Mcfarland standard

This cross-sectional laboratory-based study was conducted between June and August 2021. This study was carried out in Microbiology and Molecular Biology research laboratories of the Catholic University of Health and Allied Sciences (CUHAS) in Mwanza, Tanzania.
Known GNB isolates archived at −80 °C with resistance to meropenem that were contemporaneously isolated from clinical (urine and blood), colonization (rectal swabs), and hospital environmental (patients’ bed swabs) samples from a previous study [15 (link)] were recovered for this study.
Isolates were recovered by subculturing on MacConkey agar (MCA; HiMedia, India) plates, which were incubated aerobically at 37 °C for 18–20 h. One to two colonies from culture plates were suspended in 5 mL of 0.85% sterile normal saline. Then, suspensions were adjusted to 0.5 McFarland standard (Densicheck; bioMérieux, Grassina, Italy) for phenotypic detection of carbapenemases production.

In this study, we used the biofilm-producing DFI strains Staphylococcus aureus Z25.2 and Pseudomonas aeruginosa Z25.1, co-isolated from a diabetic foot ulcer. The strains were selected from a bacterial collection obtained from patients with Diabetes mellitus and infected foot ulcers, as described in a previous study by Mendes et al. [6 (link)] and characterized by Mottola et al. [64 (link)]. All isolates were stored at –80 °C in buffered peptone water supplemented with 20% (v/v) of glycerol. When needed, cells were streaked and grown on Mueller Hinton Cation-Adjusted (MH-CA) agar medium (Becton, Dickinson and Company, USA) at 37 °C for 24 h. Bacterial suspensions were prepared at 10⁸ cfu/mL directly from plate cultures using a 0.5 McFarland standard (BioMérieux) in sterile normal saline (Scharlau) and diluted in fresh MH-CA broth to obtain 10⁷ cfu/mL suspensions for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, and of 10⁶ cfu/mL for minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) assays. Dual-microbial suspensions containing equal concentrations of each pathogen were also prepared.

In disc-diffusion method, Mueller-Hinton agar (VWR Chemicals, Radnor, PA, USA) was prepared and placed in Petri dishes. For E. coli and S. aureus inoculum of 1.5 × 10⁸ CFU (0.5 McFarland standard, BioMérieux SA, Marcy-l’Etoile, France) of bacteria/mL was used for completed seeding of the agar, and inoculum of 0.75 × 10⁸ and 3 × 10⁸ CFU of bacteria/mL was used for B. cereus and L. plantarum, respectively. Filter paper discs of 6.0 mm in diameter (Liofilchem, Roseto degli Abruzzi, Italy) were impregnated with several concentrations of infusion, decoction and essential oil, placed in the seeded agar and incubated at 37 °C for 24 h. For solvent control, DMSO 15% (solvent used for essential oil dissolution) was used. The diameter of the inhibition zone around the filter paper disc was measured in millimetres. All the determinations were performed in triplicate, and the results were expressed as mean ± standard deviation (SD).

In the nested study, MICs for CIP were determined by the E-test method on isolates that were CIP non-susceptible by disc diffusion per CLSI guidelines [16 ]. Bacterial colonies were suspended in 0.85% normal saline to a turbidity equivalent to 0.5 McFarland standard (bioMérieux, Inc, Durham, NC, United States). The bacterial suspension was inoculated on Mueller Hinton agar (Oxoid, Hants, United Kingdom) plates, and the CIP E-strips were placed at the center of the agar followed by incubation at 35 °C for 16 -18 h. Concentration ranges for MICs for E-test strips for CIP (0.002–32 µg/mL) (bioMérieux Marcy l’Etoile, France) and non-susceptibility interpreted according to 2021 CLSI guidelines [16 ]. MIC results were classified as follows: susceptible (≤ 0.25 µg/mL), intermediate (0.5 µg/mL), or resistant (≥ 1 µg/mL). E. coli ATCC 25922 was used as quality control for determining MICs by the E-test method.

Detached healthy jackfruit pulps (Tekam Yellow J33 variety) were inoculated with 10 mL of 10⁸ CFU/mL (as determined by McFarland Standard, BioMérieux, Marcyl'Etoile, France) of 24 to 48 h pure P. stewartii subsp. stewartii suspensions. Sterile distilled water was inoculated to control healthy jackfruit pulps. 4 replicates were performed per strain. All 28 bacterial strains treatments were put inside sterile petri dish (properly sealed) and incubated at 28 ºC in a controlled chamber. The evaluation of jackfruit-bronzing symptoms was recorded daily up to two weeks of inoculation.

E. coli O101 freeze-drying powder (CVCC3749) was bought from China Veterinary Microbial Culture Collection (Beijing, China). The powder was grown in Tryptone Soy Broth (TSB; Hangzhou Microbial Reacent Co. Ltd., China). After overnight incubation at 37°C with shaking at 180 rmp/min, bacteria were diluted with fresh TSB followed by another overnight incubation. The bacterial cells were harvested by centrifugation at 5000g for 10 min at 4°C, and the pellet was washed twice and resuspended in sterile physiological saline (0.9%, w/v). The concentration of bacterial cell suspension was adjusted to different McFarland standard (bioMerieux, France) for a final density of approximately 5 × 10⁸ CFU/mL, 3 × 10⁸ CFU/mL, 2 × 10⁸ CFU/mL, and 1 × 10⁸ CFU/mL, respectively.