Carolyn McGann

Klebsiella pneumoniae ( Kpn ) is a major cause of infant mortality worldwide, with most transmission occurring among hospitalized neonates in low- and middle-income countries where infections caused by multidrug-resistant Kpn (MDR- Kpn ) are increasingly common. We hypothesized that integrating laboratory surveillance for neonatal colonization and infection, real-time epidemiologic investigations, and whole-genome sequencing (WGS) could identify transmission pathways to guide targeted infection prevention and control (IPC) strategies. We conducted Kpn surveillance in a 36-bed neonatal unit in Botswana over 12 months (2022–2023). WGS was performed on Kpn isolates from bloodstream infections (BSIs), and MDR- Kpn isolates collected from environmental sampling during outbreaks and twice-monthly colonization screenings (skin and perirectal swabs) using culture media selective for MDR- Kpn (CHROMagar Extended-spectrum beta-lactamase [ESBL]/SuperCarba). WGS data were analyzed using multilocus sequence typing (MLST), pangenome and reference-based single-nucleotide polymorphism (SNP) analyses, and Bayesian phylogenetics. We identified 55 Kpn BSIs during the 12-month surveillance period and the median prevalence of MDR- Kpn colonization was 28%. Kpn was recovered from multi-use intravenous (IV) fluid bags during a Kpn outbreak (41 BSIs, 10 deaths), which was controlled by implementing a 24-hour discard policy for IV medications . Among 270 Kpn isolates available (28 BSI, 232 colonizing, 10 environmental [six IV fluid, four sink drain]), WGS confirmed over half of BSI genomes (n = 17) and all six IV fluid isolates belonged to ST1414 and were closely related (Kpn colonization screening. This study reinforces the value of integrating WGS with real-time epidemiologic investigations to understand transmission dynamics and guide IPC. Colonization surveillance focused solely on MDR- Kpn may overlook drug-susceptible but outbreak-prone strains.

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: Klebsiella pneumoniae causes ~20% of sepsis in neonates, with ~40% crude mortality. A vaccine administered to pregnant women, protecting against ≥70% of K. pneumoniae infections, could avert ~400,000 cases and ~80,000 deaths annually, mostly in Africa and South Asia. Vaccine formulations targeting the capsular polysaccharide (K) or lipopolysaccharide (O) antigens are in development. Global K. pneumoniae populations display extensive K and O diversity, necessitating a polyvalent vaccine targeted to the serotypes associated with neonatal disease in relevant geographical regions. We investigated the prevalence of K and O types associated with neonatal sepsis in Africa and South Asia to inform maternal vaccine design. Methods and findings: We analysed 1,930 K. pneumoniae neonate blood isolates from 13 surveillance studies across 35 sites in 13 countries. We used pathogen whole-genome sequencing to predict K and O serotypes and adjust for local transmission clusters, and Bayesian hierarchical meta-analysis to estimate K and O prevalence overall and per region, treating site as a random effect. Eighty-seven K loci were identified. KL2, KL102, KL25, KL15, and KL62 accounted for 49% of isolates. We estimate that 20 K loci, combining the eight most prevalent per region, could cover 72.9% of all infections (95% credible interval: [69.4%, 76.5%]) and ≥70% in each of Eastern, Western, and Southern Africa and South Asia. Preliminary findings from three sites suggested sufficient temporal stability of K loci to maintain 20-valent K vaccine coverage over 5–10 years, but more longitudinal data are needed to support this prediction. O types were far less diverse ( n = 14 types). We estimate the top-5 (O1⍺β,2⍺, O1⍺β,2β, O2⍺, O2β, and O4) would cover 86.2% [82.6, 89.9%] of total infections (76%–92% per region), while the top-10 would cover ~99% of infections in all four regions. The main limitations of our study are the reliance on genome sequences to predict K and O serotypes (as serological typing is not available) and a lack of longitudinal data to explore stability of antigen prevalence over time. Conclusions: Neonatal sepsis is associated with diverse K and O types, with substantial geographic and temporal variation even after adjusting for localised transmission clusters. Despite this, a single 20-valent K vaccine could theoretically cover ≥70% of infections in all target regions. Locally-targeted vaccines could achieve higher coverage with lower valency, but are less feasible. In principle, very high coverage could be achieved with lower valency O-based vaccines, however, the protective efficacy against disease of antibodies targeting the O antigen remains uncertain. Further research is needed on cross-reactivity, antigen exposure, and stability of antigens over time, to better inform vaccine development.

