Genomic landscape of extended-spectrum β-lactamase resistance in Escherichia coli from an urban African setting

Objectives: Efforts to treat Escherichia coli infections are increasingly being compromised by the rapid, global spread of antimicrobial resistance (AMR). Whilst AMR in E. coli has been extensively investigated in resource-rich settings, in sub-Saharan Africa molecular patterns of AMR are not well described. In this study, we have begun to explore the population structure and molecular determinants of AMR amongst E. coli isolates from Malawi. Methods: Ninety-four E. coli isolates from patients admitted to Queen’s Hospital, Malawi, were whole-genome sequenced. The isolates were selected on the basis of diversity of phenotypic resistance profiles and clinical source of isolation (blood, CSF and rectal swab). Sequence data were analysed using comparative genomics and phylogenetics. Results: Our results revealed the presence of five clades, which were strongly associated with E. coli phylogroups A, B1, B2, D and F. We identified 43 multilocus STs, of which ST131 (14.9%) and ST12 (9.6%) were the most common. We identified 25 AMR genes. The most common ESBL gene was blaCTX-M-15 and it was present in all five phylogroups and 11 STs, and most commonly detected in ST391 (4/4 isolates), ST648 (3/3 isolates) and ST131 [3/14 (21.4%) isolates]. Conclusions: This study has revealed a high diversity of lineages associated with AMR, including ESBL and fluoroquinolone resistance, in Malawi. The data highlight the value of longitudinal bacteraemia surveillance coupled with detailed molecular epidemiology in all settings, including low-income settings, in describing the global epidemiology of ESBL resistance

Effect of intra-partum azithromycin on the development of the infant nasopharyngeal microbiota: A post hoc analysis of a double-blind randomized trial.

BACKGROUND: Sepsis is a leading cause of neonatal death. Intrapartum azithromycin reduces neonatal nasopharyngeal carriage of potentially pathogenic bacteria, a prerequisite for sepsis. Early antibiotic exposure has been associated with microbiota perturbations with varying effects. This study aims to understand the effect of intrapartum azithromycin intervention on the developing nasopharyngeal microbiota of the child. METHODS: Using 16S rRNA gene sequencing, we analysed the microbiota of 343 nasopharyngeal samples collected from birth to 12 months from 109 healthy infants selected from a double-blind randomized placebo-controlled clinical trial conducted in the Gambia (PregnAnZI-1). In the trial, 829 women were given 2g oral azithromycin or placebo (1:1) during labour with the objective of reducing bacterial carriage in mother and child during the neonatal period. The post-hoc analysis presented here assessed the effect of the intervention on the child nasopharyngeal microbiota development. FINDINGS: 55 children were from mothers given azithromycin and 54 from mothers given placebo. Comparing arms, we found an increase in alpha-diversity at day-6 (p = 0·018), and a significant effect on overall microbiota composition at days 6 and 28 (R2 = 4.4%, q = 0·007 and R2 = 2.3%, q = 0·018 respectively). At genus level, we found lower representation of Staphylococcus at day-6 (q = 0·0303) and higher representation of Moraxella at 12 months (q = 0·0443). Unsupervised clustering of samples by microbial community similarity showed different community dynamics between the intervention and placebo arms during the neonatal period. INTERPRETATION: These results indicate that intrapartum azithromycin caused short-term alterations in the nasopharyngeal microbiota with modest overall effect at 12 months of age. Further exploration of the effects of these variations on microbiome function will give more insight on the potential risks and benefits, for the child, associated with this intervention. FUNDING: This work was jointly funded by the Medical Research Council (UK) (MC_EX_MR/J010391/1/MRC), Bill & Melinda Gates Foundation (OPP1196513), and MRCG@LSHTM Doctoral Training Program

Early signals of vaccine driven perturbation seen in pneumococcal carriage population genomic data

