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.

Changes in serotype prevalence of Streptococcus pneumoniae in Southampton, UK between 2006 and 2018.

Streptococcus pneumoniae continues to cause significant disease burden. Whilst pneumococcal conjugate vaccines (PCV) have substantially reduced this burden, serotype replacement partially negates this success due to increased disease associated with non-vaccine serotypes (NVTs). Continued surveillance is therefore essential to provide crucial epidemiological data. Annual cross-sectional surveillance of paediatric pneumococcal carriage was started in Southampton, UK following PCV7 roll-out in 2006. Nasopharyngeal swabs were collected from children < 5 years old each winter (October to March) from 2006/07 and for each consecutive year until 2017/18. Pneumococcal serotype was inferred from whole genome sequencing data. A total of 1429 (32.5%) pneumococci were isolated from 4093 children. Carriage ranged from 27.8% (95%CI 23.7-32.7) in 2008/09 to 37.9% (95%CI 32.8-43.2) in 2014/15. Analyses showed that carriage increased in children aged 24-35 months (p < 0.001) and 47-60 months (p < 0.05). Carriage of PCV serotypes decreased markedly following PCV7 and/or PCV13 introduction, apart from serotype 3 where the relative frequency was slightly lower post-PCV13 (pre-PCV13 n = 7, 1.67%; post-PCV13 n = 13, 1.27%). Prevalence of NVTs implicated in increased disease was low with 24F (n = 19, 1.4%) being the most common followed by 9N (n = 11, 0.8%), 8 (n = 7, 0.5%) and 12F (n = 3, 0.2%).

13-valent pneumococcal conjugate vaccine (PCV13).

The thirteen valent pneumococcal conjugate vaccine (PCV13, Prevenar 13(TM)) is the broader coverage successor to the highly effective seven valent vaccine (PCV7, Prevenar(TM)) which has reduced rates of pneumococcal disease in many countries. Despite the success of PCV7, pneumococcal disease due to non-PCV7 serotypes remains a threat in many settings, in particular many developing countries with a high burden of pneumococcal disease where serotype 1 and 5 are among the most common serotypes. Disease due to certain non-PCV7 serotypes, in particular serotype 19A has also begun to increase in incidence in countries with widespread use of PCV7. PCV13 consists of thirteen pneumococcal capsular polysaccharides individually conjugated to the diphtheria-derived protein carrier CRM(197). In addition to serotypes 4, 6B, 9V, 14, 18C, 19F and 23F included in PCV7, PCV13 also includes serotypes 1, 3, 5, 6A, 7F and 19A. PCV13 was licensed on the basis of non-inferiority trials and has proved to be at least as safe and effective as PCV7. PCV13 replaced PCV7 in the childhood immunisation schedules of the USA and UK in 2010 and is being rolled out to an increasing number of developing countries during 2011. Here we review the current literature regarding this vaccine, describing safety, efficacy, global serotype coverage and use and future directions.

Microbial epidemiology and carriage studies for the evaluation of vaccines.

Respiratory tract infections are responsible for over 2.8 million deaths per year worldwide. Colonization is the first step in the process of microbes occupying the respiratory tract, which may lead to subsequent infection. Carriage, in contrast, is defined as the occupation of microbial species in the respiratory tract. The duration of carriage may be affected by host immunity, the composition and interactions between members of the microbial community, and the characteristics of colonizing bacteria, including physiology associated with being present in a bacterial biofilm. Numerous vaccines have been implemented to control infections caused by bacteria that can colonize and be subsequently carried. Such vaccines are often species-specific and may target a limited number of strains thereby creating a vacant niche in the upper respiratory tract. Epidemiological changes of bacteria found in both carriage and disease have therefore been widely reported, since the vacant niche is filled by other strains or species. In this review, we discuss the use of carriage-prevalence studies in vaccine evaluation and argue that such studies are essential for (1) examining the epidemiology of carriage before and after the introduction of new vaccines, (2) understanding the dynamics of the respiratory tract flora and (3) identifying the disease potential of emerging strains. In an era of increasing antibiotic resistance, bacterial carriage-prevalence studies are essential for monitoring the impact of vaccination programmes.

PclA, a pneumococcal collagen-like protein with selected strain distribution, contributes to adherence and invasion of host cells.

Analysis of Streptococcus pneumoniae sequenced genomes revealed a region present only in selected strains consisting of two ORFs: a putative cell wall anchored protein and a putative transcriptional regulator. The cell wall anchored protein contains large regions of collagen-like repeats, the number of which varies between strains. We have therefore named this protein PclA for pneumococcal collagen-like protein A. The second gene, spr1404, encodes a putative transcriptional regulator. We examined the strain distribution of these two genes among a collection of clinical isolates from invasive pneumococcal disease and found them to be present in 39% of the strains examined. Strains were either positive for both genes or lacked both, with the two genes always present together in the same location of the genome. RT-PCR analysis revealed that pclA is transcribed in vitro, even in the absence of spr1404. Single deletion mutants lacking either gene were not attenuated in a mouse model of invasive pneumonia. However, the pclA mutant was defective in adherence and invasion of host cells in vitro.

