Mueller hinton

Time-kill assay was performed to determine the time taken for the microorganisms to be killed by the cinnamon extract, and it was performed following the method of Rukayadi et al. [27 (link)]. Mueller–Hinton broth was used, and bacterial inoculum was adjusted between 6 to 8 log CFU/mL. The final concentrations of cinnamon were 0 MIC, 0.5 × MIC, and 1 × MIC aliquots. The cultures were then incubated at 37 °C with an agitation of 200 rpm. At predetermined intervals of 0, 0.5, 1, 2, 4, and 6 h, 1 mL aliquots were serially diluted in 1% (w/v) phosphate-buffered saline (PBS) and plated onto Mueller–Hinton (Oxoid, UK) agar plates. The plates were incubated at 37 °C for 24 h, and the number of colonies was counted. The assays were carried out in triplicate. The graph of log₁₀ CFU/mL was plotted against time.

The bacterial isolates were subjected to antimicrobial sensitivity testing by disk diffusion method as described by [15 (link)]. Briefly, test organisms were suspended in sterile normal saline to conform to 0.5 McFarland turbidity standard. With the aid of sterile cotton swab the suspended organisms were spread on Mueller-Hinton (Oxoid Ltd, Basingstoke, United Kingdom) Agar plate and the antibiotic disks dispensed. The plates were incubated at 37°C for 16-18h. Inhibition zone diameters were determined and recorded in Excel sheets and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [16 ].
The following panel of antibiotics (all from Oxoid) and their concentrations were used. For Gram negatives cefuroxime sodium 30 μg, amoxycillin\clavulanic acid 2:1 30 μg, chloramphenicol 30 μg, tetracycline 30μg, co-trimoxazole sxt 25 μg, nalidixic acid 30 μg, ampicillin 10 μg, ceftazidime 30 μg, cefotaxime 30 μg, ciprofloxacin 5 μg, norfloxacin 10μg, gentamycin 10μg, were tested. For Gram positives meropenem 10 μg, gentamycin 10μg, kanamycin 30μg, clindamycin 2μg, norfloxacin 10μg, ofloxacin 5μg, oxacillin 5μg, erythromycin 10μg, nalidixic acid 30 μg, trimethoprim-sulfamethoxazole 25μg, chloramphenicol 30 μg, tetracycline 30μg were tested.

Upon being received in the laboratory, rectal swabs were placed in selenite faecal enrichment broth and incubated at 37 ˚C overnight. From the enriched selenite F broth, streaking was done onto Xylose Lysine Deoxycholate agar (XLD) agar and Mac Conkey agar (Oxoid). Incubation was done at 37˚C for 24 hours. S.Typhi isolates were initially identified using distinguishing colony morphology characteristics such as pale-yellow colonies on MAC and brick red with black centres on XLD. Non-duplicate colonies from standard biochemical tests such as citrate, indole, urease, and Triple Sugar Iron (TSI) were examined with API20E (Biomeriux) and confirmed with serology. Serotyping tests were done using the slide agglutination technique using polyvalent antisera O- 9, and monovalent anti-sera d (Murex Diagnostics, Dartford, UK) [9 (link)].
Blood culture bottles were incubated in a BACTEC 9050 Culture System (Becton Dickinson, USA) at 37°C for up to seven days. A positive culture bottle with the reference strain was used to validate the results. Samples flagged as negative by the BACTEC were discarded while those flagged as positive were cultured onto MacConkey agar, Blood agar, and Chocolate agar. Isolates showing growth characteristics of S.Typhi were subcultured onto Mueller Hinton (Oxoid, Basingstoke, UK) for the growth of single discrete colonies.

Yersinia enterocolitica strains were tested for susceptibility to amoxicillin with 30 μg of clavulanic acid, 10 μg of ampicillin, 30 μg of cefotaxime, 30 μg of ceftazidime, 30 μg of cefalexin, 5 μg of ciprofloxacin, 30 μg of chloramphenicol, 10 μg of gentamycin, 30 μg of kanamycin, 30 μg of nalidixic acid, 10 μg of streptomycin, sulfamethoxazole/trimethoprim 19 : 1, and 30 μg of tetracycline (Oxoid, Thermo Scientific). These analyses were performed by the standard disc diffusion technique after 24 h incubation on Mueller-Hinton (Oxoid, Thermo Scientific) agar plates at 30°C, and they were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines [18 ].

Gliptins’ antivirulence and antibiofilm activities were evaluated using model strains P. aeruginosa (ATCC 27853) and S. aureus (ATCC 6538). Microbiological media, Mueller Hinton (MH) and Luria–Bertani (LB) broth or agar, and tryptone soy broth (TSB) were obtained from Oxoid (Hampshire, UK). All gliptins were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Dimethyl sulfoxide (DMSO) was used to dissolve the gliptins, and the DMSO and other chemicals were of analytical grade.

The antibacterial properties of the obtained PVA films loaded with plant extracts were evaluated by the Kirby–Bauer disk diffusion method [39 ]. Staphylococcus aureus ATCC 25923 (Gram-positive bacteria) (Thermo Fisher Scientific Inc., Dartford, UK) and Escherichia coli ATCC 25922 (Gram-negative bacteria) (Thermo Fisher Scientific Inc, Dartford, UK) were put in contact with the PVA film samples (6 mm). The protocol implied the preparation of a bacterial inoculum with 0.9% NaCl dilution and a turbidity of 0.5 on the McFarland scale (1.5 × 10⁸ bacterial cells/mL) for 24 h of cultured cells. The bacterial cultures were incorporated in a sterile Mueller–Hinton (Oxoid), melted and cooled to 45 °C. The PVA films loaded with plant extracts and paper discs impregnated with extracts (80 µL) were placed at a relatively equal distance between them onto one Petri dish with Mueller–Hinton agar, inoculated with bacteria suspensions. The plates were prepared in duplicate and incubated at 37 °C for 24 h. After the incubation, the area of the microbial inhibition zone for each sample was measured.

Forty one wild isolates of S. thermophilus were selected from among a collection of 106 previously obtained from raw milk samples by a selective procedure (Delgado et al., 2013 (link)). Lactobacillus plantarum ATCC 14917^T (= LMG 6907^T) and Enterococcus faecalis ATCC 29212 (= LMG 8222) were used as quality control strains for antibiotic susceptibility analysis. Lactobacillus delbrueckii subsp. bulgaricus LMG 6901 and CECT 4005 were used as resistance gene recipients during experimental yogurt manufacture. L. plantarum LMG 6907^T, E. faecalis LMG 8222, L. delbrueckii subsp. bulgaricus LMG 6901, and S. thermophilus LMG 9689^T were obtained from the Belgian Coordinate Collections of Microorganisms (LMG Bacteria Collection) (BCCM, University of Ghent, Belgium). L. delbrueckii subsp. bulgaricus CECT 4005 was obtained from the Spanish Collection of Microorganisms (CECT, University of Valencia, Spain). Unless otherwise stated, S. thermophilus strains were grown statically under anaerobic conditions in M17 (Oxoid, Basingstoke, UK) with 0.5% lactose (LM17) or Mueller-Hinton (Oxoid) at 37°C for 24–48 h. L. plantarum and E. faecalis were grown under aerobic conditions at 30°C in de Man Rogosa and Sharpe (MRS) broth (Merck, Darmstad, Germany). Lactobacillus delbrueckii subsp. bulgaricus strains were grown under anaerobic conditions in MRS at 37°C without shaking.