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Members of the Klebsiella pneumoniae species complex, particularly K. pneumoniae subsp. pneumoniae are antimicrobial resistance (AMR) associated pathogens of global importance, and polyvalent vaccines targeting Klebsiella O-antigens are in development. Whole-genome sequencing has provided insight into O-antigen distribution in the K. pneumoniae species complex, as well as population structure and virulence determinants, but genomes from sub-Saharan Africa are underrepresented in global sequencing efforts. We therefore carried out a genomic analysis of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae species complex isolates colonizing adults in Blantyre, Malawi. We placed these isolates in a global genomic context, and compared colonizing to invasive isolates from the main public hospital in Blantyre. In total, 203 isolates from stool and rectal swabs from adults were whole-genome sequenced and compared to a publicly available multicounty collection and previously sequenced Malawian and Kenyan isolates from blood or sterile sites. We inferred phylogenetic relationships and analysed the diversity of genetic loci linked to AMR, virulence, capsule and LPS O-antigen (O-types). We find that the diversity of Malawian K. pneumoniae subsp. pneumoniae isolates represents the species’ population structure, but shows distinct local signatures concerning clonal expansions. Siderophore and hypermucoidy genes were more frequent in invasive versus colonizing isolates (present in 13 % vs 1 %) but still generally lacking in most invasive isolates. O-antigen population structure and distribution was similar in invasive and colonizing isolates, with O4 more common (14%) than in previously published studies (2–5 %). We conclude that host factors, pathogen opportunity or alternate virulence loci not linked to invasive disease elsewhere are likely to be the major determinants of invasive disease in Malawi. Distinct ST and O-type distributions in Malawi highlight the need to sample locations where the burden of invasive Klebsiella disease is greatest to robustly define secular trends in Klebsiella diversity to assist in the development of a useful vaccine. Colonizing and invasive isolates in Blantyre are similar, hence O-typing of colonizing Klebsiella isolates may be a rapid and cost-effective approach to describe global diversity and guide vaccine development.

Shortening courses of antimicrobials has been proposed to reduce risk of antimicrobial resistant (AMR) infections, but acquisition and selection dynamics under antimicrobial pressure at the individual level are poorly understood. We combine multi-state modelling and whole-genome sequencing to understand colonisation dynamics of extended-spectrum beta-lactamase producing Enterobacterales (ESBL-E) in Malawian adults. We demonstrate prolonged post-exposure antibiotic effect, meaning short courses exert similar colonisation pressure to longer ones. Genome data does not identify widespread hospital-associated ESBL-E transmission, hence apparent acquisitions may be selected from the patient microbiota by antimicrobial exposure. Understanding ESBL-E dynamics under antimicrobial pressure is crucial for evidence-based stewardship protocols.

Drug-resistant bacteria of the order Enterobacterales which produce extended-spectrum beta-lactamase enzymes (ESBL-Enterobacterales, ESBL-E) are global priority pathogens. Antimicrobial stewardship interventions proposed to curb their spread include shorter courses of antimicrobials to reduce selection pressure but individual-level acquisition and selection dynamics are poorly understood. We sampled stool of 425 adults (aged 16–76 years) in Blantyre, Malawi, over 6 months and used multistate modelling and whole-genome sequencing to understand colonization dynamics of ESBL-E. Models suggest a prolonged effect of antimicrobials such that truncating an antimicrobial course at 2 days has a limited effect in reducing colonization. Genomic analysis shows largely indistinguishable diversity of healthcare-associated and community-acquired isolates, hence some apparent acquisition of ESBL-E during hospitalization may instead represent selection from a patient’s microbiota by antimicrobial exposure. Our approach could help guide stewardship protocols; interventions that aim to review and truncate courses of unneeded antimicrobials may be of limited use in preventing ESBL-E colonization.

