Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections methods
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Isolation of a homogeneously oxacillin resistant derivative of S. aureus 8325-4
(A) Western blot analysis of PBP2a levels in 8325-4, 8325-4 p mecA HeR and 8325-4 p mecA HoR. (B) Biofilm phenotypes of 8325-4, 8325-4 p mecA HeR, 8325-4 p mecA HoR and 8325-4 p mecA HoR (cured) grown for 24 h at 37°C in BHI, BHI NaCl and BHI glucose in hydrophilic 96-well polystyrene plates. (C-F) Dispersal of 8325-4 (C), 8325-4 p mecA HeR (D), 8325-4 p mecA HoR (E), 8325-4 p mecA HoR (cured) (F) biofilms grown for 24 h in BHI, BHI NaCl and BHI glucose by sodium metaperiodate and proteinase K. (G) Biofilm formation by 8325-4, 8325-4 p mecA HeR, 8325-4 p mecA HoR and 8325-4 p mecA HoR (cured) grown for 24 h in BHI glucose in the absence or presence of 500 µg/ml PAS, 0.25 mg/ml DNase I and heat-inactivated (HI) 0.25 mg/ml DNase I. Biofilm assays were repeated at least three times and standard deviations are indicated. * indicates a statistically significant difference p...
Repression of extracellular protease production and Agr activity in 8325-4 p mecA HoR
(A) Protease activity in culture supernatants of 8325-4, 8325-4 p mecA HeR, 8325-4 p mecA HoR and 8325-4 p mecA HoR (cured) grown for 8 h at 37°C in BHI glucose. (B) Comparison of relative RNAIII transcription by real time RT-PCR in 8325-4, 8325-4 p mecA HeR, 8325-4 p mecA HoR and 8325-4 p mecA HoR (cured). Total RNA was extracted from cultures grown at 37°C to A 600 = 1 (early exponential phase) and for 8 hours (late exponential/early stationary phase; A 600 ≈8) in BHI glucose. Experiments were repeated at least three times and standard deviations are indicated. Statistical significance (p value) is indicated.
Homogeneous methicillin resistance attenuates virulence in 8325-4
Implanted catheter segments were injected with 1 × 10 7 8325-4 and 8325-4 p mecA HoR and animals ( n = 8 mice per group) were sacrificed after 18 hours. Colony forming units (CFU) per catheter (A), per g peri-catheter tissue (B), per g liver (C), per ml blood (D), per g spleen (E) and per g kidney (F) recovered from animals infected with 8325-4 and 8325-4 p mecA HoR. Statistical significance (p values) is indicated.
Materials and Methods
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mouse protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Nebraska Medical Centre. All surgery was performed under anesthesia and all efforts were made to minimize suffering.
Genetic techniques
Genomic and plasmid DNA was prepared using Wizard Genomic DNA and plasmid purification kits (Promega). Prior to DNA extraction cells were pre-treated with 5-10 µl of a 1 mg/ml concentration of lysostaphin (Ambi products, New York) in 100 µl 50 mM EDTA to facilitate lysis. Restriction and DNA modifying enzymes (Roche, UK and New England Biolabs, MA) were used according to the manufacturer's instructions. The serine residue at amino acid 403 in the active site of PBP2a was mutated to alanine using Phusion polymerase (NEB) and the primers mecAS403A_For and mecAS403A_Rev ( ). Plasmid p mecA was used as the template. Successful mutagenesis of the DNA sequence encoding the S403 residue resulted in the introduction of a new Dra III restriction enzyme site and candidate plasmids harbouring the mutation were digested with this enzyme before being confirmed by sequencing. The mu...
