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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

SmaI macrorestriction patterns in PFGE of 42E. faecalisST40 isolates. The phylogenetic analysis was generated with BioNumerics 6.0. The scale bar represents the level of similiarity in % using default settings (tolerance 1.0; optimization 0.5). The dotted vertical line at 82% similarity delineates the breakpoint for defining related fragment patterns/strains according to an international agreement. Certain sub-clusters indicated a comparably high level of clonal relatedness. Sequenced isolates are marked with a black square. Legend: H, human; A, animal; F, food; 1 reference strain; B, blood culture; CSF, cerebrospinal fluid; C, colonizer; E, endocarditis; F, food; M, bovine mastitis; PF, peritoneal fluid; U, urine; W, wound. Origin: D, Germany; DK, Denmark; PL, Poland; IS, Island; GR, Greece; ESP, Spain.
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Fig1: SmaI macrorestriction patterns in PFGE of 42E. faecalisST40 isolates. The phylogenetic analysis was generated with BioNumerics 6.0. The scale bar represents the level of similiarity in % using default settings (tolerance 1.0; optimization 0.5). The dotted vertical line at 82% similarity delineates the breakpoint for defining related fragment patterns/strains according to an international agreement. Certain sub-clusters indicated a comparably high level of clonal relatedness. Sequenced isolates are marked with a black square. Legend: H, human; A, animal; F, food; 1 reference strain; B, blood culture; CSF, cerebrospinal fluid; C, colonizer; E, endocarditis; F, food; M, bovine mastitis; PF, peritoneal fluid; U, urine; W, wound. Origin: D, Germany; DK, Denmark; PL, Poland; IS, Island; GR, Greece; ESP, Spain.

Mentions: The ST40 strains were compared for specific phenotypic and genotypic characteristics in order to select representative isolates for genome sequencing. Strains were typed by SmaI macrorestriction in PFGE and sub-clusters of closely related strains were identified which were grouped independently of their geographical and temporal origin or clinical/non-clinical context (Figure 1). Distribution of phenotypic antibiotic susceptibilities was completed by determination of the corresponding genes by PCR (Table 3; Additional file 1: Table S1). Results were mainly congruent. Two discrepancies were detected; one isolate was aadE-positive but not streptomycin-high-level resistant and another isolate was aac6’-aph2”-negative but gentamicin-resistant. Presence and expression of putative virulence-associated genes encoded within the E. faecalis PAI and/or on the chromosome were investigated by PCR and partly confirmed by phenotypic in vitro assays (Table 4). The PAI-associated aggregation substance asc-10 gene was found in 16.7% of the isolates. Prevalence of the cytolysin (cyl) operon in 33.3% of the strains was associated with the evidence of β-haemolysis in vitro. The enterococcal surface protein gene esp was detected in 78.6% of the ST40 strains. All isolates harboured the gelE (gelatinase) and fsr (major accessory gene regulator) genes and showed in vitro gelatinase expression (Table 4; Additional file 1: Table S1).Figure 1


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)

SmaI macrorestriction patterns in PFGE of 42E. faecalisST40 isolates. The phylogenetic analysis was generated with BioNumerics 6.0. The scale bar represents the level of similiarity in % using default settings (tolerance 1.0; optimization 0.5). The dotted vertical line at 82% similarity delineates the breakpoint for defining related fragment patterns/strains according to an international agreement. Certain sub-clusters indicated a comparably high level of clonal relatedness. Sequenced isolates are marked with a black square. Legend: H, human; A, animal; F, food; 1 reference strain; B, blood culture; CSF, cerebrospinal fluid; C, colonizer; E, endocarditis; F, food; M, bovine mastitis; PF, peritoneal fluid; U, urine; W, wound. Origin: D, Germany; DK, Denmark; PL, Poland; IS, Island; GR, Greece; ESP, Spain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: SmaI macrorestriction patterns in PFGE of 42E. faecalisST40 isolates. The phylogenetic analysis was generated with BioNumerics 6.0. The scale bar represents the level of similiarity in % using default settings (tolerance 1.0; optimization 0.5). The dotted vertical line at 82% similarity delineates the breakpoint for defining related fragment patterns/strains according to an international agreement. Certain sub-clusters indicated a comparably high level of clonal relatedness. Sequenced isolates are marked with a black square. Legend: H, human; A, animal; F, food; 1 reference strain; B, blood culture; CSF, cerebrospinal fluid; C, colonizer; E, endocarditis; F, food; M, bovine mastitis; PF, peritoneal fluid; U, urine; W, wound. Origin: D, Germany; DK, Denmark; PL, Poland; IS, Island; GR, Greece; ESP, Spain.
Mentions: The ST40 strains were compared for specific phenotypic and genotypic characteristics in order to select representative isolates for genome sequencing. Strains were typed by SmaI macrorestriction in PFGE and sub-clusters of closely related strains were identified which were grouped independently of their geographical and temporal origin or clinical/non-clinical context (Figure 1). Distribution of phenotypic antibiotic susceptibilities was completed by determination of the corresponding genes by PCR (Table 3; Additional file 1: Table S1). Results were mainly congruent. Two discrepancies were detected; one isolate was aadE-positive but not streptomycin-high-level resistant and another isolate was aac6’-aph2”-negative but gentamicin-resistant. Presence and expression of putative virulence-associated genes encoded within the E. faecalis PAI and/or on the chromosome were investigated by PCR and partly confirmed by phenotypic in vitro assays (Table 4). The PAI-associated aggregation substance asc-10 gene was found in 16.7% of the isolates. Prevalence of the cytolysin (cyl) operon in 33.3% of the strains was associated with the evidence of β-haemolysis in vitro. The enterococcal surface protein gene esp was detected in 78.6% of the ST40 strains. All isolates harboured the gelE (gelatinase) and fsr (major accessory gene regulator) genes and showed in vitro gelatinase expression (Table 4; Additional file 1: Table S1).Figure 1

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