Rifampicin

The mutagenic effect of OEO was determined by calculating the rate of mutants resistant to rifampicin due to point mutations in the rpoB gene [10 (link),59 (link)]. Overnight culture of SaWT was diluted 1:10,000 into 50 mL TSBYE and incubated at 37 °C and 130 rpm for 24 h. This culture was then diluted 1:3 in tubes containing 10 mL TSBYE with 750 µL/L of OEO (same concentration used in the evolution assay) and without OEO. This experiment was also carried out with carvacrol (Sigma-Aldrich) at 50 µL/L as control of natural compounds from previous study [10 (link)] and with rifampicin (Sigma-Aldrich) at 0.01 mg/L concentration as a positive control of mutagenesis. Those suspensions were grown at 37 °C and 130 rpm for 24 h (ca. 2 × 10⁹ CFU/mL). Samples of the culture were serially diluted in PBS and pour-plated on TSAYE in the presence and absence of 100 mg/L rifampicin (Sigma-Aldrich). Plates were incubated at 37 °C for 48 h and colonies were counted. Mutation rates were calculated by dividing the number of colonies present in rifampicin plates (mutation events) by the number of colonies present in plates without antibiotic [60 (link)].

L. reuteri and EHEC strains used in this study are listed in S1 Table. The EHEC strain FCH6 (O157:H7, eae⁺, stx1^-, stx2⁺) had been isolated from a case of HUS in 2004 due to the consumption of raw milk cheese. EHEC strains were routinely cultured in Luria Bertani (LB) broth (Biokar, Beauvais, France). L. reuteri strains were routinely cultured in De Man, Rogosa and Sharpe (MRS) broth (Biokar, Beauvais, France). Spontaneous rifampicin-resistant mutants of L. reuteri and EHEC strains were isolated on MRS agar (pH 6.8) and LB agar plates containing 100 μg/mL of rifampicin (Sigma-Aldrich, St Quentin Fallavier, France), respectively. Each wild-type strain and its corresponding spontaneous Rif^R mutant showed identical growth patterns. Sensitivity of the rumen microbiota to rifampicin was confirmed by spotting 100 μL of rumen fluid (RF) samples on MRS agar plate containing rifampicin (50 μg/mL) before incubation at 39°C for 24 hours. Survival of L. reuteri in RF was tested by incubating RF samples (from O₂-free, CO₂-saturated sterile flasks) inoculated with L. reuteri Rif^R under anaerobiosis (39°C). The next day, the RF samples were 10-fold serially diluted in sterile phosphate buffered saline (PBS) buffer (pH 7.4), plated on MRS agar plates containing rifampicin and incubated overnight under anaerobiosis at 37°C for CFU counting. The experiments were replicated three times.

Rifampicin (Sigma Aldrich, Rockville, MD, USA) was used as a drug of choice, sodium alginate (Sigma Aldrich, Rockville, MD, USA) and aloe vera powder (MINATURE, MARUDHRA IMPEX, Ahmadabad, India) were utilized as matrix former, L-leucine (Sigma Aldrich, Rockville, MD, USA) was added as spray drying excipients. Phosphate buffer saline (Sigma Aldrich, Rockville, MD, USA) was used as a dissolution medium to mimic lung fluid. Methanol (Sigma Aldrich, Rockville, MD, USA) was used as a solubilizing medium for Rifampicin. All other chemicals used were of analytical grade and utilized without any further purification.

The endophytic colonization of grapevine tissues by Pseudomonas was studied using a laboratory-induced rifampicin-resistant (RifMut) strain from BCA17 (P. poae). The RifMut strain was developed following the methods of Adorada et al. [72 (link)] and West et al. [27 (link)]. A single colony of BCA17 was streaked onto NA amended with 1 ppm of rifampicin (Sigma Chemical Co., St. Louis, MO, USA) and resulting colonies subsequently exposed to increasing concentrations of rifampicin (5, 10, 50, and 100 pm). The final resistant strain, now referred to as RifMut, was re-inoculated onto NA with 100 ppm rifampicin on five subsequent occasions to ensure the stability of the resistance.

Conjugation experiments were performed with the plasmid-free recipient strain E. coli HK225 (Strep^r, Rif^r; Kayser et al., 1982 (link)). Briefly, single colonies of the donor and recipient were inoculated in LB broth (Difco Laboratories) and grown overnight at 37°C. Subsequently, equal volumes of the donor and recipient cultures were mixed and incubated overnight at 37°C without shaking. Serial dilutions were then plated on LB agar (Difco Laboratories) selection plates supplemented with a combination of 600 μg/ml streptomycin (Sigma–Aldrich, Buchs, Switzerland) 100 μg/ml rifampicin (Sigma–Aldrich) and 10 μg/ml cefotaxime (Sigma–Aldrich).
The conjugation frequency per donor was determined by plating serial dilutions of the mating on selective plates on which the donor strain and the transconjugant can grow (LB-agar supplemented with 10 μg/ml cefotaxime) as well as on plates on which only the transconjugants can grow (LB-agar supplemented with 600 μg/ml streptomycin, 100 μg/ml rifampicin, and 10 μg/ml cefotaxime). The transfer frequency was calculated as the quotient of the number of transconjugants over the number of transconjugants plus donors.

Feces and large-intestine scraping were cultivated with selective medium on TSA Tryptic Soy Agar (Tryptic Soy Agar, DIFCO, Taiwan, China, cat no. 211043) supplemented with 5% sheep blood, 6.25 mg/μL rifampicin (rifampicin, Sigma-Aldrich, St. Louis, MO, USA, cat no. R3501), and 800 mg/L spectinomycin (Sigma-Aldrich, cat no. S9007), 25 mg/μL vancomycin (vancomycin, Sigma-Aldrich, cat no. V2002), 25 mg/L of colistin (colistin, Sigma-Aldrich, cat no. C1511) [32 (link)], and incubated for at least three days at 42 °C in jars with an anaerobic atmosphere. Anaerobic conditions were generated using a vacuum pump filled with a mixture of N₂ (80%), CO₂ (10%), and H₂ (10%) gases. To obtain pure colonies, several passages were performed until isolation. The isolates were stored in a freezer at −80 °C.
For differential diagnosis, at the end of the experiment, mucosal scrapings from the small intestine were cultivated on blood agar and MacConkey to evaluate the growth of enterotoxigenic Escherichia coli and scrapings from the large intestine mucosa were cultivated in Rappaport broth and Hectoein agar for Salmonella spp.