Biofilm and protease assays
Semi-quantitative measurements of biofilm formation were determined using Nunclon tissue culture treated (Δ surface) 96-well polystyrene plates (Nunc, Denmark), based on the methods of Christensen et al. and Ziebuhr et al. with the following modification. Bacteria were grown in individual wells of 96-well plates at 37°C in BHI medium or BHI supplemented with 4% NaCl or 1% glucose. The serine protease inhibitor dichloroisocoumarin was added to BHI glucose media at concentrations of 0.004-0.5 mM, where indicated. After 24 h of growth, the plates were washed vigorously three times with distilled H 2 O to remove unattached bacteria and dried for 1 hour at 60°C, as recommended by Gelosia et al. as described previously,. The absorbance of the adhered, stained biofilms was measured at A 492 using a microtitre plate reader. Each strain was tested at least three times an...
Biofilm and protease assays
Bacteria were grown on Congo red agar (CRA) plates, which are composed of BHI agar supplemented with 5% sucrose (Sigma) and 0.8 mg of Congo red/ml (Sigma) to distinguish between PNAG-producing (black, dry colony morphology) and non-PNAG-producing (red, smooth colony morphology) phenotypes as described previously,.
Biofilm and protease assays
To analyze biofilm formation under flow conditions, we utilized the BioFlux 1000 microfluidic system (Fluxion Biosciences Inc., South San Francisco, CA) which allows automated image acquisition within specialized multi-well plates. To grow biofilms, the microfluidic channels were primed with 50% BHI supplemented with 4% NaCl or 1% glucose at 10.0 dyn/cm 2. Channels were seeded at 2 dyn/cm 2 with 10 7 CFU from overnight cultures of 8325-4 p mecA HeR and 8325-4 p mecA HoR. The plate was then incubated at 37°C for 1 hour to allow cells to adhere. Excess inoculums were removed and 2 ml of 50% BHI supplemented with 4% NaCl or 1% glucose was added to the input wells. Biofilms were grown at 37°C with a flow of fresh media at a constant shear of 0.7 dyn/cm 2. Images were taken every 5 minutes for 18 hours at 200 × magnification under brightfield.
Measurement outputs
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Protein expression by western blot
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Evidence quotes (8)
(A) Western blot analysis of PBP2a levels in 8325-4, 8325-4 p mecA HeR and 8325-4 p mecA HoR. (B) Biofilm phenotypes of 8325-4, 8325-4 p mecA HeR, 8325-4 p mecA HoR and 8325-4 p mecA HoR (cured) grown for 24 h at 37°C in BHI, BHI NaCl and BHI glucose in hydrophilic 96-well polystyrene plates. (C-F) Dispersal of 8325-4 (C), 8325-4 p mecA HeR (D), 8325-4 p mecA HoR (E), 8325-4 p mecA HoR (cured) (F) biofilms grown for 24 h in BHI, BHI NaCl and BHI glucose by sodium metaperiodate and proteinase K. (G) Biofilm formation by 8325-4, 8325-4 p mecA HeR, 8325-4 p mecA HoR and 8325-4 p mecA HoR (cured) grown for 24 h in BHI glucose in the absence or presence of 500 µg/ml PAS, 0.25 mg/ml DNase I and heat-inactivated (HI) 0.25 mg/ml DNase I. Biofilm assays were repeated at least three times and standard deviations are indicated. * indicates a statistically significant difference p<0.01.
(A) Protease activity in culture supernatants of 8325-4, 8325-4 p mecA HeR, 8325-4 p mecA HoR and 8325-4 p mecA HoR (cured) grown for 8 h at 37°C in BHI glucose. (B) Comparison of relative RNAIII transcription by real time RT-PCR in 8325-4, 8325-4 p mecA HeR, 8325-4 p mecA HoR and 8325-4 p mecA HoR (cured). Total RNA was extracted from cultures grown at 37°C to A 600 = 1 (early exponential phase) and for 8 hours (late exponential/early stationary phase; A 600 ≈8) in BHI glucose. Experiments were repeated at least three times and standard deviations are indicated. Statistical significance (p value) is indicated.
