Mainstreams of Horizontal Gene Exchange in Enterobacteria: Consideration of the Outbreak of Enterohemorrhagic E. coli O104:H4 in Germany in 2011

Escherichia coli O104:H4 caused a severe outbreak in Europe in 2011. The strain TY-2482 sequenced from this outbreak allowed the discovery of its closest relatives but failed to resolve ways in which it originated and evolved. On account of the previous statement, may we expect similar upcoming outbreaks to occur recurrently or spontaneously in the future? The inability to answer these questions shows limitations of the current comparative and evolutionary genomics methods.status: publishe

Genomic molecular epidemiology of carbapenemase-producing Escherichia coli ST410 isolates by complete genome analysis

The circulation of carbapenemase-producing Escherichia coli (CPEC) in our society is a serious concern for vulnerable patients in nosocomial environments. However, the genomic epidemiology of the circulation of CPEC bacteria among companion animals remains largely unknown. In this study, epidemiological analysis was conducted using complete genome identification of CPEC ST410 isolates obtained from companion animals. To estimate the genomic distance and relatedness of the isolates, a total of 37 whole-genome datasets of E. coli ST410 strains were downloaded and comparatively analysed. As a result of the analysis, the genomic structure of the chromosomes and plasmids was identified, revealing the genomic positions of multiple resistance and virulence genes. The isolates in this study were grouped into the subclade H24/RxC, with fimH24, and substituted quinolone resistance-determining regions (QRDRs) and multiple beta-lactamases, including extended-spectrum β-lactamase (ESBL) and carbapenemase. In addition, the in silico comparison of the whole-genome datasets revealed unidentified ST410 H24/Rx subgroups, including either high pathogenicity islands (HPIs) or H21 serotypes. Considering the genetic variations and resistance gene dissemination of the isolates carried by companion animals, future approaches for preventive measurement must include the One Health perspective for public health in our society.This work was supported by the National Research Foundation (NRF- 2020R1A2C200879414), BK21 FOUR Future Veterinary Medicine Leading Education and Research Center and Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Kore

Genomic comparative analysis of the environmental Enterococcus mundtii against enterococcal representative species

Background Enterococcus mundtii is a yellow-pigmented microorganism rarely found in human infections. The draft genome sequence of E. mundtii was recently announced. Its genome encodes at least 2,589 genes and 57 RNAs, and 4 putative genomic islands have been detected. The objective of this study was to compare the genetic content of E. mundtii with respect to other enterococcal species and, more specifically, to identify genes coding for putative virulence traits present in enterococcal opportunistic pathogens. Results An in-depth mining of the annotated genome was performed in order to uncover the unique properties of this microorganism, which allowed us to detect a gene encoding the antimicrobial peptide mundticin among other relevant features. Moreover, in this study a comparative genomic analysis against commensal and pathogenic enterococcal species, for which genomic sequences have been released, was conducted for the first time. Furthermore, our study reveals significant similarities in gene content between this environmental isolate and the selected enterococci strains (sharing an “enterococcal gene core” of 805 CDS), which contributes to understand the persistence of this genus in different niches and also improves our knowledge about the genetics of this diverse group of microorganisms that includes environmental, commensal and opportunistic pathogens. Conclusion Although E. mundtii CRL1656 is phylogenetically closer to E. faecium, frequently responsible of nosocomial infections, this strain does not encode the most relevant relevant virulence factors found in the enterococcal clinical isolates and bioinformatic predictions indicate that it possesses the lowest number of putative pathogenic genes among the most representative enterococcal species. Accordingly, infection assays using the Galleria mellonella model confirmed its low virulenceFil: Repizo, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Espariz, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Suárez, Cristian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Esteban, Luis. Universidad Nacional de Rosario. Facultad de Cs.médicas. Escuela de Cs.médicas. Cátedra de Fisiología; ArgentinaFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Intraclonal genome diversity of Pseudomonas aeruginosa clones CHA and TB.