Klebsiella pneumoniae is an antimicrobial resistance (AMR) associated pathogen of global importance, and polyvalent vaccines targeting K. pneumoniae O-antigens are in development. Genomes from sub-Saharan Africa (sSA) are underrepresented in global sequencing efforts. We therefore carried out a genomic analysis of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae complex isolates colonising adults in Blantyre, Malawi, placed these isolates in a global genomic context, and compared colonising to invasive isolates from the main public hospital in Blantyre. 203 isolates from stool and rectal swabs from adults were whole-genome sequenced and compared to a publicly available multicountry collection of 484 K. pneumoniae genomes sampled to cover maximum diversity of the species, 150 previously sequenced Malawian and 66 Kenyan isolates from blood or sterile sites. We inferred phylogenetic relationships and analysed the diversity of genetic loci linked to AMR, virulence, capsule (K-) and LPS O-antigen (O-types). We find that the diversity of Malawian Klebsiella isolates is representative of the species’ population structure, but with local success and expansion of sequence types (STs) ST14, ST15, ST340 and ST307. Siderophore and hypermucoidy genes were more frequent in invasive versus carriage isolates (present in 13% vs 1%, p < 0.001) but still generally lacking in most invasive isolates. The population structure and distribution of O-antigen types was similar in Malawian invasive and carriage isolates, with O4 being more common in Malawian isolates (14%) than in previously published studies (2-5%). We conclude that host factors, pathogen opportunity or alternate virulence loci not linked to invasive disease elsewhere are likely to be the major determinants of invasive disease in Malawi. Distinct ST and O-type distributions in Malawi highlights the need for geographically aware sampling to robustly define secular trends in Klebsiella diversity. Colonising and invasive isolates in Blantyre are similar and hence O-typing of colonising Klebsiella isolates may be a rapid and cost-effective approach to describe global diversity and guide vaccine development.Data SummaryAll data and code to replicate this analysis is available as the blantyreESBL v1.0.0 R package (https://doi.org/10.5281/zenodo.5554082) available at https://github.com/joelewis101/blantyreESBL. Reads from all isolates sequenced as part of this study have been deposited in the European Nucleotide Archive, and accession numbers (as well as accession numbers of publicly available genomes used in this analysis) are provided in the R package.

Escherichia coli is a ubiquitous bacterium and one of the most prevalent Gram-negative species associated with drug resistant infections. The large number of sequenced genomes available have provided us with a consistently growing knowledge base to further understand pathogenesis and epidemiology of this organism. However, data from sub-Saharan Africa (sSA) are underrepresented in global sequencing efforts and E. coli genetic diversity from this region is poorly described. To reduce this gap, we investigated extended-spectrum beta-lactamase (ESBL)-producing E. coli colonising adults in Blantyre, Malawi to assess the bacterial diversity and AMR determinants and to place these isolates in the context of the wider population structure. We performed short-read whole-genome sequencing of 473 colonising ESBL E. coli isolated from human stool and contextualised the genomes with a previously curated multi-country species wide collection of 10,146 genomes. The most frequently identified sequence types (STs) in our collection were the globally successful ST131, ST410 and ST167, and the dominant ESBL genes were blaCTX-M, mirroring global trends. However, 37% of Malawian isolates did not cluster with any isolates in the curated multicountry collection and a core gene phylogeny was consistent with locally spreading subclades within globally dominant clones, including in ST410 and ST167. We also found Carbapenemase genes in our collection at low frequency; we used long read sequencing to characterise selected ESBL and carbapenemase-associated plasmids, demonstrating the presence of globally distributed carbapenemase carrying plasmids. Increased genomic surveillance of E. coli from Malawi and sSA is necessary to understand local, regional and global transmission of both E. coli and the AMR genes they commonly carry.Impact StatementDrug-resistant Escherichia coli producing extended-spectrum beta lactamase (ESBL) or carbapenemase enzymes have been identified by the World Health Organisation as priority pathogens of global concern, and whole genome sequencing has provided insight into mechanisms of virulence, antimicrobial resistance, and the spread of high-risk clones. However, studies analysing large numbers of E. coli using whole-genome data often focus on opportunistic use of hospital diagnostic collections in high-income settings. Understanding how the genomic epidemiology of E. coli in low- and middle-income countries (including many of the nations of sub-Saharan Africa) differs is essential to provide insight into local, and global drivers of transmission. We therefore sequenced 473 ESBL-producing E. coli genomes colonising adults in Blantyre, Malawi. We analyse determinants of antimicrobial resistance and virulence and place the isolates in wider context using a previously published global E. coli collection that was generated to represent the whole species diversity of sequences publicly available at the time of generation. We find that there is diversity in Malawian isolates not reflected in the curated global collection: widely successful antimicrobial-resistance associated E. coli sequence types are represented in Blantyre, but locally circulating subclades are apparent. Furthermore, given the high number of ESBL producing pathogens causing infections there is an unmet need for carbapenem antimicrobials which are still active against ESBL-producers but are not yet widely available in our setting. We find that carbapenemases (enzymes that can render bacteria resistant to carbapenems) in our collection are unusual but present and carried on globally disseminated plasmids. So too are globally successful, stably carbapenemase-associated E. coli lineages. Although the Malawian isolates analysed typically lacked carbapenemases, carbapenem use is increasing in Malawi and their unstewarded use will accelerate selection for carbapememases in E. coli in the future. Our study highlights the need for robust stewardship protocols and ongoing genomic surveillance as these agents are introduced.Data SummaryAll data and code to replicate this analysis are available as the blantyreESBL v1.3 R package (https://doi.org/10.5281/zenodo.5554081) available at https://github.com/joelewis101/blantyreESBL. Reads from all isolates sequenced as part of this study have been deposited in the European Nucleotide Archive, under PRJEB26677, PRJEB28522 and PRJEB36486 (short reads) and PRJNA869071 (Nanopore reads and hybrid assemblies). Accession numbers (as well as accession numbers of publicly available genomes used in this analysis) linked to sample metadata are provided in the R package and as supplementary data to this manuscript.

