OpenUCT :: Browsing by Subject "Medical Microbiology"

Browsing by Subject "Medical Microbiology"

Now showing 1 - 20 of 59

Open Access
Antimicrobial susceptibility of Acinetobacter isolates from Groote Schuur Hospital region : an investigation of the appropriateness/validity of the NCCLS zone size criteria
(2002) Ben-Ismaeil, B I; Roditi, D; Oliver, S
Since the 1970s, relatively uncommon species of non-enteric gram-negative bacilli have emerged as nosocomial colonizers and pathogens. Among them, one of the most significant genera is Acinetobacter, which has given rise to an increasing number of reports of nosocomial infections. The introduction of new broad-spectrum antimicrobials in the hospitals has been one of the main factors responsible for this development.In addition leading to a move from more susceptible towards more resistant pathogens. This occurred both between and within genera. Owing to the unpredictable antimicrobial susceptibility of Acinetobacter spp., it is prudent to test each isolate for its susceptibility profile to guide in the proper treatment of infections caused by Acinetobacter.The disk diffusion method is the technique most widely used by microbiology laboratories for the routine assessment of antimicrobial susceptibility patterns.The zone diameters obtained by this technique for individual antimicrobials are reported as susceptible, intermediate or resistant by referring to an interpretative chart.Most laboratories use interpretative charts (zone size criteria) supplied by the National Committee for Clinical Laboratory Standards (NCCLS) for this purpose.In our laboratory we have noted discrepancies between sensitivities as determined by the NCCLS zone size criteria and the local minimum inhibitory concentration (MIC) results obtained for Acinetobacter species, especially for cefepime (CPIM) and ceftazidime (CT AZ). Since Acinetobacter species contribute significantly to the increased morbidity and mortality of debilitated patients ( especially v entilated ICU patients, and the fact that clinicians rely on the antimicrobial susceptibility results in the management and treatment of these patients, it was felt necessary to investigate the appropriateness/validity of the NCCLS zone size criteria used to interpret the susceptibility test results of cefepime (CPIM), ceftazidime (CT AZ), trimethoprim-sulfamethoxazole (TMP-SULF A), and piperacillin-tazobactam (PIP-T AZO) against Acinetobacter spp.
Open Access
Association of Faecalibacterium, Lachnospira, Veillonella, and Rothia with childhood wheezing
(2019) Kanyemba, Saara; Kaba, Mamadou; Tow, Lemese Ah
Wheezing symptoms among children, present major health and economic problems globally. A recent study conducted in Canada observed a reduction in stool bacterial genera Faecalibacterium, Lachnospira, Veillonella and Rothia (FLVR) in three-month old infants with atopy-wheeze symptoms. It is not known whether this is true in different human populations worldwide. The overall aim of this dissertation was to investigate the contribution of any of the FLVR bacteria or their combination in the occurrence of infant wheezing within the Drakenstein Child Health Study (DCHS), South Africa. To address this aim, I began my thesis's project by conducting a systematic literature review which investigated the association of FLVR bacteria with the occurrence of different respiratory diseases in humans. My review provided evidence for the possible involvement of FLVR bacteria in human respiratory diseases, including asthma, pulmonary tuberculosis and pneumonia. Furthermore, this review highlighted the need for a well-designed and large study to investigate the contribution of the FLVR bacteria in respiratory diseases, in an African setting. Secondly, I optimized SYBR Green based real-time quantitative polymerase chain reaction (qPCR) as well as conventional PCR assays for the detection of FLVR bacteria. Using the optimized assays, I screened 533 stool samples collected from 140 wheezing and 140 nonwheezing infants. The optimized assays demonstrated good performance in the detection of FLVR bacteria from human stool samples. Using qPCR, Rothia, Veillonella, Faecalibacterium and Lachnospira were detected in 90% (479/533), 73% (388/533), 51% (274/533) and 14% (77/533) of the samples, respectively. Conventional PCR permitted the detection of Rothia, Veillonella, Faecalibacterium and Lachnospira in 55% (263/479), 74% (289/388), 53% (145/274) and 0% (0/77) of the qPCR positive samples, respectively. I also determined the factors associated with faecal colonization by FLVR bacteria in the first year of life. I showed that reduced colonisation by Faecalibacterium was associated with male gender (adjusted OR = 0.65, 95%CI: 0.42 - 0.98) and TC-Newman residence (adjusted OR = 0.52, 95%CI: 0.29 - 0.91). Breastfeeding was associated with less colonisation by both Lachnospira, (adjusted OR = 0.17, 95%CI: 0.05 - 0.49) and Veilonella (adjusted OR = 0.32, 95%CI: 0.10 - 0.91). Mother's tertiary education was significantly associated with high Rothia colonisation (adjusted OR = 11.73, 95%CI: 1.36 - 2.58). In the last section of my thesis, I assessed the association of FLVR bacteria with infant wheezing using logistic regression models. I found a significant association of Rothia with reduced risk of infant wheezing (adjusted odds ratio (aOR)=0.54, 95%CI: 0.28-0.93) and recurrent wheezing (aOR=0.29, 95%CI: 0.05-0.88). Using receiver operating characteristic curves (ROC), I showed that among all FLVR bacteria, Lachnospira (AUROC = 0.833, 95%CI: 0.64-1.00) and Rothia (AUC=0.707, 95%CI: 0.62-0.79) could serve as biomarkers for early prediction of infant wheezing. Overall, this is the first study on FLVR bacteria and infant wheezing to be conducted in Africa. Its findings encourage more research to be conducted in order to elucidate the potential protective role of Rothia against childhood wheeze and asthma, as well as the contribution of Lachnospira in asthma development.
