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Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

Zischka M, Künne CT, Blom J, Wobser D, Sakιnç T, Schmidt-Hohagen K, Dabrowski PW, Nitsche A, Hübner J, Hain T, Chakraborty T, Linke B, Goesmann A, Voget S, Daniel R, Schomburg D, Hauck R, Hafez HM, Tielen P, Jahn D, Solheim M, Sadowy E, Larsen J, Jensen LB, Ruiz-Garbajosa P, Quiñones Pérez D, Mikalsen T, Bender J, Steglich M, Nübel U, Witte W, Werner G - BMC Genomics (2015)

Bottom Line: Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source.D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

View Article: PubMed Central - PubMed

Affiliation: Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Burgstr. 37, D-38855, Wernigerode, Germany. melanie.zischka@googlemail.com.

ABSTRACT

Background: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type.

Results: We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).

Conclusion: Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

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Related in: MedlinePlus

Phylogenetic relationship among selectedE. faecalisstrains based on whole genome alignments. The alignment was calculated with Mugsy (http://mugsy.sourceforge.net/ [last access 16.07.2014] [57]) and only aligned regions present in all analyzed strains were extracted. These regions were concatenated and positions with gaps removed. The resulting core alignment was used to infer a Maximum Likelihood tree with RAxML. The GTRGAMMA model for nucleotide substitution and rate heterogeneity was utilized, bootstrap support values of 1000 replicates are shown at the nodes. Names of the ST40 isolates and their origin are indicated at the end of the branches. The highly related ST40 isolates were further zoomed in exemplified by the dotted line and the different scale bar. Metadata are given as follows: Strain no., year of isolation, origin, country: AC, animal colonizer; AI, animal infection; HC, human colonizer; HI, human infection; CU, Cuba; D, Germany; DK, Denmark; ES, Spain; IS, Island; PL, Poland; USA.
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Fig4: Phylogenetic relationship among selectedE. faecalisstrains based on whole genome alignments. The alignment was calculated with Mugsy (http://mugsy.sourceforge.net/ [last access 16.07.2014] [57]) and only aligned regions present in all analyzed strains were extracted. These regions were concatenated and positions with gaps removed. The resulting core alignment was used to infer a Maximum Likelihood tree with RAxML. The GTRGAMMA model for nucleotide substitution and rate heterogeneity was utilized, bootstrap support values of 1000 replicates are shown at the nodes. Names of the ST40 isolates and their origin are indicated at the end of the branches. The highly related ST40 isolates were further zoomed in exemplified by the dotted line and the different scale bar. Metadata are given as follows: Strain no., year of isolation, origin, country: AC, animal colonizer; AI, animal infection; HC, human colonizer; HI, human infection; CU, Cuba; D, Germany; DK, Denmark; ES, Spain; IS, Island; PL, Poland; USA.

Mentions: A phylogenetic tree resulting from an alignment of concatenated sequences, present in all analysed strains and after elimination of existing gaps, is shown in Figure 4. It revealed a very high level of genomic similarity of unrelated ST40 strains, despite their diverse origins and the time interval from <1960 to 2009. Of note, strains of a similar origin were not arranged in the same clusters. When we focused on the ST40 isolates, the core-genome based phylogenetic tree also showed an exceptional position of D32 in relation to the other sequenced ST40 isolates and furthermore its close relationship with the other Danish porcine isolate UW7742. As expected, the completely closed and publicly available E. faecalis genomes branch separately, supporting their assignment to different sequence types and clonal complexes based on MLST. In order to confirm the relationship between the 15 E. faecalis ST40 isolates with respect to their core genome, additional phylogenetic analyses were performed by mapping Solexa single reads of 14 isolates against the E. faecalis D32 reference sequence using a mapping pipeline based on bwa. As enterococci frequently undergo chromosomal rearrangements, we first excluded SNPs which were owed to recent recombination events. This yielded a total of 1481 variable positions (SNPs), which in turn served as the basis for tree reconstruction by the PhyML algorithm. The generated tree in Additional file 5: Figure S3 revealed a highly similar structure to the previous one (Figure 4) despite the different input data supporting the reliability of both approaches. Both trees revealed exactly identical subclusters of related strains. In Additional file 5: Figure S3 the separate clustering of the two pig commensal strains D1 and D32 from Denmark is highly visible and supported by a high bootstrap value (please recall that this separation is only based on the core genome and independent of the presence or absence of MGE). The separation of the two pig commensal isolates D1 and D32 based on core genome data disproves the hypothesis of highly related pig and human endocarditis isolates as derived from PFGE analysis (Figure 1 and [64]).Figure 4


Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

Zischka M, Künne CT, Blom J, Wobser D, Sakιnç T, Schmidt-Hohagen K, Dabrowski PW, Nitsche A, Hübner J, Hain T, Chakraborty T, Linke B, Goesmann A, Voget S, Daniel R, Schomburg D, Hauck R, Hafez HM, Tielen P, Jahn D, Solheim M, Sadowy E, Larsen J, Jensen LB, Ruiz-Garbajosa P, Quiñones Pérez D, Mikalsen T, Bender J, Steglich M, Nübel U, Witte W, Werner G - BMC Genomics (2015)

Phylogenetic relationship among selectedE. faecalisstrains based on whole genome alignments. The alignment was calculated with Mugsy (http://mugsy.sourceforge.net/ [last access 16.07.2014] [57]) and only aligned regions present in all analyzed strains were extracted. These regions were concatenated and positions with gaps removed. The resulting core alignment was used to infer a Maximum Likelihood tree with RAxML. The GTRGAMMA model for nucleotide substitution and rate heterogeneity was utilized, bootstrap support values of 1000 replicates are shown at the nodes. Names of the ST40 isolates and their origin are indicated at the end of the branches. The highly related ST40 isolates were further zoomed in exemplified by the dotted line and the different scale bar. Metadata are given as follows: Strain no., year of isolation, origin, country: AC, animal colonizer; AI, animal infection; HC, human colonizer; HI, human infection; CU, Cuba; D, Germany; DK, Denmark; ES, Spain; IS, Island; PL, Poland; USA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374294&req=5

Fig4: Phylogenetic relationship among selectedE. faecalisstrains based on whole genome alignments. The alignment was calculated with Mugsy (http://mugsy.sourceforge.net/ [last access 16.07.2014] [57]) and only aligned regions present in all analyzed strains were extracted. These regions were concatenated and positions with gaps removed. The resulting core alignment was used to infer a Maximum Likelihood tree with RAxML. The GTRGAMMA model for nucleotide substitution and rate heterogeneity was utilized, bootstrap support values of 1000 replicates are shown at the nodes. Names of the ST40 isolates and their origin are indicated at the end of the branches. The highly related ST40 isolates were further zoomed in exemplified by the dotted line and the different scale bar. Metadata are given as follows: Strain no., year of isolation, origin, country: AC, animal colonizer; AI, animal infection; HC, human colonizer; HI, human infection; CU, Cuba; D, Germany; DK, Denmark; ES, Spain; IS, Island; PL, Poland; USA.
Mentions: A phylogenetic tree resulting from an alignment of concatenated sequences, present in all analysed strains and after elimination of existing gaps, is shown in Figure 4. It revealed a very high level of genomic similarity of unrelated ST40 strains, despite their diverse origins and the time interval from <1960 to 2009. Of note, strains of a similar origin were not arranged in the same clusters. When we focused on the ST40 isolates, the core-genome based phylogenetic tree also showed an exceptional position of D32 in relation to the other sequenced ST40 isolates and furthermore its close relationship with the other Danish porcine isolate UW7742. As expected, the completely closed and publicly available E. faecalis genomes branch separately, supporting their assignment to different sequence types and clonal complexes based on MLST. In order to confirm the relationship between the 15 E. faecalis ST40 isolates with respect to their core genome, additional phylogenetic analyses were performed by mapping Solexa single reads of 14 isolates against the E. faecalis D32 reference sequence using a mapping pipeline based on bwa. As enterococci frequently undergo chromosomal rearrangements, we first excluded SNPs which were owed to recent recombination events. This yielded a total of 1481 variable positions (SNPs), which in turn served as the basis for tree reconstruction by the PhyML algorithm. The generated tree in Additional file 5: Figure S3 revealed a highly similar structure to the previous one (Figure 4) despite the different input data supporting the reliability of both approaches. Both trees revealed exactly identical subclusters of related strains. In Additional file 5: Figure S3 the separate clustering of the two pig commensal strains D1 and D32 from Denmark is highly visible and supported by a high bootstrap value (please recall that this separation is only based on the core genome and independent of the presence or absence of MGE). The separation of the two pig commensal isolates D1 and D32 based on core genome data disproves the hypothesis of highly related pig and human endocarditis isolates as derived from PFGE analysis (Figure 1 and [64]).Figure 4

Bottom Line: Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source.D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

View Article: PubMed Central - PubMed

Affiliation: Division of Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, Burgstr. 37, D-38855, Wernigerode, Germany. melanie.zischka@googlemail.com.

ABSTRACT

Background: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type.

Results: We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).

Conclusion: Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Show MeSH
Related in: MedlinePlus