Patrick Musicha

Background: Klebsiella pneumoniae is the leading cause of sepsis among neonates in low- and middle-income countries (LMICs) in Africa and Asia, contributing substantially to the overall burden of antimicrobial-resistant infections and mortality among neonates globally. Pathogen sequencing has been used to investigate case clusters and confirm nosocomial transmission in a small number of neonatal units. Here we utilise pathogen sequence data to estimate the fraction of K. pneumoniae neonatal sepsis attributable to nosocomial transmission in African and South Asian countries. Methods and findings: We estimated the proportion of invasive K. pneumoniae disease involved in nosocomial transmission clusters in a given neonatal unit, using single-linkage clustering based on pairwise temporal and genetic distances estimated from bacterial whole-genome sequences aggregated from 10 contributing studies. Analysing 1,523 K. pneumoniae isolates from 27 units in 13 countries in Africa and South Asia between 2013 and 2023, we inferred 156 nosocomial transmission clusters, ranging from 2 to 188 neonates each (83 of the clusters comprised ≥3 cases). Overall, we estimated that 1,035 neonatal infections (68.0%) were part of nosocomial transmission clusters. Excluding the first infection in each cluster as a potential index case, we estimate at least 879 (57.7%) infections were acquired via nosocomial transmission. Sensitivity analyses showed that results were robust to the choice of genetic distance estimation methods and thresholds used to define clusters, and cluster estimates were stable over temporal distance thresholds ranging from 2 to 8 weeks. Isolates were mostly extended-spectrum beta-lactamase (ESBL) producers (90.9%) and included 172 multi-locus sequence types (STs). Fourteen STs, including several globally recognised multidrug-resistant lineages, were associated with transmission clusters at multiple units, and these were collectively responsible for two-thirds of all infections. Carriage of carbapenemase genes (adjusted odds ratio, aOR = 2.08 [95% confidence interval, CI: 1.04, 4.14]; p = 0.04) and ESBL genes (aOR = 2.48 [95% CI: 1.26, 4.90]; p = 0.006) were significantly positively associated with transmission in a logistic regression model with site as a covariate. Limitations of this study include the lack of sufficient clinical data to allow high-resolution investigation of transmission dynamics and lack of facility-level data to investigate contributors to the observed differences in transmission burden across sites. Conclusions: Nosocomial transmission contributes to a substantial proportion of K. pneumoniae sepsis in neonatal care units in Africa and South Asia. Reducing transmission within these settings through improved infection prevention and control and other measures could substantially reduce the neonatal sepsis burden. A high burden of transmission clusters is associated with the same drug-resistant lineages that are recognised as high-risk clones associated with hospital outbreaks in high-income countries, indicating global connectivity of the antimicrobial-resistant pathogen population.

