Functional studies of the otrB gene from Streptomyces rimosus

Oxytetracyline, (OTC), is a secondary metabolite antibiotic produced by the actinomycete Streptomyces rimosus. All of the structural genes for OTC synthesis are clustered on the S. rimosus chromosome and flanked by resistance determinants, otrA and otrB. OtrA mediates resistance through non-covalent modification of the bacterial ribosome whilst otrB, the subject of this thesis, acts by efflux of the drug from the cell. At the outset of this thesis otrB had previously been cloned and sequenced. During the course of the work some discrepancies with previously published data were found, parts of the gene were then re-sequenced and a revised otrB sequence submitted to the NCBI database. The OtrB protein is a transmembrane protein belonging the Major facilitator Superfamily (MFS). Sequence comparison with other members of the family shows OtrB to be part of a subfamily of antibiotic transporters and multi drug resistance proteins made up from 14 transmembrane helices (6+2+6) arrangement. OtrB contains many conserved motifs typical of the subfamily and of the MFS. otrB was cloned into E. coli and expressed. The functional activity of the cloned gene was assessed by growth on various concentrations of OTC. Substrate specificity was investigated using TET and CTC in the growth media. Isolation of OtrB from E. coli as a polyhistidine fusion was attempted, reasons why this was not successful are discussed. Tetracycline exporters from Gram-negative bacteria generally contain 12 transmembrane helices (6+6). The relevance of the two "extra" helices present in OtrB was investigated by the construction and expression in E. coli, of a deletion mutant in which the two putative central helices were absent

Clonal expansion within pneumococcal serotype 6C after use of seven-valent vaccine

Streptococcus pneumoniae causes invasive infections, primarily at the extremes of life. A seven-valent conjugate vaccine (PCV7) is used to protect against invasive pneumococcal disease in children. Within three years of PCV7 introduction, we observed a fourfold increase in serotype 6C carriage, predominantly due to a single clone. We determined the whole-genome sequences of nineteen S. pneumoniae serotype 6C isolates, from both carriage (n = 15) and disease (n = 4) states, to investigate the emergence of serotype 6C in our population, focusing on a single multi-locus sequence type (MLST) clonal complex 395 (CC395). A phylogenetic network was constructed to identify different lineages, followed by analysis of variability in gene sets and sequences. Serotype 6C isolates from this single geographical site fell into four broad phylogenetically distinct lineages. Variation was seen in the 6C capsular locus and in sequences of genes encoding surface proteins. The largest clonal complex was characterised by the presence of lantibiotic synthesis locus. In our population, the 6C capsular locus has been introduced into multiple lineages by independent capsular switching events. However, rapid clonal expansion has occurred within a single MLST clonal complex. Worryingly, plasticity exists within current and potential vaccine-associated loci, a consideration for future vaccine use, target selection and design

Carriage rates, circulating serotypes and antibiotic resistance among Streptococcus pneumoniae in healthy infants in Yei, South Sudan

The carriage of Streptococcus pneumoniae, serotypes, antimicrobial susceptibility patterns and disease development are poorly understood in Yei. Availability of affordable antibiotics over the counter, lack of laboratory infrastructure and high rates of penicillin resistance have the potential to aggravate rates of childhood mortality associated with Streptococcus pneumoniae. There is an urgent need to strengthen microbiological and public health services

Genetic diversity of Streptococcus pneumoniae causing meningitis and sepsis in Singapore during the first year of PCV7 implementation.