BACKGROUND: Pneumococcal conjugate vaccines (PCV) have reduced pneumococcal diseases globally. Pneumococcal genomic surveys elucidate PCV effects on population structure but are rarely conducted in low-income settings despite the high disease burden. METHODS:We undertook whole genome sequencing of 660 pneumococcal isolates collected through surveys from healthy carriers two years from PCV14 introduction and one-year post-rollout in northern Malawi. We investigated changes in population structure, within-lineage serotype dynamics, serotype diversity, and frequency of antibiotic resistance (ABR) and accessory genes. RESULTS:In the under-fives, frequency and diversity of vaccine serotypes (VT) decreased significantly post-PCV but no significant changes occurred in over-fives. Clearance of VT serotypes was consistent across different genetic backgrounds (lineages). There was an increase of non-vaccine serotypes (NVT) namely 7C, 15B/C, 23A in under-fives but 28F increased in both age groups. While carriage rates have been recently shown to remain stable post-PCV due replacement serotypes, there was no change in diversity of NVTs. Additionally, frequency of intermediate-penicillin-resistant lineages decreased post-PCV. While frequency of ABR genes remained stable, other accessory genes especially those associated with MGEs and bacteriocins showed changes in frequency post-PCV. CONCLUSIONS:We demonstrate evidence of significant population restructuring post-PCV driven by decreasing frequency of vaccine serotypes and increasing frequency of few NVTs mainly in under-fives. Continued surveillance with WGS remains crucial to fully understand dynamics of the residual VTs and replacement NVT serotypes post-PCV

Whole genome sequence analysis of Shigella from Malawi identifies fluoroquinolone resistance

Increasing antimicrobial resistance and limited alternative treatments have led to fluoroquinolone-resistant Shigella strain inclusion on the WHO global priority pathogens list. In this study we characterized multiple Shigella isolates from Malawi with whole genome sequence analysis, identifying the acquirable fluoroquinolone resistance determinant qnrS1

The effect of live attenuated influenza vaccine on pneumococcal colonisation densities among children aged 24-59 months in The Gambia: a phase 4, open label, randomised, controlled trial.

BACKGROUND: Influenza and other respiratory viruses promote Streptococcus pneumoniae proliferation in the upper respiratory tract. We sought to investigate for what we believe is the first time, the effect of intranasal live attenuated influenza vaccine (LAIV) on nasopharyngeal S pneumoniae density in a low-income to middle-income country population with high pneumococcal carriage rates. METHODS: In an open-label, randomised, controlled trial in The Gambia, 330 healthy children aged 24-59 months were randomly assigned 2:1 to receive one trivalent LAIV dose at enrolment (day 0, intervention) or at the end of active follow-up (day 21, control). The investigator team were initially masked to block size and randomisation sequence to avoid allocation bias. Group allocation was later revealed to the investigator team. The primary outcome was PCR-quantified day 7 and 21 pneumococcal density. Asymptomatic respiratory viral infection at baseline and LAIV strain shedding were included as covariates in generalised mixed-effects models, to assess the effect of LAIV and other variables on pneumococcal densities. The study is registered at ClinicalTrials.gov, NCT02972957, and is closed to recruitment. FINDINGS: Between Feb 8 and April 12, 2017, and Jan 15 and March 28, 2018, of 343 children assessed for eligibility, 213 in the intervention group and 108 in the control group completed the study and were included in the final analysis. Although no significant differences were seen in pneumococcal carriage or density at each timepoint when comparing groups, changes from baseline were observed in the LAIV group. The baseline S pneumoniae carriage prevalence was high in both LAIV and control groups (75%) and increased by day 21 in the LAIV group (85%, p=0·0037), but not in the control group (79%, p=0·44). An increase in pneumococcal density from day 0 amounts was seen in the LAIV group at day 7 (+0·207 log10 copies per μL, SE 0·105, p=0·050) and day 21 (+0·280 log10 copies per μL, SE 0·105, p=0·0082), but not in the control group. Older age was associated with lower pneumococcal density (-0·015 log10 copies per μL, SE 0·005, p=0·0030), with the presence of asymptomatic respiratory viruses at baseline (+0·259 log10 copies per μL, SE 0·097, p=0·017), and greater LAIV shedding at day 7 (+0·380 log10 copies per μL, SE 0·167, p=0·024) associated with higher pneumococcal density. A significant increase in rhinorrhoea was reported in the LAIV group compared with the control group children during the first 7 days of the study (103 [48%] of 213, compared with 25 [23%] of 108, p<0·0001), and between day 7 and 21 (108 [51%] of 213, compared with 28 [26%] of 108, p<0·0001). INTERPRETATION: LAIV was associated with a modest increase in nasopharyngeal pneumococcal carriage and density in the 21 days following vaccination, with the increase in density lower in magnitude than previously described in the UK. This increase was accelerated when LAIV was administered in the presence of pre-existing asymptomatic respiratory viruses, suggesting that nasopharyngeal S pneumoniae proliferation is driven by cumulative mixed-viral co-infections. The effect of LAIV on pneumococcal density is probably similar to other respiratory viral infections in children. Our findings provide reassurance for the use of LAIV to expand influenza vaccine programmes in low-income to middle-income country populations with high pneumococcal carriage. FUNDING: Wellcome Trust

