Jennifer E. Cornick

Genetic Characterisation of Malawian Pneumococci Prior to the Roll-Out of the PCV13 Vaccine Using a High-Throughput Whole Genome Sequencing Approach

Background Malawi commenced the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the routine infant immunisation schedule in November 2011. Here we have tested the utility of high throughput whole genome sequencing to provide a high-resolution view of pre-vaccine pneumococcal epidemiology and population evolutionary trends to predict potential future change in population structure post introduction. Methods One hundred and twenty seven (127) archived pneumococcal isolates from randomly selected adults and children presenting to the Queen Elizabeth Central Hospital, Blantyre, Malawi underwent whole genome sequencing. Results The pneumococcal population was dominated by serotype 1 (20.5% of invasive isolates) prior to vaccine introduction. PCV13 is likely to protect against 62.9% of all circulating invasive pneumococci (78.3% in under-5-year-olds). Several Pneumococcal Molecular Epidemiology Network (PMEN) clones are now in circulation in Malawi which were previously undetected but the pandemic multidrug resistant PMEN1 lineage was not identified. Genome analysis identified a number of novel sequence types and serotype switching. Conclusions High throughput genome sequencing is now feasible and has the capacity to simultaneously elucidate serotype, sequence type and as well as detailed genetic information. It enables population level characterization, providing a detailed picture of population structure and genome evolution relevant to disease control. Post-vaccine introduction surveillance supported by genome sequencing is essential to providing a comprehensive picture of the impact of PCV13 on pneumococcal population structure and informing future public health interventions.

Recombination in Streptococcus pneumoniae Lineages Increase with Carriage Duration and Size of the Polysaccharide Capsule

ABSTRACT Streptococcus pneumoniae causes a high burden of invasive pneumococcal disease (IPD) globally, especially in children from resource-poor settings. Like many bacteria, the pneumococcus can import DNA from other strains or even species by transformation and homologous recombination, which has allowed the pneumococcus to evade clinical interventions such as antibiotics and pneumococcal conjugate vaccines (PCVs). Pneumococci are enclosed in a complex polysaccharide capsule that determines the serotype; the capsule varies in size and is associated with properties including carriage prevalence and virulence. We determined and quantified the association between capsule and recombination events using genomic data from a diverse collection of serotypes sampled in Malawi. We determined both the amount of variation introduced by recombination relative to mutation (the relative rate) and how many individual recombination events occur per isolate (the frequency). Using univariate analyses, we found an association between both recombination measures and multiple factors associated with the capsule, including duration and prevalence of carriage. Because many capsular factors are correlated, we used multivariate analysis to correct for collinearity. Capsule size and carriage duration remained positively associated with recombination, although with a reduced P value, and this effect may be mediated through some unassayed additional property associated with larger capsules. This work describes an important impact of serotype on recombination that has been previously overlooked. While the details of how this effect is achieved remain to be determined, it may have important consequences for the serotype-specific response to vaccines and other interventions. IMPORTANCE The capsule determines >90 different pneumococcal serotypes, which vary in capsule size, virulence, duration, and prevalence of carriage. Current serotype-specific vaccines elicit anticapsule antibodies. Pneumococcus can take up exogenous DNA by transformation and insert it into its chromosome by homologous recombination. This mechanism has disseminated drug resistance and generated vaccine escape variants. It is hence crucial to pneumococcal evolutionary response to interventions, but there has been no systematic study quantifying whether serotypes vary in recombination and whether this is associated with serotype-specific properties such as capsule size or carriage duration. Larger capsules could physically inhibit DNA uptake, or given the longer carriage duration for larger capsules, this may promote recombination. We find that recombination varies among capsules and is associated with capsule size, carriage duration, and carriage prevalence and negatively associated with invasiveness. The consequence of this work is that serotypes with different capsules may respond differently to selective pressures like vaccines. The capsule determines >90 different pneumococcal serotypes, which vary in capsule size, virulence, duration, and prevalence of carriage. Current serotype-specific vaccines elicit anticapsule antibodies. Pneumococcus can take up exogenous DNA by transformation and insert it into its chromosome by homologous recombination. This mechanism has disseminated drug resistance and generated vaccine escape variants. It is hence crucial to pneumococcal evolutionary response to interventions, but there has been no systematic study quantifying whether serotypes vary in recombination and whether this is associated with serotype-specific properties such as capsule size or carriage duration. Larger capsules could physically inhibit DNA uptake, or given the longer carriage duration for larger capsules, this may promote recombination. We find that recombination varies among capsules and is associated with capsule size, carriage duration, and carriage prevalence and negatively associated with invasiveness. The consequence of this work is that serotypes with different capsules may respond differently to selective pressures like vaccines.

Trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in Malawi (1998–2016): a surveillance study

Summary Background Bacterial bloodstream infection is a common cause of morbidity and mortality in sub-Saharan Africa, yet few facilities are able to maintain long-term surveillance. The Malawi-Liverpool-Wellcome Trust Clinical Research Programme has done sentinel surveillance of bacteraemia since 1998. We report long-term trends in bloodstream infection and antimicrobial resistance from this surveillance. Methods In this surveillance study, we analysed blood cultures that were routinely taken from adult and paediatric patients with fever or suspicion of sepsis admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi from 1998 to 2016. The hospital served an urban population of 920 000 in 2016, with 1000 beds, although occupancy often exceeds capacity. The hospital admits about 10 000 adults and 30 000 children each year. Antimicrobial susceptibility tests were done by the disc diffusion method according to British Society of Antimicrobial Chemotherapy guidelines. We used the Cochran-Armitage test for trend to examine trends in rates of antimicrobial resistance, and negative binomial regression to examine trends in icidence of bloodstream infection over time. Findings Between Jan 1, 1998, and Dec 31, 2016, we isolated 29 183 pathogens from 194 539 blood cultures. Pathogen detection decreased significantly from 327·1/100 000 in 1998 to 120·2/100 000 in 2016 (p<0·0001). 13 366 (51·1%) of 26 174 bacterial isolates were resistant to the Malawian first-line antibiotics amoxicillin or penicillin, chloramphenicol, and co-trimoxazole; 68·3% of Gram-negative and 6·6% of Gram-positive pathogens. The proportions of non-Salmonella Enterobacteriaceae with extended spectrum beta-lactamase (ESBL) or fluoroquinolone resistance rose significantly after 2003 to 61·9% in 2016 (p<0·0001). Between 2003 and 2016, ESBL resistance rose from 0·7% to 30·3% in Escherichia coli, from 11·8% to 90·5% in Klebsiella spp and from 30·4% to 71·9% in other Enterobacteriaceae. Similarly, resistance to ciprofloxacin rose from 2·5% to 31·1% in E coli, from 1·7% to 70·2% in Klebsiella spp and from 5·9% to 68·8% in other Enterobacteriaceae. By contrast, more than 92·0% of common Gram-positive pathogens remain susceptible to either penicillin or chloramphenicol. Meticillin-resistant Staphylococcus aureus (MRSA) was first reported in 1998 at 7·7% and represented 18·4% of S aureus isolates in 2016. Interpretation The rapid expansion of ESBL and fluoroquinolone resistance among common Gram-negative pathogens, and the emergence of MRSA, highlight the growing challenge of bloodstream infections that are effectively impossible to treat in this resource-limited setting. Funding Wellcome Trust, H3ABionet, Southern Africa Consortium for Research Excellence (SACORE).

Invasive Streptococcus pneumoniae in Children, Malawi, 2004–2006

Of 176 invasive Streptococcus pneumoniae isolates from children in Malawi, common serotypes were 1 (23%), 6A/B (18%), 14 (6%), and 23F (6%). Coverage with the 7-valent pneumococcal conjugate vaccine (PCV) was 39%; PCV10 and PCV13 increased coverage to 66% and 88%, respectively. We found chloramphenicol resistance in 27% of isolates and penicillin nonsusceptibility in 10% (by using meningitis breakpoints); all were ceftriaxone susceptible.

Mechanisms and impact of genetic recombination in the evolution of Streptococcus pneumoniae

Streptococcus pneumoniae (the pneumococcus) is a highly recombinogenic bacterium responsible for a high burden of human disease globally. Genetic recombination, a process in which exogenous DNA is acquired and incorporated into its genome, is a key evolutionary mechanism employed by the pneumococcus to rapidly adapt to selective pressures. The rate at which the pneumococcus acquires genetic variation through recombination is much higher than the rate at which the organism acquires variation through spontaneous mutations. This higher rate of variation allows the pneumococcus to circumvent the host innate and adaptive immune responses, escape clinical interventions, including antibiotic therapy and vaccine introduction. The rapid influx of whole genome sequence (WGS) data and the advent of novel analysis methods and powerful computational tools for population genetics and evolution studies has transformed our understanding of how genetic recombination drives pneumococcal adaptation and evolution. Here we discuss how genetic recombination has impacted upon the evolution of the pneumococcus.

Genomic identification of a novel co-trimoxazole resistance genotype and its prevalence amongst Streptococcus pneumoniae in Malawi

Objectives This study aimed to define the molecular basis of co-trimoxazole resistance in Malawian pneumococci under the dual selective pressure of widespread co-trimoxazole and sulfadoxine/pyrimethamine use. Methods We measured the trimethoprim and sulfamethoxazole MICs and analysed folA and folP nucleotide and translated amino acid sequences for 143 pneumococci isolated from carriage and invasive disease in Malawi (2002–08). Results Pneumococci were highly resistant to both trimethoprim and sulfamethoxazole (96%, 137/143). Sulfamethoxazole-resistant isolates showed a 3 or 6 bp insertion in the sulphonamide-binding site of folP. The trimethoprim-resistant isolates fell into three genotypic groups based on dihydrofolate reductase (encoded by folA) mutations: Ile-100-Leu (10%), the Ile-100-Leu substitution together with a residue 92 substitution (56%) and those with a novel uncharacterized resistance genotype (34%). The nucleotide sequence divergence and dN/dS of folA and folP remained stable from 2004 onwards. Conclusions S. pneumoniae exhibit almost universal co-trimoxazole resistance in vitro and in silico that we believe is driven by extensive co-trimoxazole and sulfadoxine/pyrimethamine use. More than one-third of pneumococci employ a novel mechanism of co-trimoxazole resistance. Resistance has now reached a point of stabilizing evolution. The use of co-trimoxazole to prevent pneumococcal infection in HIV/AIDS patients in sub-Saharan Africa should be re-evaluated.