International audienceBACKGROUND: Adaptation of Pseudomonas aeruginosa to different living conditions is accompanied by microevolution resulting in genomic diversity between strains of the same clonal lineage. In order to detect the impact of colonized habitats on P. aeruginosa microevolution we determined the genomic diversity between the highly virulent cystic fibrosis (CF) isolate CHA and two temporally and geographically unrelated clonal variants. The outcome was compared with the intraclonal genome diversity between three more closely related isolates of another clonal complex. RESULTS: The three clone CHA isolates differed in their core genome in several dozen strain specific nucleotide exchanges and small deletions from each other. Loss of function mutations and non-conservative amino acid replacements affected several habitat- and lifestyle-associated traits, for example, the key regulator GacS of the switch between acute and chronic disease phenotypes was disrupted in strain CHA. Intraclonal genome diversity manifested in an individual composition of the respective accessory genome whereby the highest number of accessory DNA elements was observed for isolate PT22 from a polluted aquatic habitat. Little intraclonal diversity was observed between three spatiotemporally related outbreak isolates of clone TB. Although phenotypically different, only a few individual SNPs and deletions were detected in the clone TB isolates. Their accessory genome mainly differed in prophage-like DNA elements taken up by one of the strains. CONCLUSIONS: The higher geographical and temporal distance of the clone CHA isolates was associated with an increased intraclonal genome diversity compared to the more closely related clone TB isolates derived from a common source demonstrating the impact of habitat adaptation on the microevolution of P. aeruginosa. However, even short-term habitat differentiation can cause major phenotypic diversification driven by single genomic variation events and uptake of phage DNA

Achromobacter spp. in Cystic Fibrosis Patients: A Genomic-Based Approach to Unravel Microbe-Host Adaptation

Bacteria belonging to the genus Achromobacter are widely distributed in natural environments and have been recognized as emerging nosocomial pathogens for their contribution to a wide range of human infections. Achromobacter spp. can establish chronic infections associated with inflammation, produce biofilm, resist common disinfectants, readily acquire antibiotic resistance and outcompete resident microbiota. In particular, cystic fibrosis (CF) patients with lung disease are the most frequently colonized and infected by Achromobacter species usually developing persistent respiratory tract infections. In the last five years the number of publications regarding these pathogens has doubled in comparison to the preceding five-year period and their whole genome sequencing data availability has seen a steep increase, underlining both the growing research interest for these microorganisms as well as their emergence in the clinical setting. Nonetheless, many clinical aspects and pathogenic mechanisms still remain to be elucidated. The main focus of this thesis has been to unravel underlying key processes and to investigate the adaptive mechanisms exploited by these microorganisms during lung infection in CF patients. This has been pursued by analysing both genomic and phenotypic data of 103 Achromobacter spp. clinical isolates from 40 CF patients followed at the CF centres in Verona (Italy), Rome (Italy), and Copenhagen (Denmark). The work presented in this thesis provides new knowledge on the onset of Achromobacter spp. infections and their adaptation to the CF lung environment. With further genomic and phenotypic studies it will be possible to translate these results into the clinical setting, leading to better predictions of the infection course and improvement of treatment strategies to the benefit of CF patients

Comparative Genomic Analysis of Pathogenic and Probiotic Enterococcus faecalis Isolates, and Their Transcriptional Responses to Growth in Human Urine

Urinary tract infection (UTI) is the most common infection caused by enterococci, and Enterococcus faecalis accounts for the majority of enterococcal infections. Although a number of virulence related traits have been established, no comprehensive genomic or transcriptomic studies have been conducted to investigate how to distinguish pathogenic from non-pathogenic E. faecalis in their ability to cause UTI. In order to identify potential genetic traits or gene regulatory features that distinguish pathogenic from non-pathogenic E. faecalis with respect to UTI, we have performed comparative genomic analysis, and investigated growth capacity and transcriptome profiling in human urine in vitro. Six strains of different origins were cultivated and all grew readily in human urine. The three strains chosen for transcriptional analysis showed an overall similar response with respect to energy and nitrogen metabolism, stress mechanism, cell envelope modifications, and trace metal acquisition. Our results suggest that citrate and aspartate are significant for growth of E. faecalis in human urine, and manganese appear to be a limiting factor. The majority of virulence factors were either not differentially regulated or down-regulated. Notably, a significant up-regulation of genes involved in biofilm formation was observed. Strains from different origins have similar capacity to grow in human urine. The overall similar transcriptional responses between the two pathogenic and the probiotic strain suggest that the pathogenic potential of a certain E. faecalis strain may to a great extent be determined by presence of fitness and virulence factors, rather than the level of expression of such traits

Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Ruiz JC, D'Afonseca V, Silva A, et al. Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains. PLoS ONE. 2011;6(4): e18551.Background: Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829

Comprehensive genomic analysis reveals virulence factors and antibiotic resistance genes in Pantoea agglomerans KM1, a potential opportunistic pathogen