Genomic analysis of Klebsiella pneumoniae isolates from Malawi reveals acquisition of multiple ESBL determinants across diverse lineages

Objectives ESBL-producing Klebsiella pneumoniae (KPN) pose a major threat to human health globally. We carried out a WGS study to understand the genetic background of ESBL-producing KPN in Malawi and place them in the context of other global isolates. Methods We sequenced genomes of 72 invasive and carriage KPN isolates collected from patients admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi. We performed phylogenetic and population structure analyses on these and previously published genomes from Kenya (n = 66) and from outside sub-Saharan Africa (n = 67). We screened for presence of antimicrobial resistance (AMR) genetic determinants and carried out association analyses by genomic sequence cluster, AMR phenotype and time. Results Malawian isolates fit within the global population structure of KPN, clustering into the major lineages of KpI, KpII and KpIII. KpI isolates from Malawi were more related to those from Kenya, with both collections exhibiting more clonality than isolates from the rest of the world. We identified multiple ESBL genes, including blaCTX-M-15, several blaSHV, blaTEM-63 and blaOXA-10, and other AMR genes, across diverse lineages of the KPN isolates from Malawi. No carbapenem resistance genes were detected; however, we detected IncFII and IncFIB plasmids that were similar to the carbapenem resistance-associated plasmid pNDM-mar. Conclusions There are multiple ESBL genes across diverse KPN lineages in Malawi and plasmids in circulation that are capable of carrying carbapenem resistance. Unless appropriate interventions are rapidly put in place, these may lead to a high burden of locally untreatable infection in vulnerable populations

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

INTRODUCTION: Pneumococcal infections cause a high death toll in Sub Saharan Africa (SSA) but the recently rolled out pneumococcal conjugate vaccines (PCV) will reduce the disease burden. To better understand the population impact of these vaccines, comprehensive analysis of large collections of pneumococcal isolates sampled prior to vaccination is required. Here we present a population genomic study of the invasive pneumococcal isolates sampled before the implementation of PCV13 in Malawi. MATERIALS AND METHODS: We retrospectively sampled and whole genome sequenced 585 invasive isolates from 2004 to 2010. We determine the pneumococcal population genetic structure and assessed serotype prevalence, antibiotic resistance rates, and the occurrence of serotype switching. RESULTS: Population structure analysis revealed 22 genetically distinct sequence clusters (SCs), which consisted of closely related isolates. Serotype 1 (ST217), a vaccine-associated serotype in clade SC2, showed highest prevalence (19.3%), and was associated with the highest MDR rate (81.9%) followed by serotype 12F, a non-vaccine serotype in clade SC10 with an MDR rate of 57.9%. Prevalence of serotypes was stable prior to vaccination although there was an increase in the PMEN19 clone, serotype 5 ST289, in clade SC1 in 2010 suggesting a potential undetected local outbreak. Coalescent analysis revealed recent emergence of the SCs and there was evidence of natural capsule switching in the absence of vaccine induced selection pressure. Furthermore, majority of the highly prevalent capsule-switched isolates were associated with acquisition of vaccine-targeted capsules. CONCLUSIONS: This study provides descriptions of capsule-switched serotypes and serotypes with potential to cause serotype replacement post-vaccination such as 12F. Continued surveillance is critical to monitor these serotypes and antibiotic resistance in order to design better infection prevention and control measures such as inclusion of emerging replacement serotypes in future conjugate vaccines

