Probiotic disruption of quorum sensing reduces virulence and increases cefoxitin sensitivity in methicillin-resistant Staphylococcus aureus

Probiotic bacterial strain

A frozen glycerol stock culture of Enterococcus faecium 30616 (“Ef 30616”) (previously identified as Lactobacillus acidophilus strain DSM 13241, redesignated after MALDI-TOF microbial identification) was obtained from the Canadian Research Institute for Food Safety (University of Guelph, Ontario, Canada). This stock probiotic strain was used to produce bioactive materials containing probiotic metabolites for all experiments used in this study. A second probiotic strain, Lactococcus lactis 11454 (“Ll 11454”) was obtained from the American Type Culture Collection (ATCC) and used to produce bioactive containing material for MIC tests.

Staphylococcal bacterial strains

Two clinical isolates of S. aureus bacteria, MRSA 81M SPA t008 (“MRSA 81M”) and MRSA 414M SPA t034 (“MRSA 414M”), were obtained from the Atlantic Veterinary College (AVC) at the University of Prince Edward Island (Charlottetown, PE, Canada). The presence of methicillin resistance via the SCC mecA gene pathway in both strains was confirmed with oxacillin disk diffusion and mecA gene primers (Supplementary Table S1)⁴². The clinical isolates were maintained on tryptic soy agar (TSA) plates supplemented with 5% v/v sterile defibrinated sheep blood (Cedarlane, Burlington, Ontario, Canada). MRSA cultures were grown in BBL™ cation-adjusted Mueller Hinton media (Becton, Dickinson and Company, Sparks, MD, USA) for all experimental assays.

Preparation of probiotic bioactive material

E. faecium Ef 30616 was inoculated in 200 mL of 0.22 µm filter-sterilized modified De Man, Rogosa and Sharpe (MRS) medium (VWR International, Mississauga, Ontario, Canada). This bottle culture was incubated without shaking at 37 ± 1 °C for 18 h. The culture was then used to inoculate 4 L of chemically defined medium (CDM) with 200 g whey protein isolate (Canadian Protein) and 25 g lactose. The vessel culture was incubated statically at 37 ± 1 °C for 48 h. Following incubation, the Ef 30616 cells were isolated from the liquid phase by centrifugation at 12,000× g for 30 min at 4 °C (Avanti J-20 XPI, Beckman Coulter, Canada). The cell-free supernatant containing the probiotic metabolites was then frozen at − 80 °C and freeze dried. The dried Ef 30616 cell-free supernatant was kept in long-term storage in powder form at − 20 °C until needed.

Resuspension of probiotic bioactive material

Dried supernatant was weighed out and resuspended in cation-adjusted Mueller Hinton media at the desired concentration (5, 30 and 60 mg/mL). The pH of the resuspended material was checked with an Accumet^® AE150 pH meter (Fisher Scientific, Waltham, USA) and adjusted as needed to a pH of 7.3 ± 0.1. The resuspended solution was then centrifuged at 5000× g for 15 min at room temperature with a Centrifuge 5804 (Eppendorf, Hamburg, Germany). The remaining liquid was separated from the debris pellet and filtered through a Supor^®-800 0.45 µm membrane filter (Pall Corporation, New York, USA) with a 40/35 Synthware vacuum filtration apparatus (Kemtech America, Los Angeles, USA) to remove remaining colloidal material. The filtrate was then filter-sterilized using a Basix™ 25 mm 0.2 µm syringe filter (Fisher Scientific, Waltham, USA). The samples were stored at − 20 °C until needed.

Preparation of molecular weight cut off (MWCO 3000) fractions

Amicon^® Ultra 15 mL centrifugal filters with a Molecular Weight Cut Off (MWCO) of 3000 Da (Millipore Sigma, Burlington, USA) were used for ultrafiltration of the resuspended probiotic cell-free supernatant. Following resuspension as described above, 10 mL of the cell-free supernatant was added to the MWCO 3000 centrifugal filter tube and centrifuged at 5000 rpm for 1 h. The MWCO 3000 filtrate was collected; the remaining retentate fraction was collected by rinsing the filter head twice with 10 mL of cation-adjusted Mueller Hinton media for each rinse. Following the two rinses, an additional 10 mL of media was used to resuspend and collect the retentate. All collected fractions were then filter-sterilized with a 0.2 µm syringe filter to remove any contamination. The MWCO 3000 liquid retentate and filtrate solutions were stored at − 20 °C until needed for the experiments.