Open Access
Case report: Severe central nervous system manifestations associated with aberrant efavirenz metabolism in children: the role of CYP2B6 genetic variation
(2015) Abrams, Elaine
BackgroundEfavirenz, widely used as part of antiretroviral drug regimens in the treatment of paediatric human immunodeficiency virus infection, has central nervous system side effects. We describe four children presenting with serious, persistent central nervous system adverse events who were found to have elevated plasma efavirenz concentrations as a result of carrying CYP2B6 single nucleotide polymorphisms, known to play a role in the metabolism of EFV. None of the children had a CYP2B6 wildtype haplotype. We believe this is the first case of cerebellar dysfunction associated with efavirenz use to be described in children.Case presentationFour black African children, between the ages of 4 and 8years presenting between 1 and 20months post-efavirenz initiation, are described. Cerebellar dysfunction, generalised seizures and absence seizures were the range of presenting abnormalities. Plasma efavirenz levels ranged from 20-60mg/L, 5–15 times the upper limit of the suggested reference range. All abnormal central nervous system manifestations abated after efavirenz discontinuation.ConclusionEfavirenz toxicity should always be considered in human immunodeficiency virus-infected children with unexplained central nervous system abnormalities. Our findings further our understanding of the impact of genetic variants on antiretroviral pharmacokinetics in children across various ethnic groups. Screening for potential EFV-toxicity based on the CYP2B6 c.516 SNP alone, may not be adequate.
Open Access
Characterisation of the cold-shock response in Mycobacterium smegmatis
(1999) Shires, Karen Lesley; Steyn, Lafras M; Zappe, Harold
The response of Mycobacterium smegmatis to a cold shock was investigated in order to gain insight into the stress responses of members of the genus Mycobacterium. Mycobacterium smegmatis cultures were shocked from 37°C to 30°C, 25°C, 15°C, and 10°C and the effects on both growth (ATP concentration, culture turbidity, colony-forming units) and metabolism (incorporation of ¹⁴C-leucine and ³H-uracil) were investigated. The magnitude of the cold-shock response was found to be dependent upon the degree of the cold shock. A cold shock to 10°C had the greatest effect and resulted in a "lag period" of 24 hours in both the growth and metabolism of the culture. The synthesis of proteins was reduced 20-fold during this period, indicating at block in translation. The cold-shock response in Mycobacterium smegmatis was an adaptive response with growth eventually being resumed at the colder temperature, but at a reduced rate. Using the techniques of one-dimensional sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and two-dimensional protein gel electrophoresis, ³⁵S-methiononine-labelled proteins that were synthesised during the cold shock were analysed. At least fourteen radio-labelled proteins were induced during the first 24-hour period and these demonstrated two distinct patterns of cold-shock induced expression: transient and continuous. Depending upon the pattern of expression and size, the cold-shock proteins were classified as "cold-induced proteins", "cold-shock proteins" or "cold-acclimation proteins". CipM, a 27kDa protein, was identified as the major cold-shock protein through one-dimensional protein electrophoresis. From N-terminal sequence data generated from a protein (CipM.1) within this band, a corresponding degenerate DNA probe was used to isolate cipM.1. This gene was cold-inducible, with mRNA levels transiently increasing 5-7 fold after a 37°C to 10°c cold-shock. Homologues of this cold-shock gene are found in the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. The corresponding mycobacterial proteins showed homology at the N-terminus to the HU~ subunit of HU of Escherichia coli and possessed similar C-terminal praline, lysine and alanine degenerate repeats to the mycobacterial heparin-binding hemagglutinin. The response of several mycobacterial cold-shock gene homologues to a cold shock was also investigated, by northern-hybridisation and S1 nuclease analysis. The cspA homologue of Mycobacterium smegmatis demonstrated a 16-24 fold transient induction in mRNA levels following a 37°C to 10°C temperature-shift, while gyrA mRNA levels were maintained at a constant level throughout the cold shock. Although some similarities were demonstrated between the cold-shock response of Escherichia coli and Mycobacterium smegmatis, definite differences occur in the proteins that are involved in the adaptive stages of the response.
