GR13-type plasmids in Acinetobacter potentiate the accumulation and horizontal transfer of diverse accessory genes

Carbapenem resistance and other antibiotic resistance genes (ARGs) can be found in plasmids in Acinetobacter, but many plasmid types in this genus have not been well-characterised. Here we describe the distribution, diversity and evolutionary capacity of rep group 13 (GR13) plasmids that are found in Acinetobacter species from diverse environments. Our investigation was prompted by the discovery of two GR13 plasmids in A. baumannii isolated in an intensive care unit (ICU). The plasmids harbour distinct accessory genes: pDETAB5 contains blaNDM-1 and genes that confer resistance to four further antibiotic classes, while pDETAB13 carries putative alcohol tolerance determinants. Both plasmids contain multiple dif modules, which are flanked by pdif sites recognised by XerC/XerD tyrosine recombinases. The ARG-containing dif modules in pDETAB5 are almost identical to those found in pDETAB2, a GR34 plasmid from an unrelated A. baumannii isolated in the same ICU a month prior. Examination of a further 41 complete, publicly available plasmid sequences revealed that the GR13 pangenome consists of just four core but 1086 accessory genes, 123 in the shell and 1063 in the cloud, reflecting substantial capacity for diversification. The GR13 core genome includes genes for replication and partitioning, and for a putative tyrosine recombinase. Accessory segments encode proteins with diverse putative functions, including for metabolism, antibiotic/heavy metal/alcohol tolerance, restriction-modification, an anti-phage system and multiple toxin-antitoxin systems. The movement of dif modules and actions of insertion sequences play an important role in generating diversity in GR13 plasmids. Discrete GR13 plasmid lineages are internationally disseminated and found in multiple Acinetobacter species, which suggests they are important platforms for the accumulation, horizontal transmission and persistence of accessory genes in this genus.

EnteroBase: Hierarchical clustering of 100,000s of bacterial genomes into species/sub-species and populations

The definition of bacterial species is traditionally a taxonomic issue while defining bacterial populations is done with population genetics. These assignments are species specific, and depend on the practitioner. Legacy multilocus sequence typing is commonly used to identify sequence types (STs) and clusters (ST Complexes). However, these approaches are not adequate for the millions of genomic sequences from bacterial pathogens that have been generated since 2012. EnteroBase (http://enterobase.warwick.ac.uk) automatically clusters core genome MLST alleles into hierarchical clusters (HierCC) after assembling annotated draft genomes from short read sequences. HierCC clusters span core sequence diversity from the species level down to individual transmission chains. Here we evaluate the ability of HierCC to correctly assign 100,000s of genomes to the species/subspecies and population levels for Salmonella, Clostridoides, Yersinia, Vibrio and Streptococcus. HierCC assignments were more consistent with maximum-likelihood super-trees of core SNPs or presence/absence of accessory genes than classical taxonomic assignments or 95% ANI. However, neither HierCC nor ANI were uniformly consistent with classical taxonomy of Streptococcus. HierCC was also consistent with legacy eBGs/ST Complexes in Salmonella or Escherichia and revealed differences in vertical inheritance of O serogroups. Thus, EnteroBase HierCC supports the automated identification of and assignment to species/subspecies and populations for multiple genera.

Recurring outbreaks by the same Escherichia coli ST10 clone in a broiler unit during 18 months

The current investigation aimed at characterizing the cause of multiple disease outbreaks in the same broiler production unit during a course of 18 months. The outbreaks had mortality rates of up to 22%. Escherichia coli was diagnosed as the responsible agent. Multiple-locus variable-number tandem-repeat analysis showed that all chicken isolates had identical band patterns. Core genome comparisons demonstrated that the 36 chicken isolates differed with maximum of nine nucleotides indicating that the same E. coli clone was responsible for all seven disease outbreaks despite adherence to the all-in-all production principle and rigorous cleaning and disinfection procedures.

