Mapping of high-throughput sequencing (HTS) reads to a single arbitrary reference genome is a frequently used approach in microbial genomics. However, the choice of a reference may represent a source of errors that may affect subsequent analyses such as the detection of single nucleotide polymorphisms (SNPs) and phylogenetic inference. In this work, we evaluated the effect of reference choice on short-read sequence data from five clinically and epidemiologically relevant bacteria (Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Pseudomonas aeruginosa and Serratia marcescens). Publicly available whole-genome assemblies encompassing the genomic diversity of these species were selected as reference sequences, and read alignment statistics, SNP calling, recombination rates, dN/dS ratios, and phylogenetic trees were evaluated depending on the mapping reference. The choice of different reference genomes proved to have an impact on almost all the parameters considered in the five species. In addition, these biases had potential epidemiological implications such as including/excluding isolates of particular clades and the estimation of genetic distances. These findings suggest that the single reference approach might introduce systematic errors during mapping that affect subsequent analyses, particularly for data sets with isolates from genetically diverse backgrounds. In any case, exploring the effects of different references on the final conclusions is highly recommended.

Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, genomic and functional studies to show that other zoonotic clades are in fact present in 4 of the 5 lineages of the species. Further, we associate these clades, most of them previously but incompletely described, with the acquisition of a family of fish virulence plasmids containing genes essential for resistance to the immune system of certain teleosts of interest in aquaculture. Consequently, our results provide several pieces of evidence about the importance of this species as a zoonotic agent linked to fish farms, as well as on the relevance of these artificial environments acting as drivers that accelerate the evolution of the species.

Carbapenem-resistant Klebsiella pneumoniae is a major cause of hospital-acquired infections and the fastest-growing pathogen in Europe. Carbapenem resistance was detected at the Consorcio Hospital General Universitario de Valencia (CHGUV) in early 2015, and there has been a significant increase in carbapenem-resistant isolates since then. In this study, we collected carbapenem-resistant isolates from this hospital during the period of increase (from 2015 to 2019) and studied how K. pneumoniae carbapenem-resistant isolates emerged and spread in the hospital. A total of 225 isolates were subjected to whole-genome sequencing with Illumina NextSeq. We characterized the isolates by identifying lineages and antimicrobial resistance genes and plasmids, especially those related to reduced carbapenem susceptibility. Our findings show that the initial carbapenem resistance emergence and dissemination at the CHGUV occurred during a short period of 1 year. Furthermore, it was complex, involving six different lineages of types ST307, ST11, ST101 and ST437, different resistance-determinant factors, including OXA-48, NDM-1, NDM-23 and DHA-1, and different plasmids.

The emergence and spread of third-generation cephalosporins (3GC) and carbapenem-resistant Klebsiella pneumoniae pose a global critical challenge. Understanding the transmission dynamics within and between hospital environments is crucial to develop effective control strategies. From 2017 to 2019, we conducted a genomic surveillance program in eight hospitals of the Comunitat Valenciana, Spain, collecting and sequencing 1,768 3GC- and carbapenem-resistant isolates. We quantified the overall transmission using core genomes and assessed the contribution of national and global isolates to the spread of AMR in the region by including 11,967 database genomes in the analysis. The local collection was highly diverse, involving 188 lineages, including global high-risk clones such as ST307 and ST11, and 3GC and carbapenem resistance determinants. Half of the isolates were involved in transmission, with 70.5% occurring within hospitals. Different transmission patterns characterized the spread of 3GC- and carbapenem resistance in the region. While inter-hospital transmission played a significant role in the spread of 3GC-resistance, this was only sporadic for carbapenem resistance. Moreover, the factors behind inter-hospital spread for each type of resistance differed: while 3GC-resistance likely disseminated between hospitals through intermediate steps, carbapenem resistance was driven by more direct transmission routes. The burden of national and global cases on the ongoing regional AMR dissemination was low. Moreover, we revealed the rapid expansion in the region and globally of lineage ST307 carrying the blaCTX-M-15 gene, a main driver of local transmissions, providing a deeper understanding of the successful spread of this high-risk clone.