Open Access
Characterization of avipoxviruses for use in recombinant vaccines
(1992) Kow, Daria Karen; Dumbell, K R
Pox viruses have been demonstrated in over 60 types of wild and exotic birds as well as domestic birds. Avipox viruses have been isolated and characterised from fowls, quails, canaries, parrots and lovebirds. This work describes the first isolation of a poxvirus from Jackass penguins (Spheniscus dermersus) and the characterisation of the virus as a separate species of penguinpox virus.
Open Access
Characterization of Mycobacterium tuberculosis isolates with discordant rifampicin susceptibility test results
(2018) Ghebrekristos, Yonas; Beylis, Natalie; Nicol, Mark P
Background: The Xpert MTB/RIF assay was adopted as the initial diagnostic test for patients with presumptive tuberculosis (TB) by the South African National TB Control programme in December 2010. Rifampicin (RIF) resistance detected by the Xpert MTB/RIF (Xpert) is confirmed by a line probe assay (LPA) (GenoType MTBDRplus) and/or phenotypic (culture-based) drug susceptibility testing (DST) by MGIT (Mycobacterial Growth Indicator Tube) on the culture isolate from a 2nd specimen. Although both the Xpert and LPA target the rifampicin resistance determining region (RRDR) of the rpoB gene, discordant RIF results (Xpert RIF resistant (RIFR ), LPA RIF susceptible (RIFS )) have been reported. In addition, in cases where genotypic tests detect an rpoB mutation, inferring RIF resistance, routine phenotypic DST may report a RIF susceptible result. This is usually due to disputed rpoB mutations. Aim: The aims of this study are to determine 1) whether the discordance between Xpert and LPA is due to false RIFR by Xpert or false RIFS by LPA and to elucidate the causes of false results and 2) the frequency and types of rpoB mutations expected to test susceptible on routine phenotypic DST and their corresponding RIF MIC (minimum inhibitory concentration). Methods: Consecutive isolates with discordant Xpert RIFR and LPA RIFS results were selected during routine review. For the Xpert, parameters including bacterial DNA load and cycle threshold (Ct) of the probes were evaluated. In addition, isolates with a pattern of any absent rpoB WT band and absent MUT band on the LPA strip (“miscellaneous rpoB mutations”) were selected for MIC testing using the MGIT 960 system and EpiCenter TB eXiST software. Sanger sequencing of the rpoB gene from codon 462 to 591 was performed on all selected isolates. Results and discussion: Discordant Xpert/LPA results: From the total of 1542 patients with RIFR results by Xpert, 106 (6.9%) had a discordant LPA RIFS result. Sequencing results were available for 101 isolates of which 78 (77.2%) had no rpoB mutation detected and these were categorized as false RIFR by Xpert. Mutations were detected by sequencing in the remaining 23 (22.8%); these were categorized as false RIFS by LPA. Probe delay occurred in 56/76 (73.7%) cases compared with 104/1436 (7.2%) controls (p 4 and there is a Very Low bacterial load has a positive predictive value (PPV) of 64.2 % of being false and where the Ct max is between 4.1 and 4.9 with Very Low bacterial load, the PPV of a false result increases to 85.7%. For the false RIFS results by LPA, the majority 11/23 (47.8%) were due to technical errors. In 6/23 (26.1%) it was due to mixed infection and in 2/23 (8.7%) there was laboratory mix up. In the remaining 4/23 (17.4%) the cause could not be determined and mixed infection or a laboratory mix up could not be excluded. Discordant genotypic/phenotypic results: RIF resistance was detected in 1502 patients by LPA, of which 169 (11.3%) had a miscellaneous mutation. In addition, a further 21 isolates were selected from “Part 1” of the study, where sequencing confirmed that the rpoB mutation was not one of the high level / high confidence rpoB mutations. A total of 178 isolates had both MIC and rpoB sequencing results. In our study 140/178 (78.7%) isolates with miscellaneous rpoB mutations (n=158) or previously described disputed rpoB mutations (n=20) had MIC values ranging from ≤0.0625 µg/ml to 1.0 µg/ml. An MIC >1.0 µg/m was determined for 38/178 (21.3%) that would have tested RIFR by MGIT DST. Conclusion: Arising from this study is a laboratory based guideline that is now used within NHLS TB laboratories detailing steps on how to detect possible false RIFR results by Xpert MTB/RIF and on how to troubleshoot discordant Xpert RIFR and LPA RIFS results. A database has been created from the results obtained in this study that lists specific rpoB mutations and their corresponding MIC value and has the potential to assist clinicians in individualizing the patient TB treatment regimen.
