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    The emergence of penicillin resistance in nasopharyngeal and oral isolates of streptococcus pneumoniae and viridans streptococci following beta-lactam antibiotic exposure
    (1990-06-02) Van Den Berg, Ingrid Susan
    Increasing levels of penicillin-resistant Streptococcus pneumoniae and viridans streptococci are becoming a major cause for concern, particularly in South Africa. To investigate this problem 100 paediatric patients, admitted to Baragwanath Hospital during the period April to July 1988, were swabbed nasopharyngeally and orally. On admission 24 out of 53 (45%) pneumococcal carriers carried penicillin-resistant strains, while on discharge 33 strains out of 45 (73%) were penicillin-resistant (p = 0.003). The corresponding figures for high level penicillin-resistant strains were 7 out of 53 (13%) on admission to 20 out of 45 (44%) on discharge (p = 0.001). Treatment with 3-lactam antibiotics reduced the pneumococcal carriage rates by eliminating penicillin-susceptible strains while many children who were not carriers on admission became carriers of penicillin-resistant strains during hospitalisation. In the case of penicillin-resistant viridans streptococci in the oral cavity the use of 8-lactam antibiotics was accompanied by significant increases in both penicillin-resistant and high level resistant strains.
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    Molecular investigation on the impact of the pneumococcal polysaccharide-protein conjugates vaccine (PCV) on bacterial nasopharyngeal colonization in children
    (2017) Olwagen, Courtney Paige
    Background: Nasopharyngeal colonisation is a pre-requisite for developing bacterial respiratory and invasive disease. Immunisation of children with the pneumococcal conjugate vaccine (PCV) impacts upon colonising pneumococcal serotypes, which in turn could also affect the biome of the nasopharynx in relation to colonisation by other bacteria. Due to limitations in standard culture methods, the association between PCV-immunisation and bacterial carriage density is still unclear, including among HIV-infected children. In this study we aimed to evaluate the effect of infant vaccination with the 7-valent PCV (PCV7) on vaccine-serogroup colonisation in order to determine whether the increase in non-vaccine serotype (NVT) colonisation was due to unmasking of previously low density colonising serotypes or increase in acquisition of NVT. Also, we evaluated the association between PCV7 immunisation and HIV-infection on the prevalence density of nasopharyngeal colonisation by other common potentially pathogenic bacteria. Methods: A multiplex real-time qPCR assay was set up to detect 44 common pneumococcal serotypes and 5 bacterial pathogens including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pyogenes. All assays were optimised according to MIQE guidelines and their ability to detect multiple pneumococcal serotype/group and bacteria in archived nasopharyngeal swabs were evaluated. The multiplex qPCR assays were then used to evaluate vaccine-serotype, non-vaccine serotype and bacterial nasopharyngeal colonisation in achieved swabs of PCV7-vaccinated (at 6, 10 and 14 weeks of age) and PCV-unvaccinated African children at 9 and 15-16 months of age, prior to routine vaccination of children with PCV through the public immunisation program. In order to address the limitations of the qPCR assays, a nanofluidic real-time PCR assay was developed to simultaneously detect 53 pneumococcal serotypes, 6 serotypes of H. influenzae and 11 bacterial pathogens. Further, all assays were optimised and evaluated according to the MIQE guidelines and findings from Fluidigm and traditional qPCR assays were compared. Lastly, Fluidigm was used to evaluate the association of HIV-infection on the prevalence and density of nasopharyngeal colonisation at 9 and 16 months of age by common, potentially pathogenic bacteria including PCV7 pneumococcal serotypes, non-PCV7 serotypes, Haemophilus influenzae, non-typable Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Streptococcus pyogenes, Neisseria meningitidis, Neisseria lactamica, Bordetella pertussis, Bordetella parapertusis, Bordetella bronchiseptica and Bordetella holmesii in achieved nasophartngeal swabs collected from PCV7-vacciniated HIV-infected and HIV-uninfected children. Results: Molecular qPCR was more sensitive than culture in detecting multiple concurrent colonising pneumococcal serotypes as well as other common nasopharyngeal colonisers, with the majority of additional isolates detected by qPCR