The adhesins of non-typeable Haemophilus influenzae.

INTRODUCTION: Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen of the respiratory tract and the greatest contributor to invasive Haemophilus disease. Additionally, in children, NTHi is responsible for the majority of otitis media (OM) which can lead to chronic infection and hearing loss. In adults, NTHi infection in the lungs is responsible for the onset of acute exacerbations in chronic obstructive pulmonary disease (COPD). Unfortunately, there is currently no vaccine available to protect against NTHi infections. Areas covered: NTHi uses an arsenal of adhesins to colonise the respiratory epithelium. The adhesins also have secondary roles that aid in the virulence of NTHi, including mechanisms that avoid immune clearance, adjust pore size to avoid antimicrobial destruction, form micro-colonies and invoke phase variation for protein mediation. Bacterial adhesins can also be ideal antigens for subunit vaccine design due to surface exposure and immunogenic capabilities. Expert commentary: The host-pathogen interactions of the NTHi adhesins are not fully investigated. The relationship between adhesins and the extracellular matrix (ECM) play a part in the success of NTHi colonisation and virulence by immune evasion, migration and biofilm development. Further research into these immunogenic proteins would further our understanding and enable a basis for better combatting NTHi disease.

Patients with Chronic Obstructive Pulmonary Disease harbour a variation of Haemophilus species.

H. haemolyticus is often misidentified as NTHi due to their close phylogenetic relationship. Differentiating between the two is important for correct identification and appropriate treatment of infective organism and to ensure any role of H. haemolyticus in disease is not being overlooked. Speciation however is not completely reliable by culture and PCR methods due to the loss of haemolysis by H. haemolyticus and the heterogeneity of NTHi. Haemophilus isolates from COPD as part of the AERIS study (ClinicalTrials - NCT01360398) were speciated by analysing sequence data for the presence of molecular markers. Further investigation into the genomic relationship was carried out using average nucleotide identity and phylogeny of allelic and genome alignments. Only 6.3% were identified as H. haemolyticus. Multiple in silico methods were able to distinguish H. haemolyticus from NTHi. However, no single gene target was found to be 100% accurate. A group of omp2 negative NTHi were observed to be phylogenetically divergent from H. haemolyticus and remaining NTHi. The presence of an atypical group from a geographically and disease limited set of isolates supports the theory that the heterogeneity of NTHi may provide a genetic continuum between NTHi and H. haemolyticus.

Ecology and diversity in upper respiratory tract microbial population structures from a cross-sectional community swabbing study.

PURPOSE: Respiratory tract infections (RTIs) are responsible for over 2.8 million deaths per year worldwide with pathobiont carriage a required precursor to infection. We sought to determine carriage epidemiology for both bacterial and viral respiratory pathogens as part of a large population-based cross-sectional carriage study. METHODOLOGY: Nose self-swab samples were collected in two separate time-points, May to August 2012 (late spring/summer) and February to April 2013 (winter/early spring). The presence of six bacterial species: S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus, P. aeruginosa and N. meningitidis in addition to respiratory syncytial virus, influenza viruses A and B, rhinovirus/enterovirus, coronavirus, parainfluenza viruses 1-3 and adenovirus was determined using culture and PCR methods.Results/Key findings. Carriage was shown to vary with age, recent RTI and the presence of other species. Spatial structures of microbial communities were more disordered in the 0-4 age group and those with recent RTI. Species frequency distributions were flatter than random expectation in young individuals (X2=20.42, P=0.002), indicating spatial clumping of species consistent with facilitative relationships. Deviations from a neutral model of ecological niches were observed in summer samples and from older individuals but not in the winter or younger individuals (0-4 years), suggesting the presence of seasonal and age-dependent niche processes in respiratory community assembly. CONCLUSION: The application of epidemiological methods and ecological theory to respiratory tract samples has yielded novel insights into the factors that drive microbial community composition.

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.

The rise and fall of pneumococcal serotypes carried in the PCV era.