Molecular subtyping for source tracking of Escherichia coli using core genome multilocus sequence typing at a food manufacturing plant

When harmful bacteria are detected in the final product at a food manufacturing plant, it is necessary to identify and eliminate the source of contamination so that it does not occur again. In the current study, the source of contamination was tracked using core genome multilocus sequence typing (cgMLST) analysis in cases where Escherichia coli was detected in the final product at a food manufacturing plant. cgMLST analysis was performed on 40 strains of E. coli collected from the environment [floor (26 strains), drainage ditch (5 strains), container (4 strains), post-heating production line (1 strain)] and products [final product (3 strains) and intermediate product (1 strain)]. In total, 40 E. coli isolates were classified into 17 genogroups by cgMLST analysis. The 4 E. coli strains isolated from the intermediate and final products were classified into two genogroups (I and II). Certain isolates collected from the environment also belonged to those genogroups, it was possible to estimate the transmission of E. coli in the manufacturing plant. Thus, the dynamics of E. coli in the food manufacturing location were clarified by using cgMLST analysis. In conclusion, our results indicate that cgMLST analysis can be effectively used for hygiene management at food manufacturing locations.

The ColR/S two-component system is a conserved determinant of host association across Pseudomonas species

Members of the bacterial genus Pseudomonas form mutualistic, commensal and pathogenic associations with diverse hosts. The prevalence of host association across the genus suggests that symbiosis may be a conserved ancestral trait and that distinct symbiotic lifestyles may be more recently evolved. Here we show that the ColR/S two-component system, part of the Pseudomonas core genome, is functionally conserved between Pseudomonas aeruginosa and Pseudomonas fluorescens. Using plant rhizosphere colonization and virulence in a murine abscess model, we show that colR is required for commensalism with plants and virulence in animals. Comparative transcriptomics revealed that the ColR regulon has diverged between P. aeruginosa and P. fluorescens and deleting components of the ColR regulon revealed strain-specific, but not host specific, requirements for ColR-dependent genes. Collectively, our results suggest that ColR/S allows Pseudomonas to sense and respond to a host, but that the ColR-regulon has diverged between Pseudomonas strains with distinct lifestyles.

Genomic Characterization of Clinical Listeria monocytogenes Isolates in Beijing, China

Listeria monocytogenes is a foodborne human pathogen that affects public health worldwide. Whole-genome sequencing (WGS) can classify L. monocytogenes isolates and identify virulence islands and resistance genes potentially influencing infectivity. Herein, WGS was used to assess 151 L. monocytogenes isolates from 120 cases of clinical infection in Beijing, China, between 2014 and 2018. Most isolates were either serogroup 1/2a,3a or serogroup 1/2b,3b,7, with 25 multilocus sequence typing (MLST) types (STs) represented, of which ST8, ST87, and ST5 were the most common. Core-genome MLST (cgMLST) grouped the 151 isolates into 116 cgMLST types. The discriminatory power of cgMLST was greater than other subtypes, revealing that isolates from the same patient were highly related (only differing at one allele). Eighty-six isolates formed 30 complexes with ≤ 7 cgMLST alleles between neighboring isolates, suggesting possible outbreaks. Compared with isolates in the United States, ST8, ST121, ST619, ST87, and ST155 isolates were grouped into unified clades. All 151 isolates were positive for common virulence-associated loci, and 26 lineage I isolates harbored the pathogenicity island 3 (LIPI-3) locus, while 42 lineage I isolates harbored the complete LIPI-4 locus. Eleven ST619 isolates had both LIPI-3 and LIPI-4. Among the 151 isolates, 13 were resistant to at least one antibiotic, and no multidrug-resistant isolates were identified. Resistance phenotypes correlated with genotypes, apart from two meropenem resistance isolates. The findings provided insight into the nature of L. monocytogenes strains currently causing clinical disease in Beijing, and WGS analysis indicated possible outbreaks.