Open Access
Conditions affecting ergothioneine levels in Mycobacterium smegmatis & the attempted isolation of Î±-N, N, N-Histidine methyltransferase, the first enzyme in ergothioneine biosynthesis
(2007) Williams, Monique J; Steyn, Lafras M; Steenkamp, D J
Ergothioneine and mycothiol are the two major low molecular weight thiols present in mycobacteria. The generation of mycothiol-deficient mutants has demonstrated its role in protecting M tuberculosis against oxidative and nitrosative stress. To date, no ergothioneine-deficient mutants have been identified and the role of ergothioneine in mycobacteria remains unknown. The work in this thesis was performed with the aim of better understanding the function of ergothioneine in mycobacteria, by studying its biosynthesis and the conditions affecting its production.
Open Access
Construction and evaluation of three candidate vaccines expressing HIV-1 subtype-C mosaic Gag
(2015) Jongwe, Tsungai Ivai; Chapman, Ros; Williamson, Anna-Lise; Douglass, Niki; Chege, Gerald
Of the 35 million people living with HIV-1 globally, approximately 71.4% are in the resource-limited sub-Saharan Africa. The immense sequence diversity of HIV-1, even within subtypes, makes it challenging to develop effective vaccines that target a wide range of HIV subtypes. Mosaic immunogens have been computationally designed to specifically overcome this hurdle by maximizing the inclusion of common T cell epitopes. When compared to consensus immunogens, polyvalent mosaic immunogens of HIV-1 group M have shown increased breadth and depth of antigen-specific T-cell responses. More than 90% of HIV positive individuals in sub-Saharan Africa are infected with HIV-1 subtype C (HIV-1C). We therefore designed, constructed, and evaluated candidate vaccines expressing HIV-1C mosaic Gag (GagM) in a proof of concept study. Gag was chosen as the most appropriate target for a T cell-based vaccine as there are many studies correlating control of HIV viral load with T cell responses to Gag. The immunogen was designed by Fischer et al., 2007 (1). Three different vaccine platforms were chosen based on their different strengths to be used in prime-boost regimens to determine the immunogenicity of HIV-1C GagM in mice. The first was a pantothenic auxotroph of the tuberculosis vaccine Mycobacterium bovis Bacille Calmette Guérin (BCG). The second was a DNA vaccine vector with enhanced expression of transgenes due to a novel enhancer element from porcine circovirus type 1, which has been demonstrated to increase gene expression. The third vaccine vector selected was the well characterised poxvirus modified vaccinia Ankara (MVA).
Open Access
CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria
(2021) De Wet, Timothy; Warner, Digby
In the 20 years since the pioneering publication of the genome of Mycobacterium tuberculosis, significant efforts have been made to complete functional annotation of the genome. However, these efforts have generally been performed on a single-gene basis, ensuring slow progress and leaving large portions of the genome unannotated. High-throughput approaches to understanding the functional genome, such as transposon-insertion sequencing, have been developed and applied to mycobacteria in a variety of conditions; however, they have several limitations, particularly in their ability to study genes essential for viability. The recent optimisation of inducible CRISPR-interference for mycobacteria offers the potential to expand the high-throughput functional genomic toolkit. This thesis utilises CRISPR-interference for the development and validation of two high-throughput functional genomic approaches in the model mycobacterium M. smegmatis. The first approach combines large-scale pooled oligonucleotide synthesis and nextgeneration sequencing, and is termed CRISPRi-Seq. A pooled library of 11 367 mutants, targeting 2 385 M. smegmatis genes with M. tuberculosis homologues, was constructed and used to infer gene essentialities which were compared with corresponding predictions from transposon-insertion sequencing data. This process validated the CRISPRi-Seq technique and identified practical considerations for its future use. The second approach utilises data derived from CRISPRi-Seq to create an arrayed library of 263 individual M. smegmatis inducible CRISPRi mutants targeting essential genes. This library is applied to a quantitative imaging pipeline to produce detailed data-driven profiles of the morphological impact of essential gene suppression. These morphological profiles are used to statistically predict genetic function, as well as antimicrobial mechanism-of-action. The two novel approaches developed in this work represent valuable technical advances and produce large datasets of functional genomic data which are available interactively online. Taken individually, or in combination, these methodologies can be utilised to increase fundamental understanding of mycobacteria, including the pathogenic M. tuberculosis
Open Access
Disabling the intrinsic resistome of Mycobacterium tuberculosis: elucidating hierarchies of DNA repair and mutagenesis that undermine current antibiotic efficacy
(2021) Gobe, Irene; Warner, Digby; Mizrahi, Valerie; Iorger, Thomas R
