prototype laboratory strains  (ATCC)

Journal: Applied and Environmental Microbiology

Article Title: Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

doi: 10.1128/AEM.01182-17

Figure Lengend Snippet: Polymicrobial biofilm tests of P. aeruginosa and E. faecalis. (A) Colony observation of cocultured P. aeruginosa and E. faecalis (red arrows). The growth experiment of P. aeruginosa (gray triangle) and E. faecalis (black square), grown individually (B) or together (C), in a shaking incubator at 37°C. The viable count assay was used to determine the CFU per milliliter. (D) The CV biofilm assay of the monospecies and the dual-species biofilms. *, P < 0.001 versus the biofilm levels of PAO1 and PAO1 plus E. faecalis (PAEF) or E. faecalis (EF). (E) Viscosity measurements of the mono- and dual-species biofilms. Viscosity appears in units of centipoise (cP). *, P < 0.0001 versus viscosity of PAO1 and PAEF or EF. (F) SEM images of the mono- and dual-species biofilms of P. aeruginosa and/or E. faecalis. The magnifications are ×5,000. (G) Images of the mono- and dual-species biofilms of P. aeruginosa and/or E. faecalis dripping out of the 96-well plates. Detailed video clips are provided in the movies in the supplemental material.

Article Snippet: PAO1 , Prototype P. aeruginosa laboratory strain , ATCC 10145.

Techniques: Biofilm Production Assay, Viscosity

Journal: Applied and Environmental Microbiology

Article Title: Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

doi: 10.1128/AEM.01182-17

Figure Lengend Snippet: Extracellular DNA detection and DNase I treatment of mono- and dual-species P. aeruginosa and E. faecalis biofilms. The monospecies biofilms of E. faecalis (A), P. aeruginosa (B), and the dual-species biofilm of E. faecalis and P. aeruginosa (C) were stained for intracellular DNA (Syto60) and extracellular DNA (TOTO-1). Wavelengths of 652 nm and 514 nm were used for Syto60 and TOTO-1, respectively. (D) Crystal violet biofilm assay results when two different concentrations of DNase I (10 μg/ml and 100 μg/ml) were used to treat 32-h-old mature biofilms.

Article Snippet: PAO1 , Prototype P. aeruginosa laboratory strain , ATCC 10145.

Techniques: Staining, Biofilm Production Assay

Journal: Applied and Environmental Microbiology

Article Title: Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

doi: 10.1128/AEM.01182-17

Figure Lengend Snippet: Scheme of the dual-species P. aeruginosa and E. faecalis biofilm development. (A) P. aeruginosa and E. faecalis attach to a surface without spatial separation. (B) During the early stages of dual-species biofilm formation, the attached cells proliferate and start producing EPS, Pel (blue straight line), and Psl (purple curved line). (C) P. aeruginosa and E. faecalis start to exhibit distinct spatial distributions. (D) In the maturation stage of the dual-species biofilm, P. aeruginosa forms a structured biofilm on top of the E. faecalis biofilm: Psl is associated with the surface and between P. aeruginosa, while Pel is associated with interspecies interactions between P. aeruginosa and E. faecalis.

Article Snippet: PAO1 , Prototype P. aeruginosa laboratory strain , ATCC 10145.

Techniques:

Journal: Applied and Environmental Microbiology

Article Title: Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

doi: 10.1128/AEM.01182-17

Figure Lengend Snippet: Polymicrobial biofilm tests of P. aeruginosa and E. faecalis. (A) Colony observation of cocultured P. aeruginosa and E. faecalis (red arrows). The growth experiment of P. aeruginosa (gray triangle) and E. faecalis (black square), grown individually (B) or together (C), in a shaking incubator at 37°C. The viable count assay was used to determine the CFU per milliliter. (D) The CV biofilm assay of the monospecies and the dual-species biofilms. *, P < 0.001 versus the biofilm levels of PAO1 and PAO1 plus E. faecalis (PAEF) or E. faecalis (EF). (E) Viscosity measurements of the mono- and dual-species biofilms. Viscosity appears in units of centipoise (cP). *, P < 0.0001 versus viscosity of PAO1 and PAEF or EF. (F) SEM images of the mono- and dual-species biofilms of P. aeruginosa and/or E. faecalis. The magnifications are ×5,000. (G) Images of the mono- and dual-species biofilms of P. aeruginosa and/or E. faecalis dripping out of the 96-well plates. Detailed video clips are provided in the movies in the supplemental material.