Streptococcus pneumoniae is a major cause of meningitis, sepsis and pneumonia worldwide. Vaccination using pneumococcal conjugate vaccines (PCV) has therefore been part of the UK's childhood immunisation programme since 2006. Here we describe pneumococcal carriage rates in children under five years of age attending the paediatric department of a large UK hospital in response to vaccine implementation over seven winter seasons from 2006 to 2013. S. pneumoniae (n=696) were isolated from nasopharyngeal swabs (n=2267) collected during seven consecutive winters, October to March, 2006/7 to 2012/13. This includes the period immediately following the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) in 2006 in addition to pre- and post-PCV13 introduction in 2010. We show a decrease in PCV13 vaccine serotypes (VT) in the three years following PCV13 vaccine implementation (2010/11 to 2012/13). Serotype 6A represented the only observed VT following PCV13 implementation with all others (including PCV7 serotypes) absent from carriage. Overall pneumococcal carriage, attributable to non-VT (NVT), was consistent across all sampling years with a mean of 31·1%. The ten most frequently isolated NVTs were 6C, 11A, 15B, 23B, 15A, 21, 22F, 35F, 23A and 15C. Fluctuations in the prevalence of each were however noted. Comparing prevalence at 2006/07 with 2012/13 only 15A was shown to have increased significantly (p value of 0·003) during the course of PCV implementation. These data support the increasing evidence that the primary effect of PCVs is due to population immunity by reducing or eliminating the carriage of invasive VT serotypes. With IPD being increasingly attributed to non-vaccine serotypes, surveillance of carriage data continues to act as an early warning system for vaccine design and public health policy that require continual data of both carried pneumococcal serotypes and IPD attributed serotype data.

Pneumococcal conjugate vaccine implementation in middle-income countries.

BACKGROUND: Since 2000, the widespread adoption of pneumococcal conjugate vaccines (PCVs) has had a major impact in the prevention of pneumonia. Limited access to international financial support means some middle-income countries (MICs) are trailing in the widespread use of PCVs. We review the status of PCV implementation, and discuss any needs and gaps related to low levels of PCV implementation in MICs, with analysis of possible solutions to strengthen the PCV implementation process in MICs. MAIN BODY: We searched PubMed, PubMed Central, Ovid MEDLINE, and SCOPUS databases using search terms related to pneumococcal immunization, governmental health policy or programmes, and MICs. Two authors independently reviewed the full text of the references, which were assessed for eligibility using pre-defined inclusion and exclusion criteria. The search terms identified 1,165 articles and the full texts of 21 were assessed for suitability, with eight articles included in the systematic review. MICs are implementing PCVs at a slower rate than donor-funded low-income countries and wealthier developed countries. A significant difference in the uptake of PCV in lower middle-income countries (LMICs) (71%) and upper middle-income countries (UMICs) (48%) is largely due to an unsuccessful process of "graduation" of MICs from GAVI assistance, an issue that arises as countries cross the income eligibility threshold and are no longer eligible to receive the same levels of financial assistance. A lack of country-specific data on disease burden, a lack of local expertise in economic evaluation, and the cost of PCV were identified as the leading causes of the slow uptake of PCVs in MICs. Potential solutions mentioned in the reviewed papers include the use of vaccine cost-effectiveness analysis and the provision of economic evidence to strengthen decision-making, the evaluation of the burden of disease, and post-introduction surveillance to monitor vaccine impact. CONCLUSION: The global community needs to recognise the impediments to vaccine introduction into MICs. Improving PCV access could help decrease the incidence of pneumonia and reduce the selection pressure for pneumococcal antimicrobial resistance.

Pneumococci causing invasive disease in children prior to the introduction of pneumococcal conjugate vaccine in Scotland.

This study aimed to determine the serotypes and sequence types (STs) of pneumococci causing paediatric invasive disease in Scotland prior to the introduction of pneumococcal conjugate vaccines (PCVs). All invasive pneumococci isolated between 2000 and 2004 from children aged less than 5 years in Scotland were used. The isolates were characterized by serotyping and multi-locus sequence typing. Two hundred and seventeen pneumococci were characterized into 22 different serogroups/types, the most common, in rank order, being 14, 19F, 6B, 18C, 23F, 9V, 4, 1, 19A and 6A. They were further genotyped into 77 different STs, the three most common being 9, 162 and 176. Common serotypes possessed multiple STs, but pneumococci of a particular clone were mostly associated with a particular serotype. The seven most common serotypes are included in the 7-valent polysaccharide conjugate vaccine (PCV7). Serotype coverage for PCV7 was 76.5% in those aged less than 5 years but increased to 88.9% for those aged 1 year. The introduction of PCV7 into the childhood immunization schedule would reduce the burden of pneumococcal disease in children, although continued surveillance of invasive pneumococcal disease will be required before, during and after the introduction of PCVs.

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.

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.

Vaccine preventable meningitis in Malaysia: epidemiology and management.

Worldwide bacterial meningitis accounts for more than one million cases and 135,000 deaths annually. Profound, lasting neurological complications occur in 9-25% of cases. This review confirms the greatest risk from bacterial meningitis is in early life in Malaysia. Much of the disease burden can be avoided by immunization, particularly against Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae. Despite inclusion of the Hib vaccine in the National Immunisation Programme and the licensure of pneumococcal vaccines, these two species are the main contributors to bacterial meningitis in Malaysia, with Neisseria meningitidis and Mycobacterium tuberculosis, causing a smaller proportion of disease. The high Hib prevalence may partly be due to dated, small-scale studies limiting the understanding of the current epidemiological situation. This highlights the need for larger, better quality surveillance from Malaysia to evaluate the success of Hib immunization and to help guide immunization policy for vaccines against S. pneumoniae and N. meningitidis.