DNA damage repair mechanisms are critical to the adaptive evolution of Mycobacterium tuberculosis as obligate human pathogen, including the emergence of drug-resistance during anti-tuberculosis (TB) chemotherapy. In experimental models, DnaE2-dependent translesion synthesis (TLS) and UvrB-dependent nucleotide excision repair (NER) have been identified as major mediators of DNA damage tolerance and repair, respectively. Given the inferred dominance of these pathways, this thesis aimed to elucidate otherwise cryptic repair mechanisms which might buffer loss of DnaE2 and UvrB in bacilli exposed to genotoxic stress. Using dnaE2 and uvrB deletion mutants of the model mycobacterium, M. smegmatis (MSM) mc2 155, we applied genome-wide transposon (Tn) mutagenesis to identify conditionally essential repair pathways under treatment with genotoxins of different mechanistic classes. To this end, the DNA crosslinking agent, mitomycin C (MMC), and the gyrase inhibitor and clinically relevant TB drug, moxifloxacin (MOX), were used. The goal was to reveal potential targets for co-drugs that might shorten treatment duration and reduce the risk of drug resistance by severely limiting the intrinsic capacity of MTB to tolerate lethal drugs for extended periods. Among others, our analysis identified GlgB, which is involved in glycogen biosynthesis, and mycothiol biosynthesis proteins, MSMEG_0933 and MSMEG_5261, as compensating the absence of UvrB during MMC treatment. Under MOX treatment, the absence of UvrB was compensated by the RecC/Single-strand Annealing pathway. In contrast, DnaE2 deficiency revealed the conditional essentiality of the PadR family transcriptional regulator, MSMEG_2868, under MMC exposure. Importantly, in all cases, results from the Tn screen were validated using CRISPR interference targeting the identified genes. Of particular interest, we observed that UvrB was essential to compensate loss of DnaE2, whereas the reciprocal was less definitive: while DnaE2 appeared dispensable in MMC-treated uvrB, Tn analyses suggested that dnaE2 might be essential in the untreated ∆uvrB mutant. This result, which is consistent with very recent results suggesting the co-ordination of NER and DnaE2 functions in Caulobacter crescentus, is intriguing in potentially revealing a previously unappreciated role for DnaE2 in mycobacterial NER function. Taken together, these results support the utility of Tn-based whole-genome screens in revealing unexpected genegene interaction networks, and provide additional impetus to explore ancillary, non-essential metabolic functions as alternative targets for novel combination therapies designed to cripple intrinsic mechanisms of mycobacterial resistance.
Open Access
Distribution, frequency and contribution to the expression of antibiotic resistance gene of an IS element in Acinetobacter baumannii
(2006) Garny, Seike; Elisha, B Gay
Open Access
Dynamics of faecal bacterial populations in early infancy as determined by massively parallel sequencing
(2015) Claassen, Shantelle; Nicol, Mark; Kaba, Mamadou
Background: Meconium microbiota have recently gained great interest; however very few studies have included meconium specimens when longitudinally characterizing the infant GIT microbiota. This study therefore aimed to longitudinally characterize meconium microbiota profiles during the first seven months of life and to compare these profiles with those from maternal faecal specimens using quality controlled Illumina MiSeq sequencing data. Methods: We sampled infant meconium and maternal faecal specimens at birth, as well as two subsets of infant faecal specimens at 4-12 and 20-28 weeks of life. We extracted nucleic acid from faecal specimens using the automated QIAsymphony Â® SP instrument. Using Illumina MiSeq technology, we sequenced the V4 region of the bacterial 16S rRNA gene. We determined whether sufficient reads were sequenced using accumulation curves; whether any contamination occurred; and whether our sequencing approach was reproducible. The relative abundances of taxonomically classified operational taxonomic units (OTUs), and the Shannon diversity and Bray Curtis dissimilarity indices served to characterize faecal specimens from participants. Log ratio biplots and generalized linear mixed models served to statistically determine differences between faecal bacterial profiles. Results: Faecal specimens were collected from 90 mothers and 107 infants at birth, 72 infants at 4-12 and 36 infants at 20 28 weeks of age. We classified OTUs from two non-template controls which were indicative of potential contamination. Correcting for contamination resulted in a loss of 10 % of OTUs classified. Our reproducibility analysis correlated with increased concentrations of template used during library preparation. Based on diversity measures, meconium specimens harboured the most diverse bacterial profiles. The highest proportions of OTUs classified from meconium belonged to the phylum Proteobacteria (60 %), while the phylum Firmicutes was most abundant at 4-12 weeks (49 %) and 20-28 weeks (64 %) of life. The phylum Actinobacteria was at its highest at 4-12 weeks of age (26 %) and its increased proportions were associated with breastfeeding at 6-10 weeks of life. Firmicutes constituted the majority (79 %) of bacteria from maternal faecal specimens. No mother- infant pairs clustered at any of the time points studied, but infant bacterial profiles became more adult-like with increased age. An increase in infant age significantly affected bacterial proportions of 87 OTUs. Interestingly, we observed that infants exposed to HIV had higher proportions of the genus Leuconostoc and higher diversity indices compared to HIV unexposed infants at 4-12 weeks of age. Conclusion: Our study highlights that reproducibility may be worsened by the use of low template concentrations during library preparation, which may also skew diversity measures. We conclude that meconium is not sterile and that infant faecal bacterial profiles become more adult-like with increased age.