Article Snippet: E. faecalis was grown on brain heart infusion broth (BHIB) medium, and E. faecalis pMV158GFP ( EF /green) was grown on BHIB medium with tetracycline (1 μg/ml) and erythromycin (1 μg/ml) to maintain the plasmids. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Bacterial strains PAO1 Prototype P. aeruginosa laboratory strain ATCC 10145 Δ alg mutant PAO1 with in-frame deletion of alg operon (PA3540–PA3548) This study Δ pelA mutant PAO1 with in-frame deletion of PA3064 gene This study Δ psl mutant PAO1 with in-frame deletion of psl operon (PA2231–PA2242) This study Δ pelA Δ psl mutant PAO1 with in-frame deletion of PA3064 gene and psl operon This study PAO1/red PAO1 harboring pME-dsRED This study Δ alg/ red Δ alg mutant harboring pME-dsRED This study Δ pelA/ red Δ pelA mutant harboring pME-dsRED This study Δ psl/ red Δ psl mutant harboring pME-dsRED This study Δ pelA Δ psl/ red Δ pelA Δ psl mutant harboring pME-dsRED This study E. faecalis 12448 E. faecalis type strain (ATCC 19433) Korean Culture Collection of Microorganisms (KCCM) EF/ green E. faecalis OG1RF strain, harboring pMV158GFP and pAMβ1 41 Plasmids pME-dsRED Transcriptional fusion of GFP promoter with a gene encoding DsRed, Gm r This study pMV158GFP pMV158, harbors the GFP gene under the control of the P M -inducible promoter, Tet r 41 pAMβ1 Auxiliary plasmid for pMV158GFP transport, Em r 41 Open in a separate window a GFP, green fluorescent protein; Gm r , gentamicin resistance; Tet r , tetracycline resistance; Em r , erythromycin resistance.

Techniques: Biofilm Production Assay, Viscosity

Journal: Applied and Environmental Microbiology

Article Title: Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

doi: 10.1128/AEM.01182-17

Figure Lengend Snippet: CLSM image analysis of mono- and dual-species PAO1/red and EF/green biofilms. CLSM images and their relative fluorescent intensity measures along the heights of biofilms of PAO1/red (A), PAO1/red and EF/green (B), and EF/green (C). White dotted lines indicate the locations of sagittal sections for height representation of the biofilms. The relative fluorescence intensity (RFI) of 13 was considered the threshold for biofilm detection. The biofilms were incubated at 37°C for 48 h. The relative fluorescence intensities were measured using the ImageJ program.

Article Snippet: E. faecalis was grown on brain heart infusion broth (BHIB) medium, and E. faecalis pMV158GFP ( EF /green) was grown on BHIB medium with tetracycline (1 μg/ml) and erythromycin (1 μg/ml) to maintain the plasmids. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Bacterial strains PAO1 Prototype P. aeruginosa laboratory strain ATCC 10145 Δ alg mutant PAO1 with in-frame deletion of alg operon (PA3540–PA3548) This study Δ pelA mutant PAO1 with in-frame deletion of PA3064 gene This study Δ psl mutant PAO1 with in-frame deletion of psl operon (PA2231–PA2242) This study Δ pelA Δ psl mutant PAO1 with in-frame deletion of PA3064 gene and psl operon This study PAO1/red PAO1 harboring pME-dsRED This study Δ alg/ red Δ alg mutant harboring pME-dsRED This study Δ pelA/ red Δ pelA mutant harboring pME-dsRED This study Δ psl/ red Δ psl mutant harboring pME-dsRED This study Δ pelA Δ psl/ red Δ pelA Δ psl mutant harboring pME-dsRED This study E. faecalis 12448 E. faecalis type strain (ATCC 19433) Korean Culture Collection of Microorganisms (KCCM) EF/ green E. faecalis OG1RF strain, harboring pMV158GFP and pAMβ1 41 Plasmids pME-dsRED Transcriptional fusion of GFP promoter with a gene encoding DsRed, Gm r This study pMV158GFP pMV158, harbors the GFP gene under the control of the P M -inducible promoter, Tet r 41 pAMβ1 Auxiliary plasmid for pMV158GFP transport, Em r 41 Open in a separate window a GFP, green fluorescent protein; Gm r , gentamicin resistance; Tet r , tetracycline resistance; Em r , erythromycin resistance.

Techniques: Fluorescence, Incubation

Journal: Applied and Environmental Microbiology

Article Title: Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

doi: 10.1128/AEM.01182-17

Figure Lengend Snippet: Alginate is not responsible for the elevated biofilm matrix thickness in the dual-species biofilms. The matrix thickness of biofilms was tested by flipping the biofilms upside down. Biofilms of PAO1 Δalg mutant of its own (A) and with E. faecalis (B) were formed and tested. (C) Quantification of alginate production in PAO1 monospecies and PAO1 plus E. faecalis dual-species biofilms. *, P < 0.05 versus the alginate levels of PAO1 and PAO1 plus E. faecalis (PAEF).