having a low carriage density (<104 CFU/ml). Further, qPCR identified a lower prevalence of PCV7-serotype colonisation among PCV7-vaccinated compared to PCV-unvaccinated children at 9 and 16 months of age [adjusted Odds Ratio (aOR): 0.37; 95% CI; 0.19-0.7 and 0.41; 95% CI; 0.26-0.63, respectively]; and an increase in NVT-serotype [aOR: 1.88; 95% CI; 1.02-3.48 and 2.2; 95% CI; 1.18-4.1] colonisation respectively. The increase in NVT carriage among PCV7-vaccinees was driven by serotype 19A, which increased by 53.4% (p=0.021) and 70.7% (p<0.001) at 9 and 16 months of age respectively. Further, 19A had a higher density of colonisation in PCV7-vaccinated groups compared to PCV-unvaccinated groups and was more likely to be identified as a primary than non-primary isolate in PCV7-vaccinated children alone. PCV immunisation was also associated with an increased prevalence of H. influenzae at 9 months (55.8% vs. 66.3%, p<0.001) and 16 months (72% vs. 62%, p=0.017) of age, while a temporary increase in the carriage prevalence of S. aureus was found in PCV7-vaccinated (18.9%) compared to PCV-unvaccinated children (11.1%, aOR 2.1; 95% CI 1.0-1.4; p=0.049) at 9 months of age only. The density of pneumococcus (4.68 vs. 4.28 CFU/ml; p=0.007), H. influenzae (3.86 vs. 4.34 CFU/ml; p=0.008), M. catarrhalis (2.98 vs. 3.52 CFU/ml; p<0.001) and S. aureus (3.06 vs. 4.02 CFU/ml; p=0.02) were also higher among PCV7-vaccinated compared to PCV-unvaccinated children at 9 months age, although this difference diminished with increasing age. There was excellent concordance between the qPCR and Fluidigm for carriage prevalence and density of the majority of assays, with Fluidigm identifying an additional 7 pneumococcal serotypes and 11 bacterial species above those detected by qPCR. Further, discordant results between the two PCR methods were strongly associated with a low carriage density (<102 CFU/ml). Using molecular Fluidigm, a lower carriage prevalence of overall pneumococci (58.6% vs. 69.9%; p=0.02), non-vaccine serotypes (27.8% vs. 40%; p=0.047) and H. influenzae (64.2% vs. 42.3%; p=0.01) was identified in HIV-infected children compared to HIV-uninfected children who were immunised with PCV7 at 9 months of age. No difference in the carriage prevalence of overall pneumococci was however found at 16 months of age (p=0.20), although the carriage prevalence of non-vaccine serotypes (50.9% vs. 60.4%; p=0.049) and H. influenzae (56% vs. 73.4%; p=0.02) was lower in HIV-infected children at 16 months of age. In addition, the density of overall pneumococcus was found to be higher in HIV-infected children (4.81 vs. 4.44 CFU/ml; p=0.014), despite the lower carriage prevalence at 9 months of age, which was driven by a higher density of vaccine serotypes/serogroups (4.21 vs. 3.72 CFU/ml; p=0.04). By 16 months of age, there was no difference in density of pneumococcal colonisation between the HIV-infected and HIV-uninfected children (p=0.89). No difference in the density of H. influenzae was found between HIV-infected and HIV-uninfected infants at 9 months of age (p=0.08); however, by 16 months of age, HIV-uninfected children had a higher density of overall H. influenzae colonisation (4.95 vs. 4.32 CFU/ml; p<0.001), which was largely due to the higher carriage density of NThinf in HIV-uninfected children (5.0 vs. 4.23 CFU/ml; p<0.001). Conclusion: Molecular qPCR assays were shown to be a promising alternative to WHO recommended culture in that multiple pneumococcal serotypes and other bacterial pathogens could be simultaneously detected as well as the bacterial load of each colonising bacteria quantified. The mechanism behind the vaccine effect was shown to be a combination of both serotype replacement and unmasking; however, the reduction in PCV7-serotype colonisation impacted on colonisation prevalence and density of other bacterial species of the nasopharynx and the clinical relevance of this needs further exploration in relation to mucosal and invasive disease outcomes, as well as for higher valence vaccines. While the higher carriage density of overall pneumococcus in HIV-infected children, despite the lower carriage prevalence might explain the higher invasive disease burden in HIV-infected compared to HIV-uninfected children even in the era of antiretroviral therapy treatment and PCV immunisation, future studies are required to provide clarity. Nevertheless, the findings from this thesis highlight the importance of continued surveillance of the circulation of pneumococcal serotypes as well as other bacterial pathogens especially in a population with a high burden of HIV-1 infection.