Preparation of size-exclusion chromatography (SEC) fractions

Dried supernatant was resuspended in Milli-Q water to a concentration of 50 mg/mL and sonicated for 15 min to improve solubilization. Following resuspension, the samples were centrifuged for 20 min at 8000× g and the supernatant filter sterilized with a 0.22 µm syringe filter. Samples were separated on a XK 26/100 preparative column packed with Superdex 30 and run using a AKTA Explorer (Cytiva Life Sciences, Marlborough, USA). Material was eluted with 10% acetonitrile and 0.1% trifluoracetic acid at a flow rate of 3 mL/min. The elution curve was monitored by measuring the pH and absorbance at 280 and 214 nm. The desired elution’s were collected and pooled into defined fractions then frozen at − 80 °C followed by freeze drying. Lyophilized material was stored at − 80 °C until needed.

Preparation of cefoxitin for MIC testing

The antibiotic selected for minimum inhibitory concentration (MIC) testing in the two MRSA strains was cefoxitin as outlined in the Clinical and Laboratory Standards Institute (CLSI) guidelines for MIC testing of Staphylococcal species⁴³. Cefoxitin in powder form (Alfa Aesar, Haverhill, USA) was weighed with an analytical balance and resuspended in methanol at 10 mg/mL and stored at − 20 °C until needed.

Minimum inhibitory concentration (MIC) testing

MIC testing was performed with respect to the Clinical and Laboratory Standards Institute (CLSI) guidelines for MIC testing of Staphylococcal species⁴³. Overnight cultures of each respective clinical MRSA strain were diluted 1000-fold to obtain approximately 10⁶ CFU/mL as the starting inoculate. Dilution ranges for cefoxitin were from 0 to 100 μg/mL. Clinical MRSA strain MICs with cefoxitin were determined by broth microdilution with cefoxitin in cation-adjusted Mueller Hinton media in Costar^® clear 96-well standard flat-bottom microplates (Corning^® Inc., Corning, USA). Media-only aliquots were added as sterility checks and were used as background control, while the 100 µg/mL cefoxitin well served as a positive control of antibiotic-mediated killing. All test sample volumes were 200 µL per well with duplicates. Following sample preparation and aliquoting, the MIC test plates were sealed with parafilm and incubated at 35 ± 1 °C and 200 rpm shaking for 24 h in a VWR^® Incubating Mini Shaker and temperature was monitored with a Fisherbrand™ digital thermometer (Fisher Scientific, Waltham, USA). Following incubation, the plates were cooled to room temperature and turbidity was measured using a SpectraMax M2 microplate reader (Molecular Devices, San Jose, USA) at a wavelength of 600 nm. The lowest concentrations of cefoxitin that resulted in either 90% or greater reduction in turbidity compared to the respective positive-growth controls were defined as the MIC. All MIC tests were performed in three biological replicates. Following MIC checkerboard testing, as a clinical strain with a high MIC, MRSA 81M was selected for testing of ultrafiltration and SEC fractions of the bioactive material to better elucidate the bioactives’ sizes and chemical identity. The MWCO 3000 and SEC fraction MIC tests were performed identically to the MIC test method outlined above.

Fractional inhibitory concentration (FIC) index

Checkerboard MIC analyses were performed for three pre-determined probiotic bioactive material concentrations to determine the synergistic, additive, or antagonistic combinatory effects with cefoxitin against the clinical MRSA strains. Dilution ranges of the bioactive material were 5, 30 and 60 mg/mL of freeze dried Ef 30616 cell-free supernatant, or 5, 15 and 30 mg/mL of freeze dried Ef 30616 and Ll 11454 SEC fractions. Dilution ranges for cefoxitin used in FIC testing were 0–100 µg/mL for bulk material, and 0–140 µg/mL for SEC fractions. The FIC tests were performed identically to the MIC test method outlined above. The FIC index was determined as previously described³⁰. Equations (1) and (2) was used to determine the FIC values for cefoxitin or each bioactive material, and Eq. (3) was used to calculate the FIC index to determine the combinatory effect of the two components.

where FIC_A and FIC_B are the FIC values for drug A and drug B, respectively. MIC_A and MIC_B are the respective MICs of drug A and drug B alone. MIC_C is the MIC of drug A and drug B in combination. The FIC index (FIC_i) is the sum of FIC_A and FIC_B.