Background: Klebsiella pneumoniae causes ~20% of sepsis in neonates, with ~40% crude mortality. A vaccine administered to pregnant women, protecting against 70% of K. pneumoniae infections, could avert ~400,000 cases and ~80,000 deaths annually, mostly in Africa and South Asia. Vaccine formulations targeting the capsular polysaccharide (K) or lipopolysaccharide (O) antigens are in development. Global K. pneumoniae populations display extensive K and O diversity, necessitating a polyvalent vaccine targeted to the serotypes associated with neonatal disease in relevant geographical regions. We investigated the prevalence of K and O types associated with neonatal sepsis in Africa and South Asia to inform maternal vaccine design. Methods and Findings: We analysed n=1930 K. pneumoniae neonate blood isolates from 13 surveillance studies across 35 sites in 13 countries. We used pathogen sequencing to predict K and O serotypes and adjust for local transmission clusters, and Bayesian hierarchical meta-analysis to estimate K and O prevalence overall and per region, treating site as a random effect. Eighty-seven K loci were identified. KL2, KL102, KL25, KL15 and KL62 accounted for 49% of isolates. We estimate that 20 K loci, combining the eight most prevalent per region, could cover 72.9% of all infections [95% credible interval, 69.4&—76.5%] and ≥70% in each of Eastern, Western and Southern Africa and South Asia. Preliminary findings from three sites suggested sufficient temporal stability of K loci to maintain 20-valent K vaccine coverage over 5-10 years, but more longitudinal data are needed to support this prediction. O types were far less diverse (n=14 types). We estimate the top-5 (O1⍺β,2⍺, O1⍺β,2β, O2⍺, O2β and O4) would cover 86.2% [82.6—89.9%] of total infections (76—92% per region), while the top-10 would cover 99% of infections in all four regions. Conclusions: Neonatal sepsis is associated with diverse K and O types, with substantial geographic and temporal variation even after adjusting for localised transmission clusters. Despite this, a single 20-valent K vaccine could theoretically cover ≥70% of infections in all target regions. Locally-targeted vaccines could achieve higher coverage with lower valency, but are less feasible. In principle, very high coverage could be achieved with lower valency O-based vaccines, however protective efficacy of antibodies targeting the O antigen remains uncertain. Further research is needed on cross-reactivity, antigen exposure and stability of antigens over time, to better inform vaccine development.

Sepsis is the leading postnatal cause of neonatal mortality worldwide. Globally Klebsiella pneumoniae is the leading cause of sepsis in hospitalized neonates. This study reports the development and evaluation of an ELISA for anti-Klebsiella IgG using dried blood spot (DBS) samples and evaluates the association of anti-Klebsiella IgG (anti-Kleb IgG) antibodies in maternal and neonatal samples with the risk of neonatal sepsis. Neonates and their mothers were enrolled at 0–96 hours of life in the neonatal unit of a tertiary referral hospital in Gaborone, Botswana and followed until death or discharge to assess for episodes of blood culture-confirmed neonatal sepsis. Neonates with sepsis had significantly lower levels of Kleb-IgG compared to neonates who did not develop sepsis (Mann-Whitney U, p = 0.012). Similarly, samples from mothers of neonates who developed sepsis tended to have less Kleb-IgG compared to mothers of controls. The inverse correlation between Kleb-IgG levels and all-cause bacteremia suggests that maternal Kleb-IgG may be protective through cross-reactivity with common bacterial epitopes. These data support the continued use of immunoglobulin assays using DBS samples to explore the role of passive immunity on neonatal sepsis risk and reaffirm the critical need for research supporting the development of maternal vaccines for neonatal sepsis.