Current methods for capsular typing of Streptococcus pneumoniae.

Streptococcus pneumoniae is a major respiratory tract pathogen causing pneumococcal disease mainly in children aged less than five years and in the elderly. Ninety-eight different capsular types (serotypes) of pneumococci have been reported, but pneumococcal conjugate vaccines (PCV) include polysaccharide antigens against only 7, 10 or 13 serotypes. It is therefore important to track the emergence of serotypes due to the clonal expansion of non-vaccine serotypes. Increased numbers of carried and disease-causing pneumococci are now being analysed as part of the post-PCV implementation surveillance studies and hence rapid, accurate and cost-effective typing methods are important. Here we describe serotyping methods published prior to 10th November 2014 for pneumococcal capsule typing. Sixteen methods were identified; six were based on serological tests using immunological properties of the capsular epitopes, eight were semi-automated molecular tests, and one describes the identification of capsular type directly from whole genome data, which also allows for further intra and inter-genome analyses. There was no single method that could be recommended for all pneumococcal capsular typing applications. Although the Quellung reaction is still considered to be the gold-standard, laboratories should take into account the number of pneumococcal isolates and the type of samples to be used for testing, the time frame for the results and the resources available in order to select the most appropriate method. Most likely, a combination of phenotypic and genotypic methods would be optimal to monitor and evaluate the impact of pneumococcal conjugate vaccines and to provide information for future vaccine formulations.

Five winters of pneumococcal serotype replacement in UK carriage following PCV introduction.

The seven-valent pneumococcal conjugate vaccine (PCV7) was added to the UK national immunisation programme in September 2006. PCV13 replaced PCV7 in April 2010. As carriage precedes disease cases this study collected carried pneumococci from children each winter from 2006/7 to 2010/11 over PCV introduction. Conventional microbiology and whole genome sequencing were utilised to characterise pneumococcal strains. Overall prevalence of pneumococcal carriage remained stable. Vaccine serotypes (VT) decreased (p<0.0001) with concomitant increases in non-vaccine serotypes (NVT). In winter 2010/11 only one isolate of PCV7 VT was observed (6B). PCV13 unique VTs decreased between winters immediately preceding and following PCV13 introduction (p=0.04). Significant decreases for VTs 6B, 19F, 23F (PCV7) and 6A (PCV13) and increases for NVT 21, 23B, 33F and 35F were detected. The serotype replacement was accompanied by parallel changes in genotype prevalence for associated sequence types with clonal expansion contributing to replacement. By winter 2010/11, serotype coverage of PCV7 and PCV13 was 1% and 11% respectively. VT replacement was observed for PCV7 and PCV13 serotypes. Conjugate vaccine design and use requires continuous monitoring and revision.

Automated pneumococcal MLST using liquid-handling robotics and a capillary DNA sequencer.

Multilocus sequence typing (MLST) is used by the Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) as a routine method for the characterization of certain bacterial pathogens. The SMPRL recently started performing MLST on strains of Streptococcus pneumoniae, and here we describe a fully automated method for MLST using a 96-well-format liquid-handling robot and a 96-capillary automated DNA sequencer.

Lifestyle risk factors for invasive pneumococcal disease: a systematic review.

OBJECTIVE: To systematically review the literature for evidence of smoking and alcohol intake as independent risk factors for invasive pneumococcal disease (IPD). DESIGN: Systematic review. METHODS: MEDLINE (1946-May 2012) and EMBASE (1947-May 2012) were searched for studies investigating alcohol or smoking as risk factors for acquiring IPD and which reported results as relative risk. Studies conducted exclusively in clinical risk groups, those assessing risk factors for outcomes other than acquisition of IPD and studies describing risk factors without quantifying a relative risk were excluded. RESULTS: Seven observational studies were identified and reviewed; owing to the heterogeneity of study design, meta-analysis was not attempted. Five of six studies investigating smoking reported an increased risk of IPD in the range 2.2-4.1. Four of the six studies investigating alcohol intake reported a significant increased risk for IPD ranging from 2.9 to 11.4, while one reported a significant protective effect. CONCLUSIONS: Overall, these observational data suggest that smoking and alcohol misuse may increase the risk of IPD in adults, but the magnitude of this risk remains unclear and should be explored with further research. The findings of this review will contribute to the debate on whether pneumococcal vaccine should be offered to smokers and people who misuse alcohol in addition to other clinically defined risk groups.