MRSA culture growth

For the following physiological and qPCR assays, each respective MRSA strain was grown as follows. Each MRSA strain was inoculated into 10 mL of cation-adjusted Mueller Hinton media (“untreated”) and 10 mL of cation-adjusted Mueller Hinton media supplemented with 30 mg/mL of probiotic bioactive material (“treated”) then incubated at 37 ± 1 °C and 200 rpm shaking for 24 h unless otherwise stated.

Carotenoid measurements

The samples were prepared as described above. Following incubation, Staphylococcal carotenoids were extracted using a previously established methanol extraction protocol²³. The supernatant containing the extracted carotenoids was pipetted in 200 µL (with duplicates) into a standard 96-well plate and the absorbance at a wavelength of 450 nm (A450) was measured with a SpectraMax M2 microplate reader (Molecular Devices, San Jose, USA).

Oxidant susceptibility assays

The samples were prepared as described above. Following incubation, samples were centrifuged at 4000 rpm for 15 min, washed in 1× Dulbecco’s PBS solution (VWR Life Science, Radnor, USA), and resuspended at approximately 10⁹–10¹⁰ CFU/mL in 1.5% v/v hydrogen peroxide solution. Oxidant susceptibility assays were then performed following a previously established protocol²³.

Blood agar growth testing

The samples were prepared as described. Serial dilutions of the samples were performed and 100 µL of diluted sample was plated in duplicate on TSA plates supplemented with 5% v/v sterile defibrinated sheep blood (Cedarlane, Burlington, Ontario, Canada). Plates were incubated for 15–20 h at 37 ± 1 °C and analyzed for hemolysis and CFU/mL counts.

RNA extraction, cDNA synthesis and qPCR testing

The samples were prepared as described above, and then incubated at 37 ± 1 °C and 200 rpm shaking for 4 h and 6 h for MRSA 414M samples and 5 h and 6 h for MRSA 81M samples. RNA extractions of the samples were performed according to the illustra™ RNAspin kit (GE Healthcare, Buckinghamshire, UK). Reverse transcriptions and cDNA syntheses were performed according to the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, USA). qPCR experiments were performed using a CFX96™ Real-Time System C1000™ Thermal Cycler and data analyses were performed with the accompanying CFX Manager 3.1 software system (Bio-Rad, Hercules, USA). The selected genes and their primer pairs are listed in Supplementary Table S1.

Scanning electron microscopy and light microscopy

The samples were prepared as described above, and then incubated at 37 ± 1 °C and 200 rpm shaking for 4 h, 6 h and 24 h for both 414M and 81M samples. Following incubation, samples were stained with Crystal Violet, prepared for light microscopy and observed using the Upright Revolve 4 Microscope equipped with an iPad Pro (ECHO Inc., San Diego, USA). SEM samples were prepared as previously described⁴⁴. The samples were mounted on stubs using double sided tape and sputter-coated with gold using/by a Denton Vacuum system and observed with a Hitachi TM3000 Scanning Electron Microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) operated at 15 kV.

Statistical methods

MIC data were analyzed by two-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism version 9 (GraphPad software, San Diego, California). All qPCR data were analyzed by ANOVA using CFX Manager 3.1 software system (Bio-Rad, Hercules, USA). Data for carotenoid absorption assay and SCV percent cell counts were analyzed by Mann–Whitney U statistical testing using Microsoft Excel. Hydrogen-peroxide cell killing data were analyzed by Wilcoxon signed-rank for paired statistical testing using Microsoft Excel. All stated replicates (n) are the average biological value of technical duplicates.