Region-specific diversification of the highly virulent serotype 1 Streptococcus pneumoniae.

Serotype 1 Streptococcus pneumoniae is a leading cause of invasive pneumococcal disease (IPD) worldwide, with the highest burden in developing countries. We report the whole-genome sequencing analysis of 448 serotype 1 isolates from 27 countries worldwide (including 11 in Africa). The global serotype 1 population shows a strong phylogeographic structure at the continental level, and within Africa there is further region-specific structure. Our results demonstrate that region-specific diversification within Africa has been driven by limited cross-region transfer events, genetic recombination and antimicrobial selective pressure. Clonal replacement of the dominant serotype 1 clones circulating within regions is uncommon; however, here we report on the accessory gene content that has contributed to a rare clonal replacement event of ST3081 with ST618 as the dominant cause of IPD in the Gambia.

Comparative Genomic Analysis of Meningitis- and Bacteremia-Causing Pneumococci Identifies a Common Core Genome

ABSTRACT Streptococcus pneumoniae is a nasopharyngeal commensal that occasionally invades normally sterile sites to cause bloodstream infection and meningitis. Although the pneumococcal population structure and evolutionary genetics are well defined, it is not clear whether pneumococci that cause meningitis are genetically distinct from those that do not. Here, we used whole-genome sequencing of 140 isolates of S. pneumoniae recovered from bloodstream infection (n = 70) and meningitis (n = 70) to compare their genetic contents. By fitting a double-exponential decaying-function model, we show that these isolates share a core of 1,427 genes (95% confidence interval [CI], 1,425 to 1,435 genes) and that there is no difference in the core genome or accessory gene content from these disease manifestations. Gene presence/absence alone therefore does not explain the virulence behavior of pneumococci that reach the meninges. Our analysis, however, supports the requirement of a range of previously described virulence factors and vaccine candidates for both meningitis- and bacteremia-causing pneumococci. This high-resolution view suggests that, despite considerable competency for genetic exchange, all pneumococci are under considerable pressure to retain key components advantageous for colonization and transmission and that these components are essential for access to and survival in sterile sites.

The global distribution and diversity of protein vaccine candidate antigens in the highly virulent Streptococcus pnuemoniae serotype 1

Serotype 1 is one of the most common causes of pneumococcal disease worldwide. Pneumococcal protein vaccines are currently being developed as an alternate intervention strategy to pneumococcal conjugate vaccines. Pre-requisites for an efficacious pneumococcal protein vaccine are universal presence and minimal variation of the target antigen in the pneumococcal population, and the capability to induce a robust human immune response. We used in silico analysis to assess the prevalence of seven protein vaccine candidates (CbpA, PcpA, PhtD, PspA, SP0148, SP1912, SP2108) among 445 serotype 1 pneumococci from 26 different countries, across four continents. CbpA (76%), PspA (68%), PhtD (28%), PcpA (11%) were not universally encoded in the study population, and would not provide full coverage against serotype 1. PcpA was widely present in the European (82%), but not in the African (2%) population. A multi-valent vaccine incorporating CbpA, PcpA, PhtD and PspA was predicted to provide coverage against 86% of the global population. SP0148, SP1912 and SP2108 were universally encoded and we further assessed their predicted amino acid, antigenic and structural variation. Multiple allelic variants of these proteins were identified, different allelic variants dominated in different continents; the observed variation was predicted to impact the antigenicity and structure of two SP0148 variants, one SP1912 variant and four SP2108 variants, however these variants were each only present in a small fraction of the global population (<2%). The vast majority of the observed variation was predicted to have no impact on the efficaciousness of a protein vaccine incorporating a single variant of SP0148, SP1912 and/or SP2108 from S. pneumoniae TIGR4. Our findings emphasise the importance of taking geographic differences into account when designing global vaccine interventions and support the continued development of SP0148, SP1912 and SP2108 as protein vaccine candidates against this important pneumococcal serotype.

Population genetic structure, antibiotic resistance, capsule switching and evolution of invasive pneumococci before conjugate vaccination in Malawi

Highlights • High pneumococcal population diversity in terms of serotypes and sequence types (ST).• Decline in IPD incidence pre-vaccination not associated with specific serotypes.• High prevalence and antibiotic resistance rates in serotype 1 isolates.• High levels of capsule (serotype) switching pre-vaccination.• Surveillance remains crucial to understand pneumococcal epidemiology.