Genomic analysis of Klebsiella pneumoniae causing community-acquired respiratory deaths among Zambian infants and children using targeted RNA-probe hybridization-capture metagenomics

Reviewer #1 (Public review):

Summary:

This study described the genomic features of K. pneumoniae in tissue samples from children in Zambia who had died from pneumonia, where K. pneumoniae was believed to be the causative organism. This was a substudy of a previous study which described more broadly the aetiologies of community-acquired pneumonia in this population, using minimally invasive tissue sampling.

In this study, the authors used culture-independent molecular tools to characterise the K. pneumoniae genomes from post-mortem tissue samples from 7 children who had died of pneumonia. They described a diversity of strain lineages in the 7 children, with different capsular types and virulence profiles. Notably, all strains had a wide array of antimicrobial resistance genes. This is probably not surprising given the propensity of this organism to acquire AMR genes, but it is concerning in isolates from community-acquired infections.

The study also highlights the value of using advanced culture-independent molecular techniques, and the scope of data that can be obtained. In settings where culture is not always available, storage and subsequent remote analysis of these samples is an option to better understand the microbiology (although expense is still a barrier, and culture-based microbiology should not be completely neglected).

Strengths:

Ascribing aetiologies in respiratory infections is not (yet) an exact science, and there is likely to always be some doubt about whether an organism (whether identified by culture or nucleic acid detection) is a true pathogen. However, the authors have used a robust combination of histology, microbiology, radiology, clinical assessment and verbal autopsy, and this is probably as good as we can get it at present.

The molecular techniques employed and the analysis were robust and technically sound. There are some areas where there is inconsistency between the results from two samples from the same patient, and this is likely due to the lower number of reads - important information for future similar studies. It highlights the potential limitations of this technique.

The data obtained (albeit from a small sample set) are consistent with the other data from Africa, which provides support for the value and reliability of the technique itself.

Weaknesses:

The major weakness is the small sample size. The 7 patients analysed in this study are a subset of the children in the larger study who had been identified as having died from K. pneumoniae respiratory infection. So while the findings of this study highlight the potential role of this organism as a respiratory pathogen and provide some insight into the distribution of lineages in the community, the results don't really allow for major changes to clinical or diagnostic practice as yet. The data highlight a potentially under-recognised problem and would be useful to inform future studies.

A minor weakness is more related to the journal layout, where methods are presented last. The results are not as easy to follow without reviewing the methods - in particular the description of histopathological findings and results of PCR on the biopsies. Until one realises that 6 biopsies had been taken from each of the deceased children, and a subset of these biopsies used for the study, the results seemed confusing.

Reviewer #2 (Public review):

Summary:

This manuscript applies a culture-independent hybridization-capture metagenomic sequencing approach to characterize Klebsiella pneumoniae detected in post-mortem lung tissue from fatal pediatric pneumonia cases in Lusaka, Zambia. The study addresses an important challenge in retrospective genomic investigations where cultured isolates are unavailable and demonstrates the potential of targeted sequencing to recover clinically relevant genomic information directly from archived tissue specimens. The authors report sequence types, capsular loci, antimicrobial resistance determinants, virulence-associated genes, and evidence of closely related isolates in two cases. The work is valuable as a proof-of-concept application of targeted sequencing in challenging post-mortem specimens and provides useful descriptive genomic data from a setting where such information remains limited. However, several epidemiological and public health interpretations extend beyond what can be supported by the available data. The study includes only seven successfully sequenced children from a single setting and was not designed to determine the source of acquisition, transmission pathways, or population-level distributions of antimicrobial resistance or capsular types. The manuscript would therefore be strengthened by more consistently framing the findings as a descriptive genomic investigation of K. pneumoniae detected in children who died outside hospital settings, rather than as evidence of community-acquired infection or broader epidemiological shifts.

Strengths:

The principal strength of the manuscript is its methodological contribution. The authors demonstrate that hybridization-capture metagenomic sequencing can recover informative genomic data from post-mortem lung tissue in cases where conventional culture-based sequencing is not available. This is an important technical advance for retrospective studies, minimally invasive tissue sampling platforms, and settings where sample degradation, prior antibiotic exposure, or lack of routine culture limits genomic surveillance.

The study also addresses an important public health problem. K. pneumoniae is a major cause of severe infection and antimicrobial resistance globally, yet its role in fatal pediatric pneumonia outside hospital settings remains difficult to define. The generation of sequence type, capsular locus, antimicrobial resistance, and virulence-associated gene data from post-mortem specimens is therefore useful and may inform future study designs. The identification of closely related isolates in two infants is also potentially important and raises hypotheses about shared sources or transmission that could be explored in larger studies.

Another strength is that the authors appropriately acknowledge several technical challenges, including low numbers of K. pneumoniae-assigned reads in some specimens and unresolved or discordant capsular locus calls. These issues are important for readers considering the utility of this approach in low-input or mixed-specimen contexts.

Weaknesses:

The main weakness is that the epidemiological framing is stronger than the data allow. The manuscript repeatedly refers to community-acquired K. pneumoniae pneumonia and broader community epidemiology. However, the available data do not establish community acquisition, community transmission, or an epidemiological shift from nosocomial to community disease. Several children appear to have had prior healthcare contact or other potential healthcare-associated exposures, and the study design cannot determine where acquisition occurred. The findings would be more accurately framed as K. pneumoniae detected in post-mortem lung tissue from children who died outside hospital settings.

Causal attribution also requires more careful wording. Detection of K. pneumoniae in post-mortem lung tissue, together with histopathology and DeCoDe findings, provides important supportive evidence that the organism may have been in the causal chain leading to death. However, this does not necessarily establish that K. pneumoniae was the sole or direct cause of fatal pneumonia, particularly where multiple pathogens were detected.

The small sample size and case selection strategy limit the generalizability of the findings. Only seven children were successfully sequenced, and specimens appear to have been selected partly based on molecular signal. This is technically understandable, but it may introduce selection bias by enriching for cases with higher bacterial burden, better DNA preservation, or other specimen characteristics. As a result, the observed lineage diversity, resistance gene profiles, virulence-associated loci, and capsular locus distribution should not be interpreted as representative of community-acquired infections or broader population epidemiology.

The validation of the hybridization-capture approach also requires strengthening. Comparing outputs from different genomic analysis tools applied to the same sequencing data may assess bioinformatic concordance, but it does not independently validate the method. Ideally, the approach should be benchmarked against clinical K. pneumoniae isolates or matched specimens with conventional whole-genome sequencing data. Without this, it is difficult to assess the accuracy of sequence type, capsular locus, antimicrobial resistance determinant, virulence locus, and plasmid marker recovery, especially in low-read or mixed-specimen contexts.

Species-level attribution of antimicrobial resistance, virulence-associated genes, and plasmid replicons is another important limitation. In a culture-independent metagenomic study, these features cannot automatically be assigned to the identified K. pneumoniae lineage because many such elements are shared across Enterobacterales and may originate from co-detected organisms. This affects interpretation of antimicrobial resistance, hypervirulence, and MDR-hypervirulence convergence.

Overall, the authors achieved their methodological aim of demonstrating that targeted sequencing can recover useful genomic information from challenging post-mortem specimens. However, the epidemiological, transmission, antimicrobial resistance, and vaccine-related conclusions should be tempered.