Open Access
Elucidation of mechanisms of antibiotic subversion in mycobacteria
(2015) Naran, Krupa; Warner, Digby F; Mizrahi, Valerie
The intrinsic resistance of Mycobacterium tuberculosis ( Mtb ) to antibiotics is generally attributed to multiple factors, most significantly the low permeability of the mycobacterial cell wall, the operation of various drug inactivating systems, and the activity of efflux pumps. This study aimed to investigate the role of various components of the "intrinsic resistome" that limit the efficacy of antitubercular agents. The DNA damage response: the SOS response was hypothesized to play a role in antibiotic- mediated cellular death, and that disabling the mycobacterial SOS response, by generating non-cleavable LexA mutants (lexA Ind-), could be used as a tool to validate antibiotic-mediated cell death. To this end, the M. smegmatis (Msm) cleavable LexA was shown to be essential for induced mutagenesis and damage tolerance and that an intact DNA damage repair system is required to respond to antibiotic-mediated DNA damage. In contrast, Mtb cleavable LexA was required for induced mutagenesis but not necessarily damage sensitivity. In addition, the Mtb SOS response does not contribute significantly to remediation of antibiotic-mediated DNA damage. Collectively, these data suggest that DNA repair mechanisms differ between the mycobacterial species and despite effectively inactivating the LexA-dependent DNA repair mechanism(s) in Msm and Mtb, these organisms are able to circumvent this pathway and successfully remediate damaged DNA sustained under various conditions. Furthermore, Mtb auto-bioluminescent reporter strains were generated by introducing the lux operon downstream of the recA or radA promoters. Analysis of a panel of antimicrobials against these strains allowed for the identification of true DNA-damaging agents and the evaluation of the kinetics of the DNA-damage response, in a concentration- and time-dependent manner. Efflux-mediated drug resistance: This study aimed to evaluate the interactions between pairwise combinations of selected antimicrobials and efflux pump inhibitors (EPIs), in vitro and ex vivo, and to identify a novel verapamil (VER)-analogue with improved efficacy against Mtb. Subsequently, a candidate EPI was identified, with equivalent in vitro synergistic effects to VER when used in combination with various antibiotics but with reduced cytotoxic effects, ex vivo, when compared to VER. Mycothiol-mediated protection : It was hypothesized that undetectable levels of mycothiol ( MSH ) in Mtb would potentiate the use of current antibiotics. To investigate the contribution of the cellular antioxidant, MSH, to the mitigation of antimicrobial efficacy, this study aimed to disrupt MSH production by conditionally knocking-down expression of the essential gene, mshC. The mshC knock-down mutants (in all configurations) were not anhydrotetracycline (ATC)-regulatable in liquid or on solid medium, which was subsequently validate d with quantitative gene expression analysis. These data suggest that a tetracycline (Tet)-based conditional expression system may not be applicable to mshC. In conclusion, Mtb has a multitude of inherent mechanisms to subvert the effects of antimicrobial treatment. This study has contributed to the understanding of certain aspects of the intrinsic resistome and in doing so, established tools that can be used in future drug discovery programmes.
Open Access
The epidemiology & molecular basis of fluoroquinolone resistant & susceptible isolates of Campylobacter coli
(2001) Cooper, Rhett; Elisha, B Gay; Lastovica, Albert
Fluoroquinolone susceptible and resistant Campylobacter coli were isolated from pigs on two separate pig farms. C. coli are enteric pathogens of humans and animals and although diarrhoea resulting from C. coli and C. jejuni is generally a self-limiting disease, in severe cases, fluoroquinolones are the choice antibiotic for treatment. The presence of fluoroquinolone resistant C. coli strains in the food chain is cause for concern as this may be a source of resistant strains in humans. Sixty-one isolates were included in the study: 26 were susceptible to nalidixic acid and ciprofloxacin and 35 were resistant to these antibiotics. Fifty-five strains were obtained from pigs on farm A, while 6 strains were obtained from pigs on farm B, the source farm of pigs to farm A. Serotyping and flaA typing were carried out to study the epidemiology of the isolates. Serotyping identified 0:24 (11/61) as the most frequent serotype isolated, followed by 0:5 (7/61). Common serotypes 0:48, 0:54 and 0:59 were identified in strains from both farms. A high number of the strains were non-typeable (23/61) but were distinguished by flaA typing. RFLP analysis of the flaA gene revealed 13 distinct profiles in strains from farm A, and 4 profiles in strains from farm B, of which only 1 was unique to farm B. Profile 1 was the commonest profile observed with 31 % (17 /55) of flaA typed strains in this profile. There was an association between 0:24, profile 6, and resistance. Resistant and sensitive pairs were isolated from 15 pigs; flaA profiles of each of 4 pairs were identical, suggesting selection of resistant mutants from previously sensitive populations. An investigation of the molecular basis of the fluoroquinolone resistance identified a Thr-86 to Ile mutation in GyrA, the primary target of these antibiotics.