Streptococcus pyogenes colonization in children aged 24-59 months in The Gambia: Impact of Live Attenuated Influenza Vaccine and associated serological responses

BACKGROUND: Immunity to Streptococcus pyogenes in high burden settings is poorly understood. We explored S. pyogenes nasopharyngeal colonization after intranasal live attenuated influenza vaccine (LAIV) among Gambian children aged 24-59 months, and resulting serological response to 7 antigens. METHODS: A post-hoc analysis was performed in 320 children randomized to receive LAIV at baseline (LAIV group) or not (control). S. pyogenes colonization was determined by quantitative Polymerase Chain Reaction (qPCR) on nasopharyngeal swabs from baseline (D0), day 7 (D7) and day 21 (D21). Anti-streptococcal IgG was quantified, including a subset with paired serum pre/post S. pyogenes acquisition. RESULTS: The point prevalence of S. pyogenes colonization ranged from 7-13%. In children negative at D0, S. pyogenes was detected at D7 or D21 in 18% of LAIV group and 11% of control group participants (p=0.12). The odds ratio (OR) for colonization over time was significantly increased in the LAIV group (D21 vs D0 OR 3.18, p=0.003) but not in the control group (OR 0.86, p=0.79). The highest IgG increases following asymptomatic colonization were seen for M1 and SpyCEP proteins. CONCLUSIONS: Asymptomatic S. pyogenes colonization appears modestly increased by LAIV, and may be immunologically significant. LAIV could be used to study influenza-S. pyogenes interactions

Bacterial genome-wide association study of hyper-virulent pneumococcal serotype 1 identifies genetic variation associated with neurotropism.

Funder: Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation)Hyper-virulent Streptococcus pneumoniae serotype 1 strains are endemic in Sub-Saharan Africa and frequently cause lethal meningitis outbreaks. It remains unknown whether genetic variation in serotype 1 strains modulates tropism into cerebrospinal fluid to cause central nervous system (CNS) infections, particularly meningitis. Here, we address this question through a large-scale linear mixed model genome-wide association study of 909 African pneumococcal serotype 1 isolates collected from CNS and non-CNS human samples. By controlling for host age, geography, and strain population structure, we identify genome-wide statistically significant genotype-phenotype associations in surface-exposed choline-binding (P = 5.00 × 10-08) and helicase proteins (P = 1.32 × 10-06) important for invasion, immune evasion and pneumococcal tropism to CNS. The small effect sizes and negligible heritability indicated that causation of CNS infection requires multiple genetic and other factors reflecting a complex and polygenic aetiology. Our findings suggest that certain pathogen genetic variation modulate pneumococcal survival and tropism to CNS tissue, and therefore, virulence for meningitis

Respiratory microbiota: interactions with maternal pneumococcal immunisation and live attenuated influenza vaccine (LAIV) in children