Article Snippet: E. faecalis was grown on brain heart infusion broth (BHIB) medium, and E. faecalis pMV158GFP ( EF /green) was grown on BHIB medium with tetracycline (1 μg/ml) and erythromycin (1 μg/ml) to maintain the plasmids. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Bacterial strains PAO1 Prototype P. aeruginosa laboratory strain ATCC 10145 Δ alg mutant PAO1 with in-frame deletion of alg operon (PA3540–PA3548) This study Δ pelA mutant PAO1 with in-frame deletion of PA3064 gene This study Δ psl mutant PAO1 with in-frame deletion of psl operon (PA2231–PA2242) This study Δ pelA Δ psl mutant PAO1 with in-frame deletion of PA3064 gene and psl operon This study PAO1/red PAO1 harboring pME-dsRED This study Δ alg/ red Δ alg mutant harboring pME-dsRED This study Δ pelA/ red Δ pelA mutant harboring pME-dsRED This study Δ psl/ red Δ psl mutant harboring pME-dsRED This study Δ pelA Δ psl/ red Δ pelA Δ psl mutant harboring pME-dsRED This study E. faecalis 12448 E. faecalis type strain (ATCC 19433) Korean Culture Collection of Microorganisms (KCCM) EF/ green E. faecalis OG1RF strain, harboring pMV158GFP and pAMβ1 41 Plasmids pME-dsRED Transcriptional fusion of GFP promoter with a gene encoding DsRed, Gm r This study pMV158GFP pMV158, harbors the GFP gene under the control of the P M -inducible promoter, Tet r 41 pAMβ1 Auxiliary plasmid for pMV158GFP transport, Em r 41 Open in a separate window a GFP, green fluorescent protein; Gm r , gentamicin resistance; Tet r , tetracycline resistance; Em r , erythromycin resistance.

Techniques: Mutagenesis

Journal: Applied and Environmental Microbiology

Article Title: Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

doi: 10.1128/AEM.01182-17

Figure Lengend Snippet: Role of Psl and Pel in the matrix thickening of the dual-species biofilms. The matrix thickness of biofilms was tested by flipping the biofilms upside down. PAO1 ΔpelA (A and D), Δpsl (B and E), and ΔpelA Δpsl mutants (C and F) were grown as monospecies biofilms (A to C) or together with EF (D to F) as dual-species biofilms.

Article Snippet: E. faecalis was grown on brain heart infusion broth (BHIB) medium, and E. faecalis pMV158GFP ( EF /green) was grown on BHIB medium with tetracycline (1 μg/ml) and erythromycin (1 μg/ml) to maintain the plasmids. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Bacterial strains PAO1 Prototype P. aeruginosa laboratory strain ATCC 10145 Δ alg mutant PAO1 with in-frame deletion of alg operon (PA3540–PA3548) This study Δ pelA mutant PAO1 with in-frame deletion of PA3064 gene This study Δ psl mutant PAO1 with in-frame deletion of psl operon (PA2231–PA2242) This study Δ pelA Δ psl mutant PAO1 with in-frame deletion of PA3064 gene and psl operon This study PAO1/red PAO1 harboring pME-dsRED This study Δ alg/ red Δ alg mutant harboring pME-dsRED This study Δ pelA/ red Δ pelA mutant harboring pME-dsRED This study Δ psl/ red Δ psl mutant harboring pME-dsRED This study Δ pelA Δ psl/ red Δ pelA Δ psl mutant harboring pME-dsRED This study E. faecalis 12448 E. faecalis type strain (ATCC 19433) Korean Culture Collection of Microorganisms (KCCM) EF/ green E. faecalis OG1RF strain, harboring pMV158GFP and pAMβ1 41 Plasmids pME-dsRED Transcriptional fusion of GFP promoter with a gene encoding DsRed, Gm r This study pMV158GFP pMV158, harbors the GFP gene under the control of the P M -inducible promoter, Tet r 41 pAMβ1 Auxiliary plasmid for pMV158GFP transport, Em r 41 Open in a separate window a GFP, green fluorescent protein; Gm r , gentamicin resistance; Tet r , tetracycline resistance; Em r , erythromycin resistance.