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    Effect of natural colonization by Streptococcus pneumoniae on the systemic immune responses to common pneumococcal protein antigens with immune protective potential
    (2012-01-17) Ditse, Zanele
    Background: Due to the high cost and limited serotype coverage of pneumococcal conjugate vaccines (PCV), surface proteins of Streptococcus pneumoniae are being investigated for their role as potential vaccine candidates. There are limited data on natural antibody kinetics against pneumococcal surface proteins arising through exposure to pneumococcal nasopharyngeal (NP) colonization in African populations. Objectives: To characterize the natural antibody kinetics and sero-prevalence to 15 pneumococcal proteins with respect to age, PCV vaccination and HIV status as well as to explore the association between antibody titers and pneumococcal nasopharyngeal colonization in infants, older children and adults. Methods: We established a 15-plex Luminex assay for the following proteins: PspA, PspC, LytB, IgA1-proteinase, SP 0082, PdB, PcsB, PsaA, SP 0609, SP 0749, PpmA, SlrA, StkP, SP 2027 and SP 2194, and also validated the Luminex assay comparing it to a standard ELISA method for PspA, PspC, PsaA and PdB. We used the Luminex method to characterize the prevalence and dynamics of serum IgG antibodies against the pneumococcal proteins. The study involved 2 166 human subjects which included: i. A longitudinal cohort of children less than 2 years of age, who were vaccinated with the seven-valent pneumococcal conjugate vaccine (PCV-7) and were either a) HIV-exposed infected, b) HIV-exposed uninfected or c) HIV-unexposed uninfected. ii. A longitudinal cohort of PCV-7 unvaccinated children less than 2 years of age who were either: a) HIV-unexposed uninfected or b) HIV-exposed uninfected. The PCV-7 vaccinated and unvaccinated children were followed up from approximately 4 to 24 months of age. In addition, samples were also analyzed from HIV-uninfected and HIV-infected children Project ID: Pneumococcal protein antigens Student: Zanele Ditse Date: 04 October 2011 - 5 - aged between 4 to 7 years who received either a primary series of PCV-9 or placebo during infancy. Lastly, we analyzed cross-sectional samples from HIV-uninfected and HIV-infected women. Results: The multiplex Luminex assay correlated well with singleplex ELISAs for all four analyzed proteins with correlation coefficients of 0.86, 0.90, 0.87 and 0.96 for PspA, PspC, PdB and PsaA respectively. Antibody titers to PspC, PdB, LytB, SP 0082, PcsB and StkP showed increases in titer with respect to increasing age. Prevailing nasopharyngeal pneumococcal colonization in young children was associated with higher antibody titers to PspA, PspC, PdB, SP 0082, LytB, IgA1-proteinase, PpmA, PcsB and StkP. Conversely higher antibody titers to PspC, PdB, LytB, SP 0082, PcsB and StkP were associated with lower prevalence of pneumococcal colonization in older children and adults. In children under two years of age, PCV vaccination was associated with lower antibody titers to PspA, PspC, LytB, PdB, IgA1-proteinase, PcsB and StkP as well as higher antibody titers against SP 0082 and PpmA at multiple time-points. In PCV-vaccinated children under two years of age, those who were HIV-unexposed , -uninfected had higher antibody titers to PspA, PspC, SP 0082, IgA1-proteinase, PpmA and StkP compared to HIV-exposed, uninfected children. Conclusion: There was an age-related increase in antibody titers to PspA, PspC, PdB, SP 0082, LytB, IgA1-proteinase, PpmA, PcsB, and StkP in children under two years of age. PCV immunization was, however, associated with lower antibody titers to PspA, PspC, LytB, PdB, IgA1-proteinase, PcsB and StkP in young children which was not attributed to differences in the prevalence of nasopharyngeal colonization. Furthermore, HIV-infection status in young children was associated with higher antibody responses to PspA, PspC, PdB, SP 0082, LytB, IgA1-proteinase, PpmA, PcsB and StkP proteins in HIV-unexposed uninfected children compared to HIV-exposed uninfected and HIV-exposed infected children. Higher antibody concentrations to Project ID: Pneumococcal protein antigens Student: Zanele Ditse Date: 04 October 2011 - 6 - PspC, PdB, LytB, SP 0082, PcsB and StkP was negatively associated with nasopharyngeal pneumococcal colonization in older children and adults; indicating a protective role against colonization and a potential role as vaccine candidates.