Meta Analysis of the Ralstonia solanacearum species complex (RSSC) based on comparative evolutionary genomics and reverse ecology

Ralstonia solanacearum species complex (RSSC) strains are bacteria that colonize plant xylem and cause vascular wilt diseases. However, individual strains vary in host range, optimal disease temperatures, and physiological traits. To increase our understanding of the evolution, diversity, and biology of the RSSC, we performed a meta-analysis of 100 representative RSSC genomes. These 100 RSSC genomes contain 4,940 genes on average, and a pangenome analysis found that there are 3,262 genes in the core genome (∼60% of the mean RSSC genome) with 13,128 genes in the extensive flexible genome. Although a core genome phylogenetic tree and a genome similarity matrix aligned with the previously named species (R. solanacearum, R. pseudosolanacearum, R. syzygii) and phylotypes (I-IV), these analyses also highlighted an unrecognized sub-clade of phylotype II. Additionally, we identified differences between phylotypes with respect to gene content and recombination rate, and we delineated population clusters based on the extent of horizontal gene transfer. Multiple analyses indicate that phylotype II is the most diverse phylotype, and it may thus represent the ancestral group of the RSSC. Additionally, we also used our genome-based framework to test whether the RSSC sequence variant (sequevar) taxonomy is a robust method to define within-species relationships of strains. The sequevar taxonomy is based on alignments of a single conserved gene (egl). Although sequevars in phylotype II describe monophyletic groups, the sequevar system breaks down in the highly recombinogenic phylotype I, which highlights the need for an improved cost-effective method for genotyping strains in phylotype I. Finally, we enabled quick and precise genome-based identification of newly sequenced Ralstonia strains by assigning Life Identification Numbers (LINs) to the 100 strains and by circumscribing the RSSC and its sub-groups in the LINbase Web service.IMPACT STATEMENTThe Ralstonia solanacearum species complex (RSSC) includes dozens of economically important pathogens of many cultivated and wild plants. The extensive genetic and phenotypic diversity that exists within the RSSC has made it challenging to subdivide this group into meaningful subgroups with relevance to plant disease control and plant biosecurity. This study provides a solid genome-based framework for improved classification and identification of the RSSC by analyzing one hundred representative RSSC genome sequences with a suite of comparative evolutionary genomic tools. The results also lay the foundation for additional in-depth studies to gain further insights into evolution and biology of this heterogeneous complex of destructive plant pathogens.DATA SUMMARYThe authors confirm that all raw data and code and protocols have been provided within the manuscript. All publicly available sequencing data used for analysis have been supplemented with accession numbers to access the data. The assembled genome of strain 19-3PR_UW348 was submitted to NCBI under Bioproject PRJNA775652 Biosample SAMN22612291. This Whole Genome Shotgun project has been deposited at GenBank under the accession JAJMMU000000000. The version described in this paper is version JAJMMU010000000.

Comparative Genomics of Novel Agrobacterium G3 Strains Isolated From the International Space Station and Description of Agrobacterium tomkonis sp. nov.

Strains of Agrobacterium genomospecies 3 (i.e., genomovar G3 of the Agrobacterium tumefaciens species complex) have been previously isolated from diverse environments, including in association with plant roots, with algae, as part of a lignocellulose degrading community, from a hospital environment, as a human opportunistic pathogen, or as reported in this study, from a surface within the International Space Station. Polyphasic taxonomic methods revealed the relationship of Agrobacterium G3 strains to other Agrobacterium spp., which supports the description of a novel species. The G3 strains tested (n = 9) were phenotypically distinguishable among the strains from other genomospecies of the genus Agrobacterium. Phylogenetic analyses of the 16S rRNA gene, gyrB gene, multi-locus sequence analysis, and 1,089-gene core-genome gene concatenate concur that tested G3 strains belong to the Agrobacterium genus and they form a clade distinct from other validly described Agrobacterium species. The distinctiveness of this clade was confirmed by average nucleotide identity (ANI) and in silico digital DNA–DNA hybridization (dDDH) comparisons between the G3 tested strains and all known Agrobacterium species type strains, since obtained values were considerably below the 95% (ANI) and 70% (dDDH) thresholds used for the species delineation. According to the core-genome phylogeny and ANI comparisons, the closest relatives of G3 strains were Agrobacterium sp. strains UGM030330-04 and K599, members of a novel genomospecies we propose to call genomovar G21. Using this polyphasic approach, we characterized the phenotypic and genotypic synapomorphies of Agrobacterium G3, showing it is a bona fide bacterial species, well separated from previously named Agrobacterium species or other recognized genomic species. We thus propose the name Agrobacterium tomkonis for this species previously referred to as Agrobacterium genomospecies 3. The type strain of A. tomkonis is IIF1SW-B1T (= LMG 32164 = NRRL B-65602). Comparative genomic analysis show A. tomkonis strains have species-specific genes associated with secretion of secondary metabolites, including an exopolysaccharide and putative adhesins and resistance to copper. A. tomkonis specific gene functions notably relate to surface adhesion and could be involved to colonize nutrient-poor and harsh habitats. The A. tomkonis strains from the ISS showed presence of a 40-kbp plasmid and several other potential mobile genetic elements detected that could also be part of conjugative elements or integrated prophages.