Streptococcus pneumoniae is a major cause of sepsis, meningitis and respiratory disease worldwide. Pneumococcal conjugate vaccines (PCVs) have now been implemented in many countries worldwide, including Singapore. To evaluate the effectiveness of these vaccines, pneumococcal surveillance studies are required. Detailed and unified pneumococcal epidemiology data are currently scarce in South East Asia. Thus, we present data on invasive pneumococcal (IPD) isolates from Singapore that could assist in evaluating the effectiveness of pneumococcal vaccine in Singapore. One hundred and fifty-nine invasive pneumococcal disease isolates were received by the National Public Health Laboratory in Singapore between June 2009 and August 2010. Isolates were characterized using serotyping and multilocus sequence typing. Twenty-four different serotypes were found, the most common of which were 19A, 3, 7F, 23F, 6B, 14, 8 and 19F (in rank order). One hundred and two sequence types were observed, of which 38 were novel due to new alleles or new combinations of already existing alleles. Based on the Simpson's Index of Diversity, serotypes 3, 6B and 19A were the most genetically diverse. Novel sequence types were more prevalent among conjugate vaccine serotypes 3, 19F and 23F and non-conjugate vaccine serotype 8, serogroup 15 and in non-typable isolates. We have demonstrated considerable genetic diversity among invasive pneumococci before and during the widespread use of conjugate vaccines in Singapore. Approximately half of all novel IPD clones identified in this study were non-conjugate vaccine serotypes. Although PCVs would target the most common serotypes, the high genetic diversity in non-vaccine serotypes would require further surveillance studies

Pre-vaccine serotype composition within a lineage signposts its serotype replacement – a carriage study over 7 years following pneumococcal conjugate vaccine use in the UK

Serotype replacement has been reported in carriage and disease after pneumococcal conjugate vaccine (PCV) introductions in the UK and globally. We previously described concurrent expansion and decline of sequence types associated with serotype replacement over 5 years following PCV introductions in the UK. Here we use whole-genome sequencing to fully characterise the population structure of pneumococcal isolates collected over seven winters encompassing PCV7 and PCV13 introductions in the UK, investigating the importance of lineages in serotype replacement. We analysed 672 pneumococcal genomes from colonised children of 4 years old or less. The temporal prevalence of 20 lineages, defined by hierarchical Bayesian analysis of population structure (BAPS), was assessed in the context of serotype replacement. Multiple serotypes were detected in the primary winter of sampling within three vaccine-type (VT) lineages BAPS4, BAPS10 and BAPS11, in which serotype replacement were observed. In contrast, serotype replacement was not seen in the remaining three VT lineages (BAPS1, BAPS13 and BAPS14), that expressed a single serotype (6B, 6A and 3, respectively) in the primary winter. One lineage, BAPS1 serotype 6B was undetectable in the population towards the end of the study period. The dynamics of serotype replacement, in this UK population, was preceded by the presence or absence of multiple serotypes within VT lineages, in the pre-PCV population. This observation could help predict which non-vaccine types (NVTs) may be involved in replacement in future PCV introductions here and elsewhere. It could further indicate whether any antibiotic resistance associated with the lineages is likely to be affected by replacement.</p

Death or survival from invasive pneumococcal disease in Scotland: associations with serogroups and multilocus sequence types

We describe associations between death from invasive pneumococcal disease (IPD) and particular serogroups and sequence types (STs) determined by multilocus sequence typing (MLST) using data from Scotland. All IPD episodes where blood or cerebrospinal fluid (CSF) culture isolates were referred to the Scottish Haemophilus, Legionella, Meningococcal and Pneumococcal Reference Laboratory (SHLMPRL) from January 1992 to February 2007 were matched to death certification records by the General Register Office for Scotland. This represented 5959 patients. The median number of IPD cases in Scotland each year was 292. Deaths, from any cause, within 30 days of pneumococcal culture from blood or CSF were considered to have IPD as a contributing factor. Eight hundred and thirty-three patients died within 30 days of culture of Streptococcus pneumoniae from blood or CSF [13.95%; 95% confidence interval (13.10, 14.80)]. The highest death rates were in patients over the age of 75. Serotyping data exist for all years but MLST data were only available from 2001 onward. The risk ratio of dying from infection due to particular serogroups or STs compared to dying from IPD due to all other serogroups or STs was calculated. Fisher's exact test with Bonferroni adjustment for multiple testing was used. Age adjustment was accomplished using the Cochran-Mantel-Haenszel test and 95% confidence intervals were reported. Serogroups 3, 11 and 16 have increased probability of causing fatal IPD in Scotland while serogroup 1 IPD has a reduced probability of causing death. None of the 20 most common STs were significantly associated with death within 30 days of pneumococcal culture, after age adjustment. We conclude that there is a stronger association between a fatal outcome and pneumococcal capsular serogroup than there is between a fatal outcome and ST