Background: Use of oral cholera vaccine (OCV) is globally recommended as a public health response to cholera outbreaks, alongside water, sanitation and hygiene (WASH) interventions. Estimating vaccine effectiveness during emergencies in low- and middle-income countries is challenging because vaccination campaigns are often implemented over short time frames, while individual-level data are frequently incomplete due to constraints in infrastructure, resources and data systems. There is a need for pragmatic approaches that can generate timely, policy-relevant evidence using routinely collected data. Methods: We analysed routine surveillance data from a large 2022–2023 cholera outbreak in Blantyre District, Malawi. The EpiEstim framework was used to generate estimates of the time-varying reproduction number (Rₜ) from line-listed case data. We modelled changes in 𝑅 _???? as a function of cumulative OCV coverage using a log-linear framework and propagated uncertainty through posterior sampling. Lagged WASH exposure variables were incorporated in the model to generate adjusted vaccine effectiveness estimates and to explore potential interaction effects. Sensitivity analyses assessed robustness to alternative lag structures. Findings: The Blantyre outbreak was characterised by an initial period of low-level transmission followed by a sharp increase in cases from late November 2022, after which transmission declined steadily through April 2023. This decline coincided with the implementation of a reactive OCV campaign. The majority of the cases were among middle-aged men living in urban Blantyre. The unadjusted vaccine-associated reduction in transmission was estimated at 53.52% (95% credible interval (CrI):42.5–64.1%). After adjusting for a 7-day rolling average WASH activity, total vaccine effectiveness increased to 62.1% (95% CrI: 49.3–74.9%). Sensitivity analyses using alternative lag structures for WASH exposure produced comparable adjusted estimates. Interpretation: Implementation of OCV contributed to a substantial reduction in cholera transmission during the outbreak. This study demonstrates a feasible approach for estimating vaccine-attributable impact whilst accounting for public health and social measures, such as WASH interventions. The methods described will be useful in outbreaks where classical observational designs are not possible, providing actionable evidence to policy makers for outbreak response in resource-limited settings. Funding: MRC Discovery Medicine North (DiMeN) Doctoral Training Partnership (UKRI), National Institute for Health and Care Research (NIHR) Global Health Research Group on Gastrointestinal Infections and Wellcome through the core grant to the Malawi-Liverpool-Wellcome Research Programme. Research in context: Evidence before this study: Before undertaking this analysis, our team and collaborators generated detailed epidemiological and genomic evidence describing the 2022–2023 cholera outbreak in Blantyre, Malawi. Our previous fine-scale spatial work demonstrated marked heterogeneity in cholera burden across urban Blantyre. We found that transmission concentrated in densely populated informal settlements and areas with poor water and sanitation infrastructure. Our genomic analysis of outbreak isolates showed that the epidemic was driven by a recently introduced seventh-pandemic El Tor Vibrio cholerae sub-lineage AFR15 linked to regional and international transmission events, with epidemic expansion occurring during late 2022. These studies characterised where transmission occurred and the evolutionary origin of the outbreak strain. The population-level effect of reactive oral cholera vaccination (OCV) or concurrent and improved water, sanitation and hygiene (WASH) interventions on transmission dynamics during the outbreak was not quantified. In linked work, we conducted a systematic review of post-licensure vaccine impact and effectiveness studies from sub-Saharan Africa (CRD42023436851) to assess the principal study designs and the extent to which they adjusted for concurrent public health and social measures (PHSMs), such as WASH interventions. We searched PubMed, EMBASE, MEDLINE, CINAHL, and Google Scholar for vaccine impact or effectiveness studies conducted in children under five years and screened reference lists of included studies. Across all eligible studies, none measured or adjusted for concurrent PHSMs. Added value of this study: Using surveillance, vaccination, and WASH data collected from Blantyre district by the Malawi Ministry of Health, we estimated the vaccine-associated reduction in transmission during a large reactive OCV campaign. By linking time-varying R ₜ estimates to cumulative vaccine coverage within a posterior sampling framework, we provide field-relevant effectiveness estimates from a setting where traditional individual-level designs were not feasible. We further evaluated whether concurrent WASH activity materially altered vaccine effect estimates. Implications of all the available evidence: Taken together with prior spatial and genomic analyses of the same outbreak, our findings provide a more complete picture of cholera control in Malawi. The earlier work clarified transmission geography and pathogen introduction. The present analysis quantifies the population-level impact of reactive vaccination under real-world programme conditions. This integrated evidence base supports ministries of health in interpreting vaccine performance during rapidly evolving outbreaks and in strengthening the routine data systems required for timely evaluation of interventions.

Diverse salmonellae have the potential to cause disease and may be carried asymptomatically within the intestine of many vertebrate species. The relative contribution of human, animal, and environmental hosts to the transmission of Salmonella is unknown within and between households in low-income settings, especially where humans and animals may live in close contact and sanitary infrastructure is often inadequate. Between November 2018 and December 2019, we isolated Salmonella spp. from thirty households in urban and rural locations in Malawi, sampling at three time points from the stool of humans, animals, and their household environment. Using whole genome sequencing and fine-resolution bioinformatic and phylogenetic analyses we found evidence of sharing of Salmonella species and strains between humans, animals and the environment, both within and between households. The intricate web of interconnected salmonellae within this ecosystem underscores the importance of adopting a multi-faceted ‘One Health’ strategy when considering control of Salmonella in low-intensity agricultural systems.