Implanted catheter segments were injected with 1 × 10 7 8325-4 and 8325-4 p mecA HoR and animals ( n = 8 mice per group) were sacrificed after 18 hours. Colony forming units (CFU) per catheter (A), per g peri-catheter tissue (B), per g liver (C), per ml blood (D), per g spleen (E) and per g kidney (F) recovered from animals infected with 8325-4 and 8325-4 p mecA HoR. Statistical significance (p values) is indicated.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mouse protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Nebraska Medical Centre. All surgery was performed under anesthesia and all efforts were made to minimize suffering.
Genomic and plasmid DNA was prepared using Wizard Genomic DNA and plasmid purification kits (Promega). Prior to DNA extraction cells were pre-treated with 5-10 µl of a 1 mg/ml concentration of lysostaphin (Ambi products, New York) in 100 µl 50 mM EDTA to facilitate lysis. Restriction and DNA modifying enzymes (Roche, UK and New England Biolabs, MA) were used according to the manufacturer's instructions. The serine residue at amino acid 403 in the active site of PBP2a was mutated to alanine using Phusion polymerase (NEB) and the primers mecAS403A_For and mecAS403A_Rev ( ). Plasmid p mecA was used as the template. Successful mutagenesis of the DNA sequence encoding the S403 residue resulted in the introduction of a new Dra III restriction enzyme site and candidate plasmids harbouring the mutation were digested with this enzyme before being confirmed by sequencing. The mutated plasmid designated p mecA S403A.
Semi-quantitative measurements of biofilm formation were determined using Nunclon tissue culture treated (Δ surface) 96-well polystyrene plates (Nunc, Denmark), based on the methods of Christensen et al. and Ziebuhr et al. with the following modification. Bacteria were grown in individual wells of 96-well plates at 37°C in BHI medium or BHI supplemented with 4% NaCl or 1% glucose. The serine protease inhibitor dichloroisocoumarin was added to BHI glucose media at concentrations of 0.004-0.5 mM, where indicated. After 24 h of growth, the plates were washed vigorously three times with distilled H 2 O to remove unattached bacteria and dried for 1 hour at 60°C, as recommended by Gelosia et al. as described previously,. The absorbance of the adhered, stained biofilms was measured at A 492 using a microtitre plate reader. Each strain was tested at least three times and average results are presented. Mouse monoclonal anti-PBP2a antibody (CalBioreagents, CA), DNase I (Sigma) or polyanethole sodium sulfonate (PAS) (Sigma) were added to biofilm cultures at the start of the assay as indicated. Biofilm stability against proteinase K (Sigma), sodium-meta-periodate (Sig...
Bacteria were grown on Congo red agar (CRA) plates, which are composed of BHI agar supplemented with 5% sucrose (Sigma) and 0.8 mg of Congo red/ml (Sigma) to distinguish between PNAG-producing (black, dry colony morphology) and non-PNAG-producing (red, smooth colony morphology) phenotypes as described previously,.
To analyze biofilm formation under flow conditions, we utilized the BioFlux 1000 microfluidic system (Fluxion Biosciences Inc., South San Francisco, CA) which allows automated image acquisition within specialized multi-well plates. To grow biofilms, the microfluidic channels were primed with 50% BHI supplemented with 4% NaCl or 1% glucose at 10.0 dyn/cm 2. Channels were seeded at 2 dyn/cm 2 with 10 7 CFU from overnight cultures of 8325-4 p mecA HeR and 8325-4 p mecA HoR. The plate was then incubated at 37°C for 1 hour to allow cells to adhere. Excess inoculums were removed and 2 ml of 50% BHI supplemented with 4% NaCl or 1% glucose was added to the input wells. Biofilms were grown at 37°C with a flow of fresh media at a constant shear of 0.7 dyn/cm 2. Images were taken every 5 minutes for 18 hours at 200 × magnification under brightfield.
Machine-readable layer
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