Comparative Genomics of Carriage and Disease Isolates of Streptococcus pneumoniae Serotype 22F Reveals Lineage-Specific Divergence and Niche Adaptation

Streptococcus pneumoniae is a major cause of meningitis, sepsis, and pneumonia worldwide. Pneumococcal conjugate vaccines have been part of the United Kingdom’s childhood immunization program since 2006 and have significantly reduced the incidence of disease due to vaccine efficacy in reducing carriage in the population. Here we isolated two clones of 22F (an emerging serotype of clinical concern, multilocus sequence types 433 and 698) and conducted comparative genomic analysis on four isolates, paired by Sequence Type (ST) with one of each pair being derived from carriage and the other disease (sepsis). The most compelling observation was of nonsynonymous mutations in pgdA, encoding peptidoglycan N-acetylglucosamine deacetylase A, which was found in the carriage isolates of both ST433 and 698. Deacetylation of pneumococcal peptidoglycan is known to enable resistance to lysozyme upon invasion. Althought no other clear genotypic signatures related to disease or carriage could be determined, additional intriguing comparisons between the two STs were possible. These include the presence of an intact prophage, in addition to numerous additional phage insertions, within the carriage isolate of ST433. Contrasting gene repertoires related to virulence and colonization, including bacteriocins, lantibiotics, and toxin-–antitoxin systems, were also observed

Low-Dose Nitric Oxide as Targeted Anti-biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis

Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases

Parallel evolution in streptococcus pneumoniae biofilms

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and noninvasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole-genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole-genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51 to 264 bp, one insertion resulting in a coding frameshift, and seven nonsense single-nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae and therefore may have important implications for colonization, carriage, and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development

Trends in serotypes and sequence types among cases of invasive pneumococcal disease in Scotland, 1999-2010.

INTRODUCTION: The 7-valent pneumococcal conjugate vaccine (Prevenar(®), Wyeth; PCV7) was introduced to the UK paediatric immunisation schedule in 2006. This study investigates trends in serotypes and multi locus sequence types (STs) among cases of invasive pneumococcal disease (IPD) in Scotland prior to, and following, the introduction of PCV7. METHODS: Scottish Invasive Pneumococcal Disease Enhanced Surveillance has records of all cases of IPD in Scotland since 1999. Cases diagnosed from blood or cerebrospinal fluid isolates until 2010 were analysed. Logistic and poisson regression modelling was used to assess trends prior to and following the introduction of PCV7. RESULTS: Prior to PCV7 use, on average 650 cases of IPD were reported each year; 12% occurred in those aged <5 years and 35% affected those aged over 65 years. Serotypes in PCV7 represented 47% of cases (68% in <5 year olds). The serotype and ST distribution was relatively stable with only serotype 1 and associated ST 306 showing an increasing trend. PCV7 introduction was associated with a 69% (95% CI: 50%, 80%) reduction in the incidence of IPD among those aged <5 years, a 57% (95% CI: 47%, 66%) reduction among those aged 5-64 years but no significant change among those aged 65 years and over where increases in non-PCV7 serotypes were observed. Serotypes which became more prevalent post-PCV7 are those which were associated with STs related to the PCV7 serotypes. CONCLUSIONS: Routine serotyping and sequence typing in Scotland allowed the assessment of the relationship between the capsule and the clones in the post vaccination era. Changes in the distribution of serotypes post PCV7 introduction appear to be driven by associations between serotypes and STs prior to PCV7 introduction. This has implications for the possible effects of the introduction of higher valency vaccines and could aid in predicting replacement serotypes in IPD