Background: Klebsiella pneumoniae ( Kpn ) is an important cause of healthcare-associated infections (HAI). In low and middle-income countries, HAI due to Kpn disproportionally affects neonates. In this study, we investigated the genomic changes that occurred during long-term circulation of a Kpn ST39 clone, causing a disproportionate number of infections on the neonatal ward at a tertiary healthcare facility in Malawi in 2017. Methods: We analyzed whole genome sequences of Klebsiella pneumoniae ST39 collected from Queen Elizabeth Central Hospital over a 20-year period, including generation of several high-quality hybrid genomes. We compared virulence markers, antibiotic resistance determinants, and mobile genetic elements, focusing on variable regions between strains from the outbreak clone in 2017 to genomes from other co-occurring ST39 lineages. Results: We identified eight variable genomic regions that demonstrate the plasticity of Kpn within-ST, including the role of bacteriophages in shaping the genome of ST39. Conclusions: The analyzed Klebsiella pneumoniae ST39 lineages have a highly variable genome capable of incorporating large genomic regions during prolonged hospital circulation, which may offer a selective advantage in hospital environments and provide resistance to antimicrobial agents. Data summary: All sequencing data is available in BioProject PRJEB102175; detailed accession numbers are provided in Table S1. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

BACKGROUND: Ciprofloxacin resistant Klebsiella pneumoniae is common or emerging in many geographies, and knowledge of local resistance rates is important for empirical therapy. Whilst there are known K. pneumoniae ciprofloxacin resistance determinants, there is a lack of systematic data on the effect of determinants, alone and in combination, and there are no publicly accessible tools for predicting resistance from whole genome sequence data. METHODS: The KlebNET-GSP AMR Genotype-Phenotype Group aggregated a matched genotype-phenotype dataset of n=12,167 K. pneumoniae species complex ( Kp SC) isolates from 27 countries between 2001-2021. We developed a rules-based classifier to predict ciprofloxacin resistance by categorizing the number of quinolone resistance determining regions mutations in gyrA and parC , the number of plasmid-mediated quinolone resistance genes, and the presence/absence of aac(6ʹ)-Ib-cr (which can acetylate ciprofloxacin). Predictive performance was assessed using the discovery dataset, for which we re-phenotyped discrepant isolates; and validated using externally contributed datasets (n=7,030 Kp SC isolates). RESULTS: The rules-based classifier predicted R vs S/I with categorical agreement, sensitivity, and specificity >96%, and major/very major error rates Kp SC sources (human, animal, other), species, and intra-species lineages. External validation of the classifier yielded overall 93.12% categorical agreement [95% confidence interval (CI), 92.50-93.74%], 8.65% major errors [95% CI, 7.34-9.97%], and 6.20% very major errors [95% CI, 5.51-6.90%]. We implemented the classifier in Kleborate, a command-line tool that is integrated into the Pathogenwatch web platform. Using this to assess the global distribution of ciprofloxacin resistance determinants in Kp SC genomes available in Pathogenwatch (n=31,319, from 109 countries between years 2000-2023), we observed a significant positive association between national quinolone consumption rates and predicted ciprofloxacin resistance (R ² =0.20, p=0.004). CONCLUSIONS Ciprofloxacin resistance phenotypes can be reasonably predicted from genotypes, which is sufficient for informing surveillance. However, unexplained resistance remains and accuracy is insufficient for clinical applications. We demonstrate the value of aggregating genotype-phenotype data to explore resistance mechanisms and develop predictors, but highlight complexities in combining phenotype data from different assays and standards.

