Molecular Typing

BackgroundIn Malaysia, an increase in non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae (NC-CRKP) has been observed over the years. Previously, four NC-CRKP with increased susceptibility to ciprofloxacin in the presence of phenylalanine-arginine β-naphthylamide (PAβN) were identified. However, no contribution of the PAβN-inhibited efflux pump to carbapenem resistance was observed. All four NC-CRKP harboured non-carbapenemase β-lactamase, with two also exhibiting porin loss. In this study, we further investigated the genomic features and resistance mechanisms of these four isolates.MethodsAll four NC-CRKP were subjected to whole-genome sequencing, followed by comparative genomic and phylogenetic analyses.ResultsMulti-locus sequence typing (MLST) analysis divided the four NC-CRKP into different sequence types: ST392, ST45, ST14, and ST5947. Neither major nor rare carbapenemase genes were detected. Given the presence of non-carbapenemase β-lactamase in all isolates, we further investigated the potential mechanisms of resistance by identifying related chromosomal mutations. Deletion mutation was detected in the cation efflux system protein CusF. Insertion mutation was identified in the nickel/cobalt efflux protein RcnA. Missense mutation of ompK36 porin was detected in two isolates, while the loss of ompK36 porin was observed in another two isolates.ConclusionsThis study revealed that NC-CRKP may confer carbapenem resistance through a combination of non-carbapenemase β-lactamase and potential chromosomal mutations including missense mutation or loss of ompK36 porin and/or a frameshift missense mutation in efflux pump systems, such as cation efflux system protein CusF and nickel/cobalt efflux protein RcnA. Our findings highlighted the significance of implementing whole-genome sequencing into clinical practice to promote the surveillance of carbapenem resistance mechanisms among NC-CRKP.

DNA metabarcoding is subject to observation bias associated with PCR and sequencing, which can result in observed read proportions differing from actual species proportions in the DNA extract. Here, we amplify and sequence a mock community of known composition containing marine fishes and cetaceans using four different primer sets and a variety of PCR conditions. We first compare metabarcoding observations to two different sets of expected species proportions based on total genomic DNA and on target mitochondrial template DNA. We find that calibrating observed read proportions based on template DNA concentration is most appropriate as it isolates PCR amplification bias; calibration with total genomic DNA results in bias that can be attributed to both PCR amplification bias and differing ratios of template to total genomic DNA. We then model the remaining amplification bias and find that approximately 60% can be explained by inherent species-specific DNA characteristics. These include primer-template mismatches, amplicon fragment length, and GC content, which vary somewhat across Taq polymerases. Finally, we investigate how different PCR protocols influence community composition regardless of expected proportions and find that changing protocols most strongly influence the amplification of templates with primer mismatches. Our findings suggest that using primer-template pairs without mismatches and targeting a narrow taxonomic group can yield more repeatable and accurate estimates of species' true, underlying DNA template proportions. These findings identify key factors that should be considered when designing studies that aim to apply metabarcoding data quantitatively.

Studies on the epidemiology and clinical relevance of Mycobacterium tuberculosis complex (MTBC) have immensely benefited from molecular typing methods, associated software applications, and bioinformatics tools. Over the last two decades, the Pasteur Institute of Guadeloupe has developed a range of bioinformatic resources, including databases and software, to advance understanding of TB epidemiology. Traditional methods, such as IS6110-RFLP, MIRU-VNTR typing, and spoligotyping, have been instrumental but are increasingly supplanted by more precise and high-throughput techniques. These typing methods offer relatively good discrimination and reproducibility, making them popular choices for epidemiological studies. However, the advent of whole-genome sequencing (WGS) has revolutionized Mycobacterium tuberculosis complex (MTBC) typing, providing unparalleled resolution and data analysis depth. WGS enables the identification of single nucleotide polymorphisms and other genetic variations, facilitating robust phylogenetic reconstructions, and detailed outbreak investigations. This review summarizes current molecular typing methods, as well as databases and software tools used for MTBC data analysis. A comprehensive comparison of available tools and databases is provided to guide future research on the epidemiology of TB and pathogen-associated variables (drug resistance or virulence) and public health initiatives.

Using genomic barcodes to trace bacterial lineages within a host reveals previously unobservable dynamics of infection, including the impact of infection bottlenecks, routes of bacterial dissemination, and patterns of within-host evolution. Barcoding introduces trackable diversity to otherwise isogenic bacterial populations. Comparing the barcodes within an inoculum to those within the host quantifies the "founding population," which reveals the magnitude of population collapse caused by host bottlenecks. Furthermore, comparisons of the founders between tissues can reveal the patterns of pathogen dissemination. On longer timescales, the emergence of dominant barcoded lineages can also be used to detect within-host evolution. Collectively, barcoding studies quantify the hidden parameters that underlie bacterial colonization and create a quantitative framework for modeling and preventing infectious disease.