Muller hinton broth

Antibiotic susceptibility of the isolates was tested according to the Clinical Laboratory Standards Institute [42 ] using E. coli ATCC25922 as quality control. Amoxicillin/clavulanate, ampicillin, cefoxitin, cefpodoxime, ceftazidime, cefotaxime, aztreonam, piperacillin/tazobactam, imipenem, ertapenem, meropenem, gentamicin, amikacin, tobramycin, chloramphenicol, doxycycline, tetracycline, ciprofloxacin, nalidixic acid and co-trimoxazole susceptibility was assessed by disc diffusion. Fosfomycin susceptibility was tested by agar dilution using Muller-Hinton Agar (Oxoid, Basingstoke, UK) supplemented with 25 mg/L glucose-6-phosphate. The minimum inhibitory concentration of colistin was determined by broth microdilution (BMD) in cation adjusted Muller-Hinton Broth (Oxoid, Basingstoke, UK) using colistin sulphate (Sigma-Aldrich, St. Louise, MO, USA). For colistin BMD an mcr-1 positive E. coli isolate (ABC149) [14 (link)] with colistin MIC of 4 mg/L was also included. Strains were considered multi-drug resistant if exhibiting non-susceptibility to ≥3 different classes of drugs tested.

The activities of 6-Pentyl-α-pyrone lactone and ZnONPs against MDR Enterobacterales isolates were screened by the agar well diffusion method, as described previously [21 (link)]. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of 6-pentyl-α-pyrone lactone and ZnONPs were determined by the broth microdilution technique according to Rankin [22 ]. The interaction activities of antimicrobial combination were assessed by the checkerboard method [23 (link)] using Muller–Hinton broth (Oxoid, Hampshire, UK) and a bacterial density of 5 × 10⁵ CFU/mL. Fractional inhibitory concentrations (FIC) of the antimicrobial combination were calculated as mentioned by Hsieh et al. [24 (link)]. The combination is considered synergistic when the FIC index (ΣFIC) is ≤0.5, indifferent when the ΣFIC is >0.5 to <2, and antagonistic when the ΣFIC is ≥2. The MIC 50 and MIC 90 were calculated using an orderly array method [25 (link)].

In order to isolate single cells from S. epidermidis biofilms, we combined a filtration step with mechanical disruption to separate biofilm cells from biofilm-associated factors that might impact on antibiotic efficacy. Staphylococcus epidermidis biofilms were grown in 6-well microplates with Tryptone Soya Broth (TSB, Oxoid, Hampshire, England) as described by Deighton et al.56 (link). Single biofilm cells were isolated as follows: after preparing and washing biofilms, one milliliter of Muller-Hinton broth (MHB, Oxoid, Hampshire, England) was added to each well, and the wells were scraped thoroughly with a FALCON^® cell scraper (Corning, Mexico). The resulting suspensions were mixed well by pipetting several times and transferred into a sterile tube, followed by vigorous vortexing. This step was repeated three times and the final volume of each suspension was adjusted to 25 mL with MHB. The tube was sonicated for 10 min using a sonication bath (42 KHZ, BRANSON 1510) and then vortexed at the highest speed for 2 min (30″ × 4) to break up cell aggregates. The suspensions were firmly pressed through 1.2 μm Acrodisc syringe filters. Muller-Hinton broth was chosen as it is recommended by Clinical Laboratory and Standard Institute (CLSI) for antibiotic susceptibility testing and has been used previously in persister cell studies23 (link).

Ten S. typhimurium bacteria (S1–S10) were included in the current study from the Department of Microbiology and Immunology, Tanta University. The chemicals dimethyl sulfoxide (DMSO), O-nitrophenyl-β-galactopyranoside (ONPG), 1-N-phenylnapthylamine (NPN), and methanol were from Merck, Philadelphia, PA, USA, and the media MHA, Muller–Hinton broth (MHB), and MacConkey agar were from Oxoid, Hampshire, UK.

Acetone, chloroform, ethyl acetate, ethanol (all were AR grade from Merck, South Africa), DPPH, (Sigma, Germany), Muller Hinton broth, Muller Hinton agar (Oxoid) ADC Middlebrook supplement, Middlebrook 7H9 broth (Fluka), glycerol, Iodo-nitro-terazolium chloride (Fluka), Mycoplasma agar, Mycoplasma broth, Yeast malt broth and Yeast Malt agar (Oxoid).

The EOs were prepared in DMSO (Lach-Ner SRO, Prague, Czech Republic). The broth microdilution method reported by Kocić-Tanackov et al. [28 ] was used to determine the MICs and MBCs of the EOs. Briefly, 100 µL of EO was mixed with 100 µL of Muller–Hinton broth (Oxoid, Basingstoke, UK) in the first well of a microtiter plate. After that, a 1:1 serial dilution was made, reaching the concentration 0.23 µL/mL. All wells were filled with 10 µL Y. enterocolitica suspension (10⁸ CFU/mL). Incubation was maintained at 37 °C for 24 h. Then, the content of each well was inoculated onto Muller–Hinton agar (Biokar Diagnostic, Beauvais, France) and incubated at 37 °C for 24 h. The lowest concentrations of EOs that inhibited the visible growth of Y. enterocolitica were defined as MICs, while the lowest concentrations of EOs with no growth after subculturing onto Muller–Hinton agar were defined as MBCs.

The broth microdilution method was used to determine the MIC of CS-NPs [12] . Two-fold serial dilutions of CS-NPs in Muller-Hinton broth (Oxoid, UK) were prepared to reach a final volume of 0.1 mL of each concentration in each well. A standardized inoculum using the direct colony suspension was prepared and diluted in sterile saline to obtain the suspension's final concentration of 0.5 McFarland. Then the inoculum was diluted at 1:20 to yield 5 × 10⁶ CFU/mL. Finally, 0.01 mL of this suspension was added to each well. Negative control tubes containing broth only and other negative control tubes containing CS-NPs only with different concentrations. The microtiter plate was incubated at 37 °C for 24 hrs.
The MIC of CS-NPs against P. aeruginosa clinical isolates was determined using the resazurin microtiter plate assay[15] (link). This assay uses the redox indicator resazurin (Sigma-Aldrich, Germany) that changed color from blue to pink in the presence of viable cells. The MIC was determined as the concentration at which there was no color change following 4 hrs incubation of the overnight cells with 0.015% resazurin (Figure 1). One dilution below the MIC was regarded as the sub-MIC concentration and was used to evaluate the ability of the CS-NPs to inhibit the virulence activity.