Pantoea agglomerans is a Gram-negative facultative anaerobic bacillus causing a wide range of opportunistic infections in humans including septicemia, pneumonia, septic arthritis, wound infections and meningitis. To date, the determinants of virulence, antibiotic resistance, metabolic features conferring survival and host-associated pathogenic potential of this bacterium remain largely underexplored. In this study, we sequenced and assembled the whole-genome of P. agglomerans KM1 isolated from kimchi in South Korea. The genome contained one circular chromosome of 4,039,945 bp, 3 mega plasmids, and 2 prophages. The phage-derived genes encoded integrase, lysozyme and terminase. Six CRISPR loci were identified within the bacterial chromosome. Further in-depth analysis showed that the genome contained 13 antibiotic resistance genes conferring resistance to clinically important antibiotics such as penicillin G, bacitracin, rifampicin, vancomycin, and fosfomycin. Genes involved in adaptations to environmental stress were also identified which included factors providing resistance to osmotic lysis, oxidative stress, as well as heat and cold shock. The genomic analysis of virulence factors led to identification of a type VI secretion system, hemolysin, filamentous hemagglutinin, and genes involved in iron uptake and sequestration. Finally, the data provided here show that, the KM1 isolate exerted strong immunostimulatory properties on RAW 264.7 macrophages in vitro. Stimulated cells produced Nitric Oxide (NO) and pro-inflammatory cytokines TNF-alpha, IL-6 and the anti-inflammatory cytokine IL-10. The upstream signaling for production of TNF-alpha, IL-6, IL-10, and NO depended on TLR4 and TLR1/2. While production of TNF-alpha, IL-6 and NO involved solely activation of the NF-kappa B, IL-10 secretion was largely dependent on NF-kappa B and to a lesser extent on MAPK Kinases. Taken together, the analysis of the whole-genome and immunostimulatory properties provided in-depth characterization of the P. agglomerans KM1 isolate shedding a new light on determinants of virulence that drive its interactions with the environment, other microorganisms and eukaryotic host

Ralstonia syzygii, the Blood Disease Bacterium and Some Asian R. solanacearum Strains Form a Single Genomic Species Despite Divergent Lifestyles

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer

Mobile genetic elements causing plasticity in E. faecium

The paper 3 of this thesis is not available in Munin. Paper 3: Sivertsen, A., Pedersen, T., Janice, J., Hegstad, K.: “The Enterococcus Cassette Chromosome: an SCCmec-like mobilisable element”. (Manuscript).I helseinstitusjoner vancomycin-resistente enterokokker en fryktet bakterie som kan lage alvorlig infeksjonssykdom i pasienter med dårlig immunforsvar, og er vanskelig å behandle. Avhandlingen fokuserer på vancomycin-resistente enterokokker (VRE), og hvordan disse bakteriene gjennom å overføre gener mellom hverandre kan utvikle resistens mot antibiotika. Vi har analysert to utbrudd av VRE i Sverige og i Norge, og har funnet at VRE kan være på vei til å bli et mer vanlig patogen i skandinaviske sykehus. Dagens diagnostiske verktøy er ikke i stand til å fange opp alle typer VRE. Dette har konsekvenser for både diagnostikk, resistensovervåkning og risiko for feilbehandling av svært syke pasienter. Nye gensekvenseringsteknologier kan forbedre denne type diagnostikk ved å kunne se på genotypen i tillegg, noe som er viktig da vancomycinresistensgener kan være tilstede i bakterien uten at bakterien har fenotype for dette. I begge utbruddene var diagnostikken utilfredsstillende, da bakterien kunne bli tolket som følsom. I det ene utbruddet utviklet VRE resistens under behandling på grunn av for eksempel mobile genetiske elementer kalt IS-elementer hoppet rundt i vancomycinresistensgenene og påvirket uttrykket deres. Resultater fra doktorgraden har allerede ført til en endring i rådgiving på diagnostikk av VRE hos svært syke pasienter. Vi har også funnet at mobile genetiske elementer (MGE), DNA-et som flytter seg mellom bakterier, har en stor evne til å rekombinere seg og danne varianter med ulikt geninnhold. Inntil nå har strukturen på disse elementene vært vanskelig å rekonstruere. Dermed har betydningen av MGE’er for bakterienes resistenspotensiale vært vanskelig å vurdere. Man kan observere at disse elementene oppfører seg som Babushka-dukker ved å koble seg på hverandre. Såkalte long-read sekvenseringsmetoder er i stand til å rekonstruere strukturen på slike elementer, og det at de kun har vært kommersielt tilgjengelige de siste 3-4 år gjør at der fortsatt er mye å lære om mobile genetiske elementer og deres innvirkning på resistensutvikingen av bakterier