Background: Klebsiella pneumoniae ( Kpn ) is an important cause of neonatal sepsis in sub-Saharan Africa. Kpn are intrinsically resistant to penicillins and frequently resistant to gentamicin and 3 ^rd -generation cephalosporins, key agents in the treatment of neonatal sepsis. Such infections can be prevented by effective infection prevention and control (IPC), but the most effective IPC packages for resource-limited healthcare settings are incompletely defined. Methods: We recruited a cohort of 94 neonates admitted to a Malawian neonatal unit, alongside their mothers, and utilised single colony whole genome sequencing and post-enrichment metagenomics to determine the most important transmission routes of Extended-Spectrum Beta-Lactamase producing (ESBL) Kpn . These data were analysed at the level of ST and pairwise SNP distance and combined with statistical models to infer transmission. Findings: ESBL- Kpn rapidly colonised neonates; female sex, receipt of oxygen, caesarean delivery and antibiotic use increased colonisation risk. STs causing invasive infection and stool colonisation were temporally related to those found in the ward. There was greatest circulation of ESBL- Kpn between compartments that are in contact with neonatal stool (neonate stool, mothers’ hands, cots, and the swaddling cloths). Utilising epidemiological and SNP data, Kpn appeared to be transmitted to neonates primarily from cots, ward surfaces (sinks and oxygen delivery equipment) and from other neonates. Network analysis implicated cots, antibiotics and oxygen delivery in ESBL- Kpn transmission. Interpretation: An unsafe hospital environment is strongly implicated in neonatal invasive infection and stool colonisation with ESBL Kpn . IPC interventions should focus on containing neonatal stool, hand hygiene, cot decontamination, single use oxygen delivery and surface cleaning, particularly sinks. Funding: This work was funded by the Antimicrobial Resistance Cross-Council Initiative through grants from the Medical Research Council, a Council of UK Research and Innovation and the National Institute for Health Research (MR/R015074/1 & MR/S004793/1); and the Bill and Melinda Gates Foundation (INV-005692). Malawi-Liverpool-Wellcome Research Programme (MLW) is core-fund by Wellcome (206545/Z/17/Z). Research in context: Evidence before this study: Neonatal sepsis is a leading cause of neonatal mortality, particularly in sub-Saharan Africa. Klebsiella pneumoniae ( Kpn ) has emerged as an important cause of neonatal sepsis and is difficult to treat due to increasing antimicrobial resistance. Extended-spectrum beta-lactamases are enzymes that confer resistance to ceftriaxone, and for neonates in many sub-Saharan African neonatal units this resistance determinant renders their infections untreatable. While infection prevention and control (IPC) measures are important to limit transmission, the most important IPC measures in this context remain unclear. Added value of this study: This study shows that colonisation with ESBL Kpn in the neonatal unit is common. Risk factors for colonisation include oxygen delivery, antibiotic therapy, and female gender. Isolates from invasive disease were genomically like those that caused stool colonisation and that were colonising the general ward environment. There was circulation of bacteria between the neonatal gastrointestinal tract, cots, mothers’ hands, and swaddling cloths. Transmission appeared to be from cots, ward surfaces, particularly sinks and oxygen equipment, other neonates, and the mothers’ hands. Implications of all available evidence: IPC should be prioritized to reduce colonisation and subsequent infection with ESBL Kpn , and other enteric pathogens regardless of AMR profile. Suggested IPC interventions in this context should focus on reducing bacterial load in the ward environment, with neonatal stool management, maternal hand hygiene, single-use oxygen delivery equipment, replacing sinks with hand sanitizer and cot decontamination.