Background Sepsis protocols in sub-Saharan Africa are typically extrapolated from high-income settings, yet sepsis in sub-Saharan Africa is likely caused by distinct pathogens and may require novel treatment strategies. Data to guide such strategies are lacking. We aimed to define causes and modifiable factors associated with sepsis outcomes in Blantyre, Malawi, in order to inform the design of treatment strategies tailored to sub-Saharan Africa. Methods We recruited 225 adults who met a sepsis case definition defined by fever and organ dysfunction in an observational cohort study at a single tertiary center. Etiology was defined using culture, antigen detection, serology, and polymerase chain reaction. The effect of treatment on 28-day outcomes was assessed using Bayesian logistic regression. Results There were 143 of 213 (67%) participants living with human immunodeficiency virus (HIV). We identified a diagnosis in 145 of 225 (64%) participants, most commonly tuberculosis (TB; 34%) followed by invasive bacterial infections (17%), arboviral infections (13%), and malaria (9%). TB was associated with HIV infection, whereas malaria and arboviruses with the absence of HIV infection. Antituberculous chemotherapy was associated with survival (adjusted odds ratio for 28-day death, 0.17; 95% credible interval, 0.05–0.49 for receipt of antituberculous therapy). Of those with confirmed etiology, 83% received the broad-spectrum antibacterial ceftriaxone, but it would be expected to be active in only 24%. Conclusions Sepsis in Blantyre, Malawi, is caused by a range of pathogens; the majority are not susceptible to the broad-spectrum antibacterials that most patients receive. HIV status is a key determinant of etiology. Novel antimicrobial strategies for sepsis tailored to sub-Saharan Africa, including consideration of empiric antituberculous therapy in individuals living with HIV, should be developed and trialed.

Single-cell genomics is a powerful tool for determining the genetic structure of complex communities of unicellular organisms. Patients infected with the malaria-causing parasite,Plasmodium falciparum, often carry multiple, genetically distinct parasites. Little is known about the diversity and relatedness of these lineages. We have developed an improved single-cell genomics protocol to reconstruct individual haplotypes from infections, a necessary step in uncovering parasite ecology within the host. This approach captures singly-infected red blood cells (iRBCs) by fluorescence-activated cell sorting (FACS) prior to whole genome amplification (WGA) and whole genome sequencing (WGS). Here, we demonstrate that parasites in late cell cycle stages, which contain increased DNA content, are far superior templates for generating high quality genomic data. Targeting of these cells routinely generates near-complete capture of the 23MbP. falciparumgenome (mean breadth of coverage 90.7%) at high efficiency. We used this approach to analyze the genomes of 48 individual cells from a polyclonal malaria infection sampled in Chikhwawa, Malawi. This comprehensive dataset enabled high-resolution estimation of the clonality and the relatedness of parasite haplotypes within the infection, long-standing problems in malaria biology.