Open Access
Epidemiology of extended spectrum beta-lactamase and carbapenemase-producing bacteria in stool from apparently healthy children, South Africa
(2015) Manenzhe, Rendani Innocent; Nicol, Mark; Kaba, Mamadou
Background: The prevalence of extended-spectrum beta-lactamase (ESBL) - and carbapenemase-producing Enterobacteriaceae in healthy humans in the community is largely unknown. We aimed to determine the prevalence and genetic characteristics of ESBL- and carbapenemase-producing Enterobacteriaceae in stools from healthy infants and their mothers, and to determine the risk factors associated with their carriage. Methods: This study was nested within the Drakenstein Child Health Study, a birth cohort in a semi-rural region of Western Cape Province, South Africa. Maternal and infants faecal samples (including the meconium) were collected at birth and at two additional time-points (5-12 and 20-28 weeks) from the infants only. Samples were screened for ESBLs and carbapenemase-producing organisms using ChromID ESBL and ChromID CARBA media, respectively. Identification of suspect ESBL/carbapenemase-producing isolates and antibiotic susceptibility were determined using the Vitek 2 system. ESBL production was confirmed using the combination disc test, and that of carbapenemase using the modified hodge test. Selected ESBL and carbapenemase genes were evaluated by the singleplex conventional polymerase chain reaction and Sanger sequencing. Risk factors were assessed by univariate analysis using the EPI Info version 7 software.
Open Access
Evolution of sensory neuropathy after initiation of antiretroviral therapy
(2018) Centner, Chad; Heckmann, Jeannine M
Introduction: We studied the evolution of sensory neuropathy after antiretroviral therapy (ART) in human immunodeficiency virus–infected South Africans. Methods: Enrolment commenced before ART with 6-monthly follow-ups for 24 months. Symptomatic distal sensory polyneuropathy (SDSP) was defined as one symptom and sign. Symptom/sign scores were compared between visits. Results: We enrolled 184 participants. Pre-ART, 16% had SDSP. After 18 months of ART, pain prevalence decreased in those with pre-ART SDSP (odds ratio [OR], 0.09; 95% confidence interval [95%CI], 0.03-0.29). Symptoms improved in 50% ever experiencing pain (mean improvement=-4.5 on 11-point scale). Participants SDSP-free pre-ART developed SDSP at a rate of 18 per 100 person-years. After 24 months, 18% had SDSP. Stavudine (60% of cohort) did not predict incident SDSP, but associated with increased prevalence of reduced/absent reflexes at 18 months (OR, 2.24; 95% CI, 1.08-4.65). Conclusions: Painful symptoms improved during ART. Evolving sensory neuropathy was due to increasing small and large fiber dysfunction.
Open Access
Gene expression in Mycobacteria : attenuation of gene expression by antisense methods and translation enhancement by downstream box elements
(2004) Rush, Gavin John; Steyn, Lafras M
Bibliography: leaves 172-193.
Open Access
High incidence of antimicrobial resistant organisms including extended spectrum beta-lactamase producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus in nasopharyngeal and blood isolates of HIV-infected children from Cape Town, South Africa
(BioMed Central Ltd, 2008) Cotton, Mark; Wasserman, Elizabeth; Smit, Juanita; Whitelaw, Andrew; Zar, Heather
BACKGROUND:There is little information on nasopharyngeal (NP) flora or bacteremia in HIV-infected children. Our aim was to describe the organisms and antimicrobial resistance patterns in children enrolled in a prospective study comparing daily and three times weekly trimethoprim-sulfamethoxazole (TMP-SMX) and isoniazid (INH) or placebo prophylaxis. METHODS: NP swabs were taken at baseline from HIV-infected children enrolled in the study. Standard microbiological techniques were used. Children were grouped according to previous or current exposure to TMP-SMX and whether enrolled to the study during a period of hospitalization. Blood culture results were also recorded within 12 months of baseline. RESULTS: Two hundred and three children, median age 1.8 (Interquartile [IQ]: 0.7-4) years had NP swabs submitted for culture. One hundred and eighty-four (90.7%) had either stage B or C HIV disease. One hundred and forty-one (69.8%) were receiving TMP-SMX and 19 (9.4%) were on antiretroviral therapy. The majority, 168 (82%) had a history of hospitalization and 91 (44.8%) were enrolled during a period of hospitalization. Thirty-two subjects (16.2%) died within 12 months of study entry.One hundred and eighty-one potential pathogens were found in 167 children. The most commonly isolated organisms were Streptococcus pneumoniae (48: 22.2%), Gram-negative respiratory organisms (Haemophilus influenzae and Moraxella catarrhalis) (47: 21.8%), Staphylococcus aureus (44: 20.4%), Enterobacteriaceae 32 (14.8%) and Pseudomonas 5 (2.3%).Resistance to TMP-SMX occurred in > 80% of pathogens except for M. catarrhalis (2: 18.2% of tested organisms). TMP-SMX resistance tended to be higher in those receiving it at baseline (p = 0.065). Carriage of Methicillin resistant S. aureus (MRSA) was significantly associated with being on TMP-SMX at baseline (p = 0.002). Minimal inhibitory concentrations (MIC) to penicillin were determined for 18 S. pneumoniae isolates: 7 (38.9%) were fully sensitive (MIC [less than or equal to] 0.06 mug/ml), 9 (50%) had intermediate resistance (MIC 0.12 - 1 mug/ml) and 2 (11.1%) had high level resistance (MIC [greater than or equal to]2 mug/ml). Fifty percent of Enterobacteriaceae produced extended spectrum beta-lactamases (ESBL) (resistant to third generation cephalosporins) and 56% were resistant to gentamicin. Seventy-seven percent of S. aureus were MRSA. Carriage of resistant organisms was not associated with hospitalization.On multivariate logistic regression, risk factors for colonization with Enterobacteriaceae were age [less than or equal to] one year (Odds ratio 4.4; 95% Confidence Interval 1.9-10.9; p = 0.0008) and CDC stage C disease (Odds ratio 3.6; 95% Confidence Interval 1.5-8.6; p = 0.005)Nineteen (9.4%) subjects had 23 episodes of bacteremia. Enterobacteriaceae were most commonly isolated (13 of 25 isolates), of which 6 (46%) produced ESBL and were resistant to gentamicin. CONCLUSION: HIV-infected children are colonized with potential pathogens, most of which are resistant to commonly used antibiotics. TMP-SMX resistance is extremely common. Antibiotic resistance is widespread in colonizing organisms and those causing invasive disease. Antibiotic recommendations should take cognizance of resistance patterns. Antibiotics appropriate for ESBL-producing Enterobacteriaceae and MRSA should be used for severely ill HIV-infected children in our region. Further study of antibiotic resistance patterns in HIV-infected children from other areas is needed.
Open Access
The house dust microbiota in the Drakenstein Child Health Study
(2015) Duyver, Menna; Nicol, Mark; Ah Tow, Lemese
Introduction: The indoor home environment comprises many niches that are occupied by bacterial communities. The composition of these bacterial communities may be influenced by numerous factors such as number of occupants, pets, season and location. Understanding the house dust microbial community is vital to understanding its' influence on human respiratory health. Aims: The aims of the studies described in this MSc dissertation were to: 1) evaluate the performance of ten commercial nucleic acid extraction kits on dust samples; 2) optimise dust removal from electrostatic dustfall collectors (EDC); 3) determine the bacterial composition of house dust using 16S rRNA gene sequencing and 4) determine those factors influencing the bacterial composition of house dust by performing bioinformatic and data analysis on the sequenced dust samples. Methods: In order to study the microbial content of house dust, an efficient DNA extraction protocol was required. Ten commercial nucleic acid purification protocols were evaluated on their ability to efficiently extract good quality DNA from very low quantities (20 mg) of wet bulk house dust. For the purpose of this study, EDCs were used to collect settled dust from homes of participants in the Drakenstein Child Health Study (DCHS). Electrostatic Dustfall Collectors were placed twice within the same household, approximately 6 months apart, spanning two seasons. The Z/R Fungal/Bacterial DNA MicroprepTM (ZMC) protocol was used to extract DNA from dust removed from EDCs. The V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform to determine the bacterial taxonomic composition of the house dust samples. A custom python wrapper that meshes a set of tools integrated into a computationally efficient workflow, known as the YAP pipeline was used to classify 16S rRNA sequences into bacterial taxonomies. Based on 97% sequence similarity, the pre-processed sequences were assigned to Operational Taxonomic Units (OTU). R software together with RStudio software was used for all statistical analysis and graphical representations of the data.
Open Access
The identification and characterization of a clinical isolate of a methicillin susceptible Staphylococcus aureus ST612
(2012) Moleleki, Malefu; Elisha, B Gay
A previous study of 100 methicillin-resistant Staphylococcus aureus (MRSA) isolates, collected between January 2007 and December 2008 from five Cape Town hospitals, identified ST612-MRSA-IV as the predominant MRSA. This raised the question of whether methicillin-susceptible S. aureus (MSSA) isolates with the same sequence type (ST) were present in hospitals in this city. Nineteen MSSA isolates, collected during the same period, and from four of the hospitals in Cape Town, as the previously characterized MRSA isolates, were screened for the presence of ST612-MSSA. spa typing and multi-locus sequence typing (MLST) identified one isolate, MS14, as ST612-MSSA, spa type t064.