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    Novel mechanisms of resistance to protein synthesis inhibitors in Streptococcus pneumoniae
    (2008-04-15T09:05:10Z) Wolter, Nicole
    Streptococcus pneumoniae is a leading cause of pneumonia, bacteremia, meningitis, otitis media and sinusitis, and is responsible for significant morbidity and mortality worldwide. The burden of pneumococcal disease has been greatly impacted by the high prevalence of HIV, especially in developing countries. Macrolides are commonly used for the treatment of pneumococcal infections with the resulting effect of increasing resistance. Pneumococci develop resistance to macrolides predominantly by two mechanisms; target modification and drug efflux. Target modification occurs through the acquisition of an erm(B) gene (MLSB phenotype) or through ribosomal mutation, and drug efflux occurs through the acquisition of a mef(A) gene (M phenotype). Alternative protein synthesis-inhibiting antibiotics such as linezolid and telithromycin have been developed in response to the increasing level of antibiotic resistance. In this study, novel mechanisms of resistance to protein synthesis-inhibiting antibiotics, and the current prevalence and epidemiology of macrolide resistance in South Africa were investigated. Two clinical isolates of S. pneumoniae resistant to macrolides, linezolid and chloramphenicol were identified in the PROTEKT surveillance study and the ABCs program of the CDC. The isolates were found to each contain a 6 bp deletion, resulting in the deletion of two amino acids from a highly conserved region of ribosomal protein L4 (64PWRQ67 to 64P_Q67 and 67QKGT70 to 67Q_T70). The genes encoding the mutant ribosomal proteins transformed susceptible strain R6 to macrolide, linezolid and chloramphenicol resistance, proving that the ii deletions conferred the resistance on the isolates, and indicating that these antibiotics share a common binding site. Growth studies of the R6 transformants showed increased mass doubling times, suggesting that the L4 mutations were associated with a fitness cost, but the original strains showed evidence of fitness compensation. The L4 mutations in these isolates represent a novel mechanism of cross-resistance to macrolides, linezolid and chloramphenicol. A macrolide-resistant clinical isolate of S. pneumoniae with mutations in 23S rRNA showed a heterogeneous phenotype and genotype. A mutant gene encoding 23S rRNA from this isolate transformed susceptible strain R6 to resistance. Transformants displayed similar heterogeneity to the isolate. Culture of resistant strain R6 in the presence of antibiotic maintained resistance, however culture of the strain in the absence of antibiotic pressure resulted in a reversion to susceptibility. By DNA sequencing, gene conversion was shown to occur between the wild-type and mutant 23S rRNA alleles. Growth studies indicated that the resistant phenotype was associated with a fitness cost. Therefore, under antibiotic selective pressure alleles converted to the mutant form, and in the absence of selective pressure alleles reverted to wild-type, in order to regain fitness. Through gene conversion the pneumococcus has the ability to rapidly adapt to the environment, with implications for susceptibility testing and patient treatment. A rare clinical isolate of S. pneumoniae, highly resistant to telithromycin, was received from the Canadian