Mobile genetic elements define the non-random structure of the Salmonella enterica serovar Typhi pangenome.

Bacterial relatedness measured using select chromosomal loci forms the basis of public health genomic surveillance. While approximating vertical evolution through this approach has proven exceptionally valuable for understanding pathogen dynamics, it excludes a fundamental dimension of bacterial evolution-horizontal gene transfer. Incorporating the accessory genome is the logical remediation and has recently shown promise in expanding epidemiological resolution for enteric pathogens. Employing k-mer-based Jaccard index analysis, and a novel genome length distance metric, we computed pangenome (i.e., core and accessory) relatedness for the globally important pathogen Salmonella enterica serotype Typhi (Typhi), and graphically express both vertical (homology-by-descent) and horizontal (homology-by-admixture) evolutionary relationships in a reticulate network of over 2,200 U.S. Typhi genomes. This analysis revealed non-random structure in the Typhi pangenome that is driven predominantly by the gain and loss of mobile genetic elements, confirming and expanding upon known epidemiological patterns, revealing novel plasmid dynamics, and identifying avenues for further genomic epidemiological exploration. With an eye to public health application, this work adds important biological context to the rapidly improving ways of analyzing bacterial genetic data and demonstrates the value of the accessory genome to infer pathogen epidemiology and evolution.IMPORTANCEGiven bacterial evolution occurs in both vertical and horizontal dimensions, inclusion of both core and accessory genetic material (i.e., the pangenome) is a logical step toward a more thorough understanding of pathogen dynamics. With an eye to public, and indeed, global health relevance, we couple contemporary tools for genomic analysis with decades of research on mobile genetic elements to demonstrate the value of the pangenome, known and unknown, annotated, and hypothetical, for stratification of Salmonella enterica serovar Typhi (Typhi) populations. We confirm and expand upon what is known about Typhi epidemiology, plasmids, and antimicrobial resistance dynamics, and offer new avenues of exploration to further deduce Typhi ecology and evolution, and ultimately to reduce the incidence of human disease.

Plasmid classifications.

Plasmids are universally present in bacteria and play key roles in the dissemination of genes such as antibiotic resistance determinants. Major concepts in Plasmid Biology derive from the efforts to classify plasmids. Here, we review the main plasmid classification systems, starting by phenotype-based methods, such as fertility inhibition and incompatibility, followed by schemes based on a single gene (replicon type and MOB class), and finishing with recently developed approaches that use genetic distances between whole plasmid sequences. A comparison of the latter highlights significant differences between them. We further discuss the need for an operational definition of plasmid species that reveals their biological features, akin to plasmid taxonomic units (PTUs).

COPLA, a taxonomic classifier of plasmids.

BACKGROUND: Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence. RESULTS: We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized. CONCLUSIONS: COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.

Antimicrobial Resistance Determinants in Genomes and Plasmids from Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is a Gram-negative coccoid rod species, clinically relevant as a human pathogen, included in the ESKAPE group. Carbapenem-resistant A. baumannii (CRAB) are considered by the World Health Organization (WHO) as a critical priority pathogen for the research and development of new antibiotics. Some of the most relevant features of this pathogen are its intrinsic multidrug resistance and its ability to acquire rapid and effective new resistant determinants against last-resort clinical antibiotics, mostly from other ESKAPE species. The presence of plasmids and mobile genetic elements in their genomes contributes to the acquisition of new antimicrobial resistance determinants. However, although A. baumannii has arisen as an important human pathogen, information about these elements is still not well understood. Current genomic analysis availability has increased our ability to understand the microevolution of bacterial pathogens, including point mutations, genetic dissemination, genomic stability, and pan- and core-genome compositions. In this work, we deeply studied the genomes of four clinical strains from our hospital, and the reference strain ATCC®19606TM, which have shown a remarkable ability to survive and maintain their effective capacity when subjected to long-term stress conditions. With that, our aim was presenting a detailed analysis of their genomes, including antibiotic resistance determinants and plasmid composition.

Pathways for horizontal gene transfer in bacteria revealed by a global map of their plasmids.

Plasmids can mediate horizontal gene transfer of antibiotic resistance, virulence genes, and other adaptive factors across bacterial populations. Here, we analyze genomic composition and pairwise sequence identity for over 10,000 reference plasmids to obtain a global map of the prokaryotic plasmidome. Plasmids in this map organize into discrete clusters, which we call plasmid taxonomic units (PTUs), with high average nucleotide identity between its members. We identify 83 PTUs in the order Enterobacterales, 28 of them corresponding to previously described archetypes. Furthermore, we develop an automated algorithm for PTU identification, and validate its performance using stochastic blockmodeling. The algorithm reveals a total of 276 PTUs in the bacterial domain. Each PTU exhibits a characteristic host distribution, organized into a six-grade scale (I-VI), ranging from plasmids restricted to a single host species (grade I) to plasmids able to colonize species from different phyla (grade VI). More than 60% of the plasmids in the global map are in groups with host ranges beyond the species barrier.