Techniques:

Journal: Applied and Environmental Microbiology

Article Title: Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

doi: 10.1128/AEM.01182-17

Figure Lengend Snippet: Quantitative real-time PCR analysis of pslA and pelB expression. The qRT-PCR of PAO1 monospecies (the reference samples; PA) and PAO1 plus E. faecalis dual-species (the experimental samples; PAEF) biofilms for pslA (A and B) and pelB (C and D) genes. Gene expression was measured at the early (A and C) and mature (B and D) stages of the biofilm developments. RQ represents the relative quantitation value for genetic expression. **, P < 0.001 versus gene expression of PAO1 and PAO1 plus E. faecalis biofilms. ***, P < 0.0001 versus gene expression of PAO1 and PAO1 plus E. faecalis biofilms.

Article Snippet: E. faecalis was grown on brain heart infusion broth (BHIB) medium, and E. faecalis pMV158GFP ( EF /green) was grown on BHIB medium with tetracycline (1 μg/ml) and erythromycin (1 μg/ml) to maintain the plasmids. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Bacterial strains PAO1 Prototype P. aeruginosa laboratory strain ATCC 10145 Δ alg mutant PAO1 with in-frame deletion of alg operon (PA3540–PA3548) This study Δ pelA mutant PAO1 with in-frame deletion of PA3064 gene This study Δ psl mutant PAO1 with in-frame deletion of psl operon (PA2231–PA2242) This study Δ pelA Δ psl mutant PAO1 with in-frame deletion of PA3064 gene and psl operon This study PAO1/red PAO1 harboring pME-dsRED This study Δ alg/ red Δ alg mutant harboring pME-dsRED This study Δ pelA/ red Δ pelA mutant harboring pME-dsRED This study Δ psl/ red Δ psl mutant harboring pME-dsRED This study Δ pelA Δ psl/ red Δ pelA Δ psl mutant harboring pME-dsRED This study E. faecalis 12448 E. faecalis type strain (ATCC 19433) Korean Culture Collection of Microorganisms (KCCM) EF/ green E. faecalis OG1RF strain, harboring pMV158GFP and pAMβ1 41 Plasmids pME-dsRED Transcriptional fusion of GFP promoter with a gene encoding DsRed, Gm r This study pMV158GFP pMV158, harbors the GFP gene under the control of the P M -inducible promoter, Tet r 41 pAMβ1 Auxiliary plasmid for pMV158GFP transport, Em r 41 Open in a separate window a GFP, green fluorescent protein; Gm r , gentamicin resistance; Tet r , tetracycline resistance; Em r , erythromycin resistance.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Gene Expression, Quantitation Assay

Journal: Applied and Environmental Microbiology

Article Title: Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

doi: 10.1128/AEM.01182-17

Figure Lengend Snippet: Bacterial strains and plasmids used in this investigation

Article Snippet: E. faecalis was grown on brain heart infusion broth (BHIB) medium, and E. faecalis pMV158GFP ( EF /green) was grown on BHIB medium with tetracycline (1 μg/ml) and erythromycin (1 μg/ml) to maintain the plasmids. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Bacterial strains PAO1 Prototype P. aeruginosa laboratory strain ATCC 10145 Δ alg mutant PAO1 with in-frame deletion of alg operon (PA3540–PA3548) This study Δ pelA mutant PAO1 with in-frame deletion of PA3064 gene This study Δ psl mutant PAO1 with in-frame deletion of psl operon (PA2231–PA2242) This study Δ pelA Δ psl mutant PAO1 with in-frame deletion of PA3064 gene and psl operon This study PAO1/red PAO1 harboring pME-dsRED This study Δ alg/ red Δ alg mutant harboring pME-dsRED This study Δ pelA/ red Δ pelA mutant harboring pME-dsRED This study Δ psl/ red Δ psl mutant harboring pME-dsRED This study Δ pelA Δ psl/ red Δ pelA Δ psl mutant harboring pME-dsRED This study E. faecalis 12448 E. faecalis type strain (ATCC 19433) Korean Culture Collection of Microorganisms (KCCM) EF/ green E. faecalis OG1RF strain, harboring pMV158GFP and pAMβ1 41 Plasmids pME-dsRED Transcriptional fusion of GFP promoter with a gene encoding DsRed, Gm r This study pMV158GFP pMV158, harbors the GFP gene under the control of the P M -inducible promoter, Tet r 41 pAMβ1 Auxiliary plasmid for pMV158GFP transport, Em r 41 Open in a separate window a GFP, green fluorescent protein; Gm r , gentamicin resistance; Tet r , tetracycline resistance; Em r , erythromycin resistance.

Techniques: Plasmid Preparation, Mutagenesis, Control