Sepsis is the leading postnatal cause of neonatal mortality worldwide. Globally Klebsiella pneumoniae is the leading cause of sepsis in hospitalized neonates. This study reports development and evaluation of ELISA for anti- Klebsiella IgG using dried blood spot samples and evaluates the association of anti- Klebsiella IgG (anti-Kleb IgG) antibodies in maternal and neonatal samples and the risk of neonatal sepsis. Neonates and their mothers were enrolled at 0-96 hours of life in the neonatal unit of a tertiary referral hospital in Gaborone, Botswana and followed until death or discharge to assess for episodes of blood culture-confirmed neonatal sepsis. Neonates with sepsis had significantly lower levels of Kleb- IgG compared to neonates who did not develop sepsis (Mann-Whitney U, p=0.012). Similarly, samples from mothers of neonates who developed sepsis tended to have less Kleb- IgG compared to mothers of controls (p=0.06). The inverse correlation between Kleb-IgG levels and all-cause bacteremia suggests that maternal Kleb- IgG is broadly protective through cross-reactivity with common bacterial epitopes. These data support the continued use of immunoglobulin assays using DBS samples to explore the role of passive immunity on neonatal sepsis risk and reaffirm the critical need for research supporting the development of maternal vaccines for neonatal sepsis.

In low- and middle-income countries, where antimicrobial access may be erratic and neonatal sepsis pathogens are frequently multidrug-resistant, empiric antibiotic prescribing practices may diverge from the World Health Organization (WHO) guidelines. This study examined antibiotic prescribing for neonatal sepsis at a tertiary referral hospital neonatal unit in Gaborone, Botswana, using data from a prospective cohort of 467 neonates. We reviewed antibiotic prescriptions for the first episode of suspected sepsis, categorized as early-onset (EOS, days 0–3) or late-onset (LOS, >3 days). The WHO prescribing guidelines were used to determine whether antibiotics were “guideline-synchronous” or “guideline-divergent”. Logistic regression models examined independent associations between the time of neonatal sepsis onset and estimated gestational age (EGA) with guideline-divergent antibiotic use. The majority (325/470, 69%) were prescribed one or more antibiotics, and 31 (10%) received guideline-divergent antibiotics. Risk factors for guideline-divergent prescribing included neonates with LOS, compared to EOS (aOR [95% CI]: 4.89 (1.81, 12.57)). Prematurity was a risk factor for guideline-divergent prescribing. Every 1-week decrease in EGA resulted in 11% increased odds of guideline-divergent antibiotics (OR [95% CI]: 0.89 (0.81, 0.97)). Premature infants with LOS had higher odds of guideline-divergent prescribing. Studies are needed to define the causes of this differential rate of guideline-divergent prescribing to guide future interventions.

Carbapenem-resistant Acinetobacter baumannii (CRAb) has emerged as a major and often fatal cause of bloodstream infections among hospitalized patients in low- and middle-income countries (LMICs). CRAb outbreaks are hypothesized to arise from reservoirs in the hospital environment, but outbreak investigations in LMICs are seldom able to incorporate whole genome sequencing (WGS) due to resource limitations. We performed WGS at the National Institute for Communicable Diseases (Johannesburg, South Africa) on stored A. baumannii isolates (n=43) collected during 2021–2022 from a 530-bed referral hospital in Gaborone, Botswana where CRAb infection incidence was noted to be rising. This included blood culture isolates from patients (aged 2 days – 69 years), and environmental isolates collected at the hospital’s 33-bed neonatal unit. Multilocus sequence typing (MLST), antimicrobial/biocide resistance gene identification, and phylogenetic analyses were performed using publicly accessible analysis pipelines. Single nucleotide polymorphism (SNP) matrices were used to assess clonal lineage. MLST revealed 79% of isolates were sequence type 1 (ST1), including all 19 healthcare-associated blood isolates and three out of five environmental isolates. Genes encoding for carbapenemases ( bla _NDM-1 , bla _OXA-23 ) and biocide resistance ( qacE ) were present in all 22 ST1 isolates. Phylogenetic analysis of the ST1 clade demonstrated spatial clustering by hospital unit. Nearly identical isolates spanned wide ranges in time (>1 year), suggesting ongoing transmission from environmental sources. One highly similar clade (average difference of 2.3 SNPs) contained all eight neonatal blood isolates and three environmental isolates from the neonatal unit. These results were critical in identifying environmental reservoirs (e.g. sinks) and developing remediation strategies. Using a phylogenetically informed approach, we also identified diagnostic genes useful for future tracking of outbreak clones without the need for WGS. This work highlights the power of South-South and South-North partnerships in building public health laboratory capacity in LMICs to detect and contain the spread of antimicrobial resistance.