Diversity of carbapenem-resistant Klebsiella pneumoniae ST14 and emergence of a subgroup with KL64 capsular locus in the Arabian Peninsula

To understand the reasons of successful spread of carbapenem-resistant Klebsiella pneumoniae ST14 (CRKP-ST14) in countries of the Arabian Peninsula, the resistome, capsular locus, carbapenemase carrying plasmid types, and core genome of isolates from the region were compared to global isolates. Thirty-nine CRKP-ST14 strains isolated from 13 hospitals in the United Arab Emirates, Bahrain, and Saudi Arabia were selected for whole genome sequencing on Illumina MiSeq platform based on the variety of carbapenemase genes carried and plasmids bearing these genes. Their resistome, capsular locus, and core genome MLST were compared to 173 CRKP-ST14 genomes available in public databases. The selected 39 CRKP-ST14 produced either NDM-1, OXA-48, OXA-162, OXA-232, KPC-2, or co-produced NDM-1 and an OXA-48-like carbapenemase. cgMLST revealed three clusters: 16 isolates from five UAE cities (C1), 11 isolates from three UAE cities and Bahrain (C2), and 5 isolates from Saudi Arabia (C3), respectively, and seven singletons. Resistance gene profile, carbapenemase genes, and their plasmid types were variable in both C1 and C2 clusters. The majority of CRKP-ST14 had KL2, but members of the C2 cluster and two further singletons possessed KL64 capsular locus. Based on cgMLST comparison of regional and global isolates, CRKP-ST14 with KL64 from four continents formed a distinct cluster, suggesting a recent emergence and spread of this variant. Our findings confirmed clonal transmission coupled with likely horizontal gene transfer in carbapenem-resistant Klebsiella pneumoniae ST14. Dissemination of this genetically flexible, highly resistant clone warrants further monitoring.

To catch a hijacker: abundance, evolution and genetic diversity of P4-like bacteriophage satellites

Bacteriophages (phages) are bacterial parasites that can themselves be parasitized by phage satellites. The molecular mechanisms used by satellites to hijack phages are sometimes understood in great detail, but the origins, abundance, distribution and composition of these elements are poorly known. Here, we show that P4-like elements are present in more than 30% of the genomes of Enterobacterales, and in almost half of those of Escherichia coli , sometimes in multiple distinct copies. We identified over 1000 P4-like elements with very conserved genetic organization of the core genome and a few hotspots with highly variable genes. These elements are never found in plasmids and have very little homology to known phages, suggesting an independent evolutionary origin. Instead, they are scattered across chromosomes, possibly because their integrases are often exchanged with other elements. The rooted phylogenies of hijacking functions are correlated and suggest longstanding coevolution. They also reveal broad host ranges in P4-like elements, as almost identical elements can be found in distinct bacterial genera. Our results show that P4-like phage satellites constitute a very distinct, widespread and ancient family of mobile genetic elements. They pave the way for studying the molecular evolution of antagonistic interactions between phages and their satellites. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.