation approach. Both insertional and deletion mutation methods were attempted but neither produced viable mutants in either of the upstream genes, suggesting that they may be critical for the survival of B. fragilis under normal growth conditions. The use of heterologous gene expression was subsequently employed to establish the functional role of the bcp gene and the encoded putative BCP. A similar approach for SDH was not successful. Heterologous complementation and protein expression of BCP in E. coli, with subsequent biochemical assay, showed that the B. fragilis bcp gene encoded a functional bacterioferritin co-migratory protein (BCP), which is a small thiol-specific protein with antioxidant properties. This BCP showed flexibility in its substrate preference with activity against H2O2, tet-butyl hydroperoxide and linoleic acid. The peroxidase activity of this TSA protein was dependent on the presence of one or more members of the thioredoxin group and NADPH. The BCP aided protection of the enzymatic activity of the B. fragilis redox sensitive Fe-S metalloenzyme Glutamine synthetase (GSIII) during exposure to 100 Î¼M H2O2. There was also evidence to suggest that it aided the recovery of the enzymatic activity of GSIII after exposure to 100 Î¼M H2O2. The findings of this research have resulted in the following hypothesis: The recA operon of B. fragilis acts during host invasion to contribute to the maintenance of DNA integrity and the anaerobic cellular environment until the oxyR regulated system and the pathogenicity genes are activated.
Open Access
Molecular characterization of ABC-type multidrug efflux systems in Bifidobacterium longum subsp. longumT JCM 1217
(2011) Moodley, Clinton; Reid, Sharon J; Abratt, Valerie Rose
A healthy and stable gastrointestinal microbiota is a vital feature of the innate immune system. It affords the host numerous health benefits and acts as a barrier against opportunistic gut infections. Probiotic bacterial supplements are, therefore, widely used in industry to promote good health. There is, however, a need to understand not only the factors underlying the health promoting capabilities of these bacteria, but also the intrinsic antimicrobial resistance mechanisms which these bacteria are known to harbour. These antibiotic resistance traits confer a competitive advantage on these bacteria over other bacterial species where they reside in the gut. It also allows them to survive during antibiotic therapy and they are able to continue conferring health benefits on the host. To better understand the mechanisms these bacteria utilize in conferring antibiotic resistance, genes which confer multidrug resistance by the active hydrolysis of ATP were studied here. These genes belong to the ATP-binding cassette (ABC) family of efflux transporters.
Open Access
Molecular diversity of faecal Lactobacillus species in stone- free black and white population groups and their possible role in kidney stone disease
(2008) Magwira, Cliff A; Abratt, Valerie Rose; Reid, Sharon J
Open Access
Resistance to antimicrobial agents in bifidobacteria
(2005) Price, Claire Emile; Abratt, Valerie Rose; Reid, Sharon J
For bifidobacteria to survive and achieve colonisation, they have to interact with inhibitory host-produced substances such as bile salts. Another aspect which should be studied is the safety of the probiotic bacterium and risks of acquisition of genes for resistance to antimicrobial agents. Although bifidobacteria exhibit resistance to a wide range of antibiotics, little is known about the molecular basis for this resistance. The aim of this project was, therefore, to investigate the molecular mechanisms responsible for the resistance to antibiotics and bile salts observed in bifidobacteria, and more specifically, to determine whether efflux systems are involved in this resistance. Five Bifidobacterium spp. were exposed to a range of antimicrobial agents. These included ethidium bromide, the bile salt sodium glycocholate, and a range of antibiotics.
Open Access
Rhamnose metabolism and metronidazole resistance in B. thetaiotaomicron VPI-5482
(2006) Patel, Ekta H; Abratt, Valerie Rose
Bacteroides thetaiotaomicron is an important human gut commensal organism that facilitates polysaccharide utilization, but can act as an opportunistic pathogen outside of this environment causing anaerobic bacteraemia and abscess formation. There is a growing resistance by B. thetaiotaomicron and other opportunistic pathogens to metronidazole, the leading drug of choice. This dissertation aimed to use physiological, molecular and biochemical analyses to elucidate the possible mechanism/s of metronidazole resistance in B. thetaiotaomicron, in order to extend the understanding of metronidazole resistance at a genetic level. A B. thetaiotaomicron VPI-5482 transposon-generated metronidazole resistant mutant, named B. thetaiotaomicron Tn MetR , that displayed a MIC = 8 μg/ml was isolated and characterized. Southern hybridization was used to identify the insertion of a single copy of the transposon in an intergenic region of a gene cluster involved in the uptake and catabolism of L-rhamnose. RNA hybridization studies confirmed that the genes in B. thetaiotaomicron Tn MetR were transcribed in the presence and absence of the substrate, L-rhamnose. In addition, five of the catabolic genes were expressed as an operon, rhaKlPAO. A monocistronic gene, rhaR, located downstream of the transposon insertion site had predicted amino acid sequence similarity to a group of AraC/XylS transcriptional regulators. Insertional inactivation of the coding sequence of this gene, using the pGERM suicide vector rendered this mutant, B. thetaiotaomicron rhaR", unable to utilize L-rhamnose. Introducing RhaR in this strain, on a plasmid, restored growth in the medium supplemented with L -rhamnose as the sole carbon source, confirming that rhaR is a positive regulator of the rhamnose operon. The transcriptional regulation of the rhamnose pathway was further examined by primer extension and two promoter sites, PrhaKIPAO and P rhaR were identified. The link between increased L·rhamnose metabolism and metronidazole resistance was further investigated by creating an overexpressor of the positive transcriptional regulator, named B. thetaiotaomicron (pL YLrhaR). The B. thetaiotaomicron Tn MetR and B. thetaiotaomicron (pL YLrhaR) exhibited resistance to metronidazole in medium with L-rhamnose as the sole carbon source, and displayed elevated levels of lactate dehydrogenase and decreased levels of pyruvate oxidoreductase activity. These two enzymes have been linked to the electron flux required for the intracellular anaerobic activation of metronidazole and this phenotype has previously been described in metronidazole resistant B. fragilis clinical isolates. This study demonstrated that the overexpression of the rhamnose pathway in B. thetaiotaomicron VPI-5482 resulted in metronidazole resistance and provides the first data to support this link.