Bacterial Surveillance Network and was investigated for the mechanism of resistance. The isolate was found to contain an erm(B) gene iii with a truncated control peptide, as well as a mutant ribosomal protein L4, containing a number of mutations. Transformation of susceptible strain PC13, containing a wild-type erm(B) gene, with the mutant erm(B) gene decreased the susceptibility of PC13 to telithromycin, but did not confer high-level resistance. Transformation of PC13 with the mutant L4 gene or a fragment of the L4 gene containing only the 69GTG71 to TPS mutation, conferred high-level resistance on PC13. In contrast, transformation of R6, which did not contain an erm(B) gene, with the L4 gene or L4 fragment only conferred reduced telithromycin susceptibility. High-level telithromycin resistance was therefore conferred by a combination of an erm(B) gene with a 69GTG71 to TPS mutation in a highly conserved region of ribosomal protein L4. The combination of mechanisms inhibited the binding of telithromycin to the ribosome, whereas neither mechanism individually was sufficient. A telithromycin-resistant clinical isolate of S. pneumoniae was received from the PROTEKT surveillance study and was investigated for the resistance mechanism. The isolate was found to contain a 136 bp deletion in the regulatory region of erm(B). This mutant gene was shown, by transformation studies, to confer resistance on susceptible strain PC13. Expression of erm(B) on the transcriptional level was quantified by real-time reverse transcription PCR. In the presence of erythromycin and telithromycin, erm(B) expression was significantly higher in the mutant PC13 strain than the wild-type strain. Growth studies showed that the mutant PC13 strain had a shorter lag phase than the wild-type strain in the presence of erythromycin. Telithromycin resistance was conferred by the mutant iv erm(B) gene that was expressed at a higher level than the wild-type gene, most likely resulting in higher ribosomal methylation levels sufficient to hinder telithromycin binding. Macrolide resistance in invasive pneumococcal disease in South Africa for the period 2000 to 2005 was investigated through a national laboratory-based surveillance system. Viable isolates (n=15982) collected during the six-year period were phenotypically characterised, by determination of MICs and serotyping. Two hundred and sixty random isolates from 2005 were genotypically screened for the presence of erm(B) and mef(A). Macrolide resistance increased significantly from 9% in 2000 to 14% in 2005. Resistant isolates were received from all provinces of South Africa, with Gauteng and the Western Cape having the highest incidence. Serotype 14 was the most common macrolide-resistant serotype and 96% of macrolide-resistant isolates in 2005 were serotypes included in the 7-valent pneumococcal conjugate vaccine and serotype 6A. Macrolide resistance was significantly higher in children <5 than in individuals 5 years and older. The majority of strains (75%) over the six-year period displayed the MLSB phenotype. Of the 260 strains genotypically screened, 57% were positive for erm(B), 27% were positive for mef(A), 15% contained both erm(B) and mef(A), and 1% were negative for both genes and were found to contain ribosomal mutations. Eighty percent of isolates containing both erm(B) and mef(A) were serotype 19F and were found to be clonal by PFGE and MLST. These multidrug-resistant isolates were related to the Taiwan19F-14 global clone. v Many protein synthesis-inhibiting antibiotics share overlapping binding sites on the large ribosomal subunit. Alterations in 23S rRNA and ribosomal proteins L4 and L22, within the binding pocket, confer resistance and often cross-resistance to many of these antibiotics. The ability of the pneumococcus to develop resistance and the global spread of resistant strains highlights the importance of monitoring resistance levels and understanding resistance mechanisms.