Author Correction: Human neutrophils phagocytose and kill Acinetobacter baumannii and A. pittii.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MOBscan: Automated Annotation of MOB Relaxases.

Relaxase-based plasmid classification has become popular in the past 10 years. Nevertheless, it is not obvious how to assign a query protein to a relaxase MOB family. Automated protein annotation is commonly used to classify them into families, gathering evolutionarily related proteins that likely perform the same function, while circumventing the problem of different naming conventions. Here, we implement an automated method, MOBscan, to identify relaxases and classify them into any of the nine MOB families. MOBscan is a web tool that carries out a HMMER search against a curated database of MOB profile Hidden Markov models. It is freely available at https://castillo.dicom.unican.es/mobscan/ .

Plasmid Reconstruction from Next-Gen Data: A Detailed Protocol for the Use of PLACNETw for the Reconstruction of Plasmids from WGS Datasets.

Mobile Genetic Elements (MGE) play essential roles in adaptive bacterial evolution, facilitating genetic exchange for extrachromosomal DNA, especially antibiotic resistance genes and virulence factors. For this reason, high-throughput next-generation sequencing of bacteria is of great relevance, especially for clinical pathogenic bacteria. Accurate identification of MGE from whole-genome sequencing (WGS) datasets is one of the major challenges, still hindered by methodological limitations and high sequencing costs.This chapter encompasses the protocol used for plasmid reconstruction by applying the PLACNETw methodology, from raw reads to assembled plasmids and chromosome. PLACNETw is a graphical user-friendly interface to visualize and reconstruct MGE from short-read WGS datasets. No bioinformatic background or sophisticated computational resources are required and high precision and sensitivity are achieved.

Whole-Genome Sequence of Acinetobacter baumannii HUMV-3743, Isolated from a Human Wound Exudate.

Acinetobacter baumannii strain HUMV-3743 was obtained from wound exudate from an adult patient. Here, we report its complete genome sequence using Illumina-based sequence analysis, which revealed a genome of 4 Mb, which includes 2 predicted plasmids of 78.9 and 107 kb. A total of 3,881 protein-coding genes are predicted from this assembly.

Human neutrophils phagocytose and kill Acinetobacter baumannii and A. pittii.

Acinetobacter baumannii is a common cause of health care associated infections worldwide. A. pittii is an opportunistic pathogen also frequently isolated from Acinetobacter infections other than those from A. baumannii. Knowledge of Acinetobacter virulence factors and their role in pathogenesis is scarce. Also, there are no detailed published reports on the interactions between A. pittii and human phagocytic cells. Using confocal laser and scanning electron microscopy, immunofluorescence, and live-cell imaging, our study shows that immediately after bacteria-cell contact, neutrophils rapidly and continuously engulf and kill bacteria during at least 4 hours of infection in vitro. After 3 h of infection, neutrophils start to release neutrophil extracellular traps (NETs) against Acinetobacter. DNA in NETs colocalizes well with human histone H3 and with the specific neutrophil elastase. We have observed that human neutrophils use large filopodia as cellular tentacles to sense local environment but also to detect and retain bacteria during phagocytosis. Furthermore, co-cultivation of neutrophils with human differentiated macrophages before infections shows that human neutrophils, but not macrophages, are key immune cells to control Acinetobacter. Although macrophages were largely activated by both bacterial species, they lack the phagocytic activity demonstrated by neutrophils.

Whole-Genome Sequence of Acinetobacter pittii HUMV-6483 Isolated from Human Urine.

Acinetobacter pittii strain HUMV-6483 was obtained from urine from an adult patient. We report here its complete genome assembly using PacBio single-molecule real-time sequencing, which resulted in a chromosome with 4.07 Mb and a circular contig of 112 kb. About 3,953 protein-coding genes are predicted from this assembly.

Whole-Genome Sequence of Hafnia alvei HUMV-5920, a Human Isolate.

A clinical isolate of Hafnia alvei (strain HUMV-5920) was obtained from a urine sample from an adult patient. We report here its complete genome assembly using PacBio single-molecule real-time (SMRT) sequencing, which resulted in a chromosome with 4.5 Mb and a circular contig of 87 kb. About 4,146 protein-coding genes are predicted from this assembly.