Respiratory tract infections (RTIs) are a leading cause of mortality in children under five years. The high burden of RTIs is experienced in low and middle-income countries (LMICs). RTIs are caused by bacteria and viruses that are common colonisers of the respiratory microbial ecosystem (microbiome) and generally colonize the nasopharynx asymptomatically. There is increasing evidence from high-income countries (HICs) that the respiratory microbiome plays a critical role in pathogen-resistance and containment, and immune modulation including vaccine-induced immune responses in all stages of life. In addition, there is also increasing evidence that vaccines may alter the composition of the microbiome. However, data illustrating the development of respiratory microbiota, vaccine-microbiome interactions, and the role of the respiratory microbiome in vaccine immunogenicity in children from LMIC settings is limited. Understanding these aspects presents the first steps towards the development of effective intervention strategies targeting the respiratory microbiome in settings with high-disease burden, including enhancing robust immune responses to vaccines. Current understanding asserts that in LMICs, asymptomatic carriage of pathogenic bacteria and viruses including Streptococcus pneumoniae is high and occurs rapidly in early life. In addition, poor vaccine immunogenicity is common; reasons for this are not fully understood but could be linked to the microbiome. To address these gaps, this thesis describes a series of investigations using data and materials obtained through two longitudinal clinical trials conducted in Gambian children. The first study investigates the interactions between live attenuated influenza vaccine (LAIV) and nasopharyngeal microbiota, including interactions specifically focused on S. pneumoniae, during the first 21 days following immunisation in young children (2-5 years of age). The second study investigates the development of respiratory microbiota in the first nine months of life and the effect of maternal pneumococcal-conjugate vaccination (PCV [Prevenar13™]) on this development in Gambian infants. Results from the first study showed a high baseline S. pneumoniae carriage rate in both LAIV and control groups (74.9%). By day 21, S. pneumoniae carriage rate increased in response to LAIV (84.5%), but not in controls. An increase in S. pneumoniae density was also seen in LAIV immunized children at day 7 and day 21, but not in the control group. Younger age, the presence of asymptomatic respiratory viruses at baseline, and higher LAIV shedding at day 7 were also associated with higher pneumococcal density. Microbiome analyses showed a temporary impact of LAIV administration on the overall microbial community composition at day 7, which was no longer significant at day 21. In addition, the presence of asymptomatic respiratory viruses at baseline also induced an independent effect on day 7 microbial communities. On taxa level, LAIV administration resulted in modest increases in the abundance of Psychrobacter spp and an oral bacterium, Gracilibacteria spp, with a decrease in Neisseria spp and several less abundant taxa. In children who received LAIV, baseline microbial community composition did not impact mucosal IgA response but was associated with increases in both Matrix and Nuclear protein (MNP)-specific CD4+ T-cell responses and ex vivo T Follicular Helper (Tfh)-like cells. Microbiota profiles with higher abundances of lactic acid producing bacteria including Dolosigranulum spp. and Corynebacterium propinquum at the time of LAIV receipt were found in seroconvertors, and those with MNP-specific and Tfh-like CD4+ T-cell following vaccination. Results from the second study showed that, post-partum, the nasopharynx of Gambian infants is initially colonized with mixed bacterial communities. Within the first fourteen days, there is rapid niche differentiation, initially dominated by Staphylococcus with concurrent acquisition of other bacteria including Corynebacterium, Dolosigranulum, Haemophilus, Streptococcus and Moraxella. Season of sampling, presence of young children in the household and sleeping together with other siblings influence the development of the nasopharyngeal microbiota. Interestingly, the effect of maternal PCV13 immunisation on overall microbial composition occurred later, observed at six months of age rather than earlier in life. On taxa level, maternal PCV13 immunisation was associated with modest changes in several microbial taxa during development. The findings of this thesis show that the impact of LAIV on S. pneumoniae density and nasopharyngeal microbiota in children is modest and exacerbated by pre-existing asymptomatic respiratory viral infection. In addition to illustrating the importance of asymptomatic viral-viral and viral-bacterial interactions in the respiratory tract, these findings provide reassurance for use of LAIV to expand influenza vaccine programmes in LMIC with high pneumococcal carriage. This thesis also demonstrates that nasopharyngeal microbiota composition may play a role in modulating adaptive immunity to LAIV in children. This is an important finding in the context of formulating interventions to improve immunogenicity to intranasal or mucosal administered vaccines in the future. Finally, this thesis adds to the information gap illustrating the development of the nasopharyngeal microbiome and how early life vaccine interventions may affect this process. However, further studies are required to investigate the impact of these effects on early life respiratory health