Mueller hinton agar plate

S. aureus clinical isolates were isolated from clinical specimen referred to the routine medical microbiology laboratory of the Department of Infectious Diseases, University Hospital Heidelberg, Germany. Clinical isolates were numbered according to their origin, e.g. isolates cultured from patients with invasive diseases were categorized as “INV” and those obtained from respiratory specimen of cystic fibrosis patients as “CF”. Storage of the bacterial strains at −80°C was performed in skim milk (Becton Dickinson, Heidelberg, Germany) or with cryobeads (Cryobank™, Mast Diagnostica, Reinfeld, Germany). Reference and mutant S. aureus strains are listed in Table 1. For each experiment, bacteria were prepared, thawed and cultured freshly on Columbia/5% sheep red blood agar plates (Becton Dickinson, Heidelberg, Germany), Mueller Hinton agar plates (BioMerieux, Nuertingen, Germany), or CASO-bouillon (Becton Dickinson, Heidelberg, Germany) and incubated at 37°C overnight.

Susceptibility testing for relevant anti-pseudomonal antibiotics was carried out using the disk diffusion method (Oxoid, Basingstoke, UK) and E-tests (Liofilchem, Roseto degli Abruzzi, Italy) on Mueller-Hinton agar plates (bioMérieux, Marcy-l’Étoile, France) including amikacin, ceftazidime, cefepime, ciprofloxacin, gentamicin, imipenem, levofloxacin and meropenem. Colistin susceptibility was performed using the broth microdilution assay in cation-adjusted Mueller-Hinton broth (Merlin Diagnostika GmbH, Bremen, Germany). Interpretation of the results was based on the standards and breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) v. 11.0 [38 ]. Results indicating “susceptible, increased exposure (I)” were grouped with and reported as susceptible (S) [39 ]. Classification of the isolates as MDR (resistance to at least one agent in ≥3 antibiotic groups) was based on Magiorakos et al. [40 (link)]. A multiple antibiotic resistance (MAR) index—ranging between 0 and 1—was calculated by dividing the total number of detected resistance to antimicrobials for each isolate by the total number of tested antimicrobials [41 (link)].

For the phenotypic characterization of the isolates, the combination disk test was utilized according to CLSI [15 ] and EUCAST [17 ] guidelines. In brief, an initial inoculum was prepared as already described, and Mueller Hinton agar plates (bioMérieux, France) were inoculated. Antibiotic discs (Himedia, Mumbai, India) containing cefepime (FEP 30 μg), cefepime/clavulanic acid (FEP 30 μg), ceftazidime (CAZ 30 μg), ceftazidime/clavulanic acid (CAC 30/10 μg) cefotaxime (CTX 30 μg), cefotaxime/clavulanic acid (CEC 30/10 μg), and cefoxitin (FOX 30 μg) were used. An increase of ≥5 mm in the zone diameter of a combination of the antibiotic with clavulanic acid against the zone diameter of the antibiotic alone was reported as corresponding to an ESBL phenotype. The isolates were characterized as AmpC if resistance to cefotaxime and ceftazidime was recorded without induction by clavulanic acid and exhibited a ≥5 mm increase in the inhibition zone. For quality control, the type cultures E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used.

For the disk diffusion test, E. faecalis at a concentration of 10⁸ CFU/mL in 100 uL suspension and C. albicans at a concentration of 10⁶ CFU/mL in 100 uL suspension were seeded on Mueller Hinton agar plates (Biomerieux, Lyon, France) [58 (link),59 (link)].
Seven different study groups containing calcium hydroxide and Cyprus mountain thyme oil (O. dubium EO) or garden mint oil (M. spicata EO) were tested for antimicrobial activity. For this purpose, the different groups formed were added to the sterile disks to be tested. Distilled water was chosen as a negative control for both microorganisms. Vancomycin was chosen as a positive control for E. faecalis and nystatin for C. albicans.
Agar plates for E. faecalis were incubated at 37 °C for 24, 48, and 72 h. C. albicans was incubated at 37 °C for 48 and 72 h. The inhibition zones formed around the disks on the agar plates indicated the degree of antimicrobial activity (Figure 4). Inhibition zone measurements (mm) were taken at 24, 48, and 72 h for E. faecalis and 48 and 72 h for C. albicans. Each test was repeated in triplicate to obtain reliable data.

We determined antimicrobial susceptibility of the E. coli strains using disk diffusion on Mueller-Hinton agar plates (bioMérieux, https://www.biomerieux.com), as recommended by Comité de l’Antibiogramme de la Société Française de Microbiologie (https://www.sfm-microbiologie.org) guidelines. We defined ESBL production by synergy between clavulanic acid and >1 extended-spectrum cephalosporin or aztreonam.

To quantify bacteria within the explanted samples (n = 4 per group), serial dilutions from sonicated fluids were plated onto Mueller-Hinton agar plates (Biomerieux, Marcy l'Etoile, France) and incubated for 16 h at 37°C. Briefly, sterile container containing explanted samples was filled with 1 ml of sterile saline and sonicated in an ultrasound bath (VWR, Milan, Italy) for 5 min with a frequency of 30 kHz and a power output of 300 W at room temperature. All samples were assayed by serial 10-fold dilution in sterile saline solution and then plated on solid growth medium. After incubation of the plates at 37°C for 16 h, colonies of Gram-positive catalase positive cocci, resembling those of S. aureus, were tested for coagulase activity with Coagulase Plasma (Remel Europe Ltd. Dartford, UK) and identified by means of API Staph assay (BioMerieux, Mercy L'Etoile, France). Positivity to catalase test consists in the development of gaseous oxygen when the colony was put in contact with oxygenate water. Coagulase test results positive when S.aureus colonies in contact with coagulase plasma, form a visible clot after incubation for 4–6 h at 37°C. Colonies identified as S. aureus grown on Mueller-Hinton agar plates were then counted. The detection limit (L. o. D) was ≤1.300 (Log CFU)/g of bone.