Klebsiella pneumoniae is a frequent cause of antimicrobial resistant healthcare associated infections in neonates across sub-Saharan Africa, with multiple lineages associated with neonatal sepsis. However, the full diversity of circulating strains and key reservoirs facilitating transmission within hospitals is unknown. We investigated the population structure and within- sample diversity of K. pneumoniae in a Malawian neonatal unit. We recruited 94 mother- neonate pairs and collected regular stool samples, hand swabs, cot swabs and swaddling cloth samples. Additionally, we collected ward surface-swab samples and staff hand swabs weekly. To establish within sample diversity we employed a dual sequencing approach; (i) single colony picks from Extended-Spectrum Beta-Lactamase selective chromogenic agar for short-read whole genome sequencing ; and (ii) post-enrichment metagenomics using plate sweeps from a non-selective agar. In total, we analysed 552 single-colony picks and 772 plate-sweeps from neonate, maternal and environmental samples. Comparing sequence types, surface antigens, antimicrobial resistance and virulence genes, and plasmid replicons, between sample types and sequencing approaches, we identified key advantages and limitations of post-enrichment metagenomics. Our approach revealed high diversity at both the ward and individual level, with a high proportion of the overall diversity likely due to Extended-Spectrum Beta-Lactamase negative organisms. ST15 and ST307 were found in high numbers using both methodologies, whilst ST14 was identified primarily from the non-selective post-enrichment metagenomic samples. Isolates and samples from ward surface swabs had more antimicrobial resistance genes and plasmid replicons than those isolated from human stool. This approach demonstrates the value of combining colony-based and metagenomic sequencing approaches, as a cost-effective alternative to shotgun metagenomics to study health care associated infections. Impact statement: Klebsiella pneumoniae is an important cause of healthcare-associated infections (HAIs) in neonates, particularly in sub-Saharan Africa. While multiple strains can colonise a single person or surface, the full diversity of K. pneumoniae in the hospital environment is unknown. This study investigates ward-level and within-sample diversity of K. pneumoniae in a Malawian neonatal unit. We collected stool samples from babies and their mothers, as well as swabs from ward surfaces. To characterize bacterial diversity, we compared two approaches: (i) single- colony whole-genome sequencing of isolates selected under antimicrobial pressure and (ii) post-enrichment metagenomics, where entire microbial communities were sequenced from non- selective culture plates in a single run. We found high diversity of K. pneumoniae , with considerable diversity revealed by post-enrichment metagenomics. Samples frequently had multiple strains of K. pneumoniae within them and this varied by sample type. These data highlight the need to utilize methods that account for within-sample diversity when investigating K. pneumoniae, indicate that single colony WGS is inadequate and a combination of post- enrichment metagenomics and single colony WGS is preferable.

Objectives: For 475 ESBL-producing Escherichia coli (ESBL-Ec), and 171 ESBL-producing Klebsiella pneumoniae (ESBL-Kp) collected from human carriers, the human-polluted (hp)-environment, and food: (i) to compare the antimicrobial resistance gene (ARG) content, and (ii) to assess clonal relationships between human and non-human isolates. Methods: Two prospective multicentre cohorts were assessed: colonised hospitalised index-subjects and household contacts, and long-term care facility (LTCF) residents. Additionally, linked hp-environment and food samples were collected. Presence of ARGs were assessed using pairwise comparisons and proportional similarity index (PSI). Clonal relationships were assessed using cgMLST distance visualisations and maximum likelihood phylogeny. Results: ESBL-Ec and ESBL-Kp co-occurred in 14/65 households, 3/6 LTCFs, and in 33/202 of ESBL-positive participants. Thirty-nine percent of detected ARG types were found in both species (36/93). Frequencies of beta-lactamase, ESBL, aminoglycoside, and sulfonamide ARG types from human ESBL-Ec and ESBL-Kp overlapped considerably: PSIs 0.59-0.75, and were equal or higher compared to the overlap between ESBL-Ec from humans and food isolates: PSIs 0.33-0.72. Isolates from humans and the hp-environment were frequently clonally related, indicating human contamination of the environment. Links with food isolates were observed less frequently. For ESBL-Ec both interregional and regional clonal dissemination were observed, while for ESBL-Kp clonal dissemination was mainly regional. Conclusions: ESBL-Ec and ESBL-Kp from human carriage showed considerable overlap in ARG content. Furthermore, clonal links were observed frequently between humans and hp-environment, and with lower frequency between humans and food. These findings are consistent with human-to-human transmission as an important driver of ARG spread in humans.

Recent data re-affirm antimicrobial resistance (AMR) as a One Health problem, particularly in low- and middle-income countries. Transdisciplinary and intersectoral collaboration are required if we are to improve environmental hygiene, addressing both AMR and a range of aligned development challenges. Antimicrobial resistance is a One Health problem that impacts humans, animals, and the environment. In this Comment, the authors discuss evidence for antimicrobial resistance transmission to humans, highlighting contrasting pictures between high- and low/middle-income settings.