Erythromycin

The antibiotic susceptibility profiles of the S. aureus isolates were determined by the Minimum Inhibitory Concentration (MIC) technique with the microdilution method in Mueller-Hinton broth (MH; Becton Dickinson, East Rutherford, NJ, USA), as recommended by the Clinical and Laboratory Standards Institute (2014) . The MIC tests were conducted with vancomycin, ciprofloxacin, erythromycin (MP Biomedicals, Solon, OH, USA), clarithromycin (Grünenthal Gmbh, Aachen, Germany), oxacillin, clindamycin, linezolid (Sigma-Aldrich, St. Louis, MO, USA), meropenem (AstraZeneca Pharmaceuticals LP, Wilmington, DE, USA), trimethoprim, sulfamethoxazole (Roche, Basel, Switzerland), and gentamicin (Schering-Plough Pharmaceuticals, Kenilworth, NJ, USA). To identify methicillin-resistant S. aureus clinical isolates, the bacteria were tested for oxacillin resistance by the oxacillin-salt screening method. oxacillin is a more stable antibiotic than methicillin, although they are chemically identical. S. aureus strain ATCC 29213 (American Type Culture Collection, Manassas, VA, USA) was used as a positive control.

The antibiotic susceptibility profiles for the VT-MRSA isolates were determined using the MIC technique via the microdilution method according to the CLSI 2013 [32 ], as described in the previous section. MIC tests were performed for ampicillin (Sigma-Aldrich), ceftazidime (Pfizer; México DF, México), ceftriaxone (Roche; Mexico DF, México), ciprofloxacin (Bayer; Levenkusen Westfalia, Germany), erythromycin (MP Biomedicals; Solon, OH, USA), kanamycin (Sigma-Aldrich), meropenem (Astra Zeneca; México DF, Mexico), rifampicin (MP Biomedicals), gentamicin (MP Biomedicals), and trimethoprim-sulfamethoxazole (Sigma-Aldrich).

The following antibiotics were used in these experiments: ampicillin, chloramphenicol, colistin, gentamicin, kanamycin, oxacillin, tetracycline, and vancomycin (Sigma); azithromycin and tobramycin (Tocris); daptomycin (TCI); erythromycin (MP Biochemicals); ciprofloxacin and rifampin (Applichem); meropenem (Combi-Blocks), linezolid (Chem Impex, Int.).

The serotype 1/2b strain 2011L-2858, referred to here as “2858”, was kindly obtained from Cheryl Tarr and was previously investigated for its potential to adhere and grow on cantaloupe [23 (link)], as well as the role of a penicillin-binding protein encoded by pbp4 on copper tolerance and virulence [22 (link)]. Strains B2G6 and J2E3 are non-hemolytic mutants of strain 2858 obtained as described below from screening a mariner-based mutant library on blood agar (Remel Inc., Lenexa, KS, USA) plates. Unless otherwise indicated, L. monocytogenes was grown at 37 °C in brain heart infusion (BHI) broth (Becton, Dickinson & Co., Sparks, MD, USA) or on BHI agar (BHIA, BHI with 1.2% Bacto-agar, Becton, Dickinson & Co.). When needed, erythromycin (MP Biomedicals, Solon, OH, USA) was added at 5 µg/mL in BHI (BHI-Em5) or in BHIA (BHIA-Em5) and kanamycin (Fisher Scientific, Fair Lawn, NJ, USA) was added at 10 µg/mL to BHI (BHI-Km10) and BHIA (BHIA-Km10).

The microdilution protocol was used to assess the resistance of probiotic bacteria to different types of antibiotics [23 (link)] with some modifications according to EFSA Guidance [24 (link)]. The antibiotics ampicillin, chloramphenicol, erythromycin, gentamycin, kanamycin (Sigma Aldrich, St. Louis, MO), and streptomycin (MP Biomedicals, Santa Ana, CA) and the amounts of the active compound were placed into the broth media tube: ampicillin (10 μg), chloramphenicol (30 μg), erythromycin (15 μg), gentamycin (10 μg), kanamycin (30 μg), and streptomycin (10 μg). Cultures were inoculated in MRS broth filtered through a 0.22-μm filter and incubating aerobically at 37°C overnight. Furthermore, antibiotic stock solutions were prepared according to the following formula: $\begin{array}{c}\mathrm{W}=\frac{\mathrm{1000}}{\mathrm{P}}\times \mathrm{V}\times \mathrm{C}\end{array}$ where P is potency given by the manufacturer (μg /mg), V is volume required (ml), C is final concentration of solution (multiples of 1000, mg/L), and W is weight of antibiotic (mg) to be dissolved in volume V (mL). The data was expressed after 24 hours as the following formula: $\begin{array}{c}\mathrm{S}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}\%=\frac{\log \mathrm{N}\mathrm{1}}{\log \mathrm{N}\mathrm{0}}\times \mathrm{100}\\ \end{array}$ $\begin{array}{c}\mathrm{S}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\hspace{0.17em}\hspace{0.17em}\text{of}\hspace{0.17em}\hspace{0.17em}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{o}\mathrm{t}\mathrm{i}\mathrm{c}=\mathrm{100}-\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}\%\\ \end{array}$ log⁡N1 is absorbance of culture 620 nm in MRS broth with different antibiotic types and log⁡N0 is absorbance of culture 620 nm in MRS broth as a control.