Bhi broth

Antisepsis of the patient’s face with 0.2% chlorhexidine was performed before endodontic surgery, and local anesthesia was administered in the region of the involved tooth. Before being isolated completely, the tooth was prophylaxed with pumice and Robson’s brush. After that, the surgical field was disinfected for 30 s with sterile swabs dampened with 2.5% sodium hypochlorite (NaClO). The coronary opening was performed in high rotation with a spherical diamond tip and sterile saline refrigeration. Using sterile paper cones compatible with the canal’s anatomical diameter, the biological material was collected immediately after the coronary opening and before the canal’s toxic septic content was neutralized.^{17 (link)}
To allow biological material absorption, paper cones were introduced close to the total length of the root canal as determined by diagnostic radiography, touching the internal walls and remaining in a static position for 1 min. When the RCS was dry, the cones were moistened with sterile saline solution (0.9% NaClO) before collection to ensure the collection of a viable sample, and multiple cones were collected to increase the chances of containing microorganisms. This process was repeated three times to obtain a more diverse microbiological sampling, totaling three paper cones collected for each sample. The same endodontic specialist performed all collections.
The cones were immediately removed from the root canal and placed in sterile 2-mL cryotubes containing 1.7 mL of brain heart infusion (BHI) broth (Acumedia, USA). The tubes were homogenized, refrigerated, and transported to the microbiology laboratory within 24 h for bacterial culture and isolation.

Cooked turkey ham samples were purchased at the local market in Rio de Janeiro (Brazil) and taken immediately to the analysis laboratory. The turkey ham slices were cut into a mold with the same diameter as the film (90 mm). Based on the overall results, BM film and BM/ZnO-0.5 film were selected for antimicrobial evaluation. A control treatment (C) of turkey ham samples was used.
Strains of Staphylococcus aureus (ATCC 13565) were activated in Brain Heart Infusion (BHI) broth (ACUMEDIA, MI, USA) with incubation for 24 h at 35 °C. After activation, bacterial colonies were isolated on Baird–Parker Agar (KASVI, Spain) supplemented with egg yolk tellurite (MILLIPORE, Burlington, VT, USA) with incubation for another 24 h at 37 °C. Colonies of isolated bacteria were transferred to test tubes containing 5.0 mL of 0.85% (w/v) sterile saline solution. Turbidity was compared to a standard barium sulfate solution equivalent to the 0.5 MacFarland scale, corresponding to an approximate concentration of 8 log CFU mL⁻¹ based on the Clinical and Laboratory Standards Institute guidelines (CLSI, 2006) [28 ].
The bacterial contamination assay was adapted using the method described by Dutra et al. (2022) [29 (link)]. In sterilized trays, 90 μL of a S. aureus suspension diluted to 6 log CFU ⁻¹ was inoculated individually onto the turkey ham slices (10 g). The cultures were spread individually on the surface of the turkey ham samples with a sterile Drigalski spatula and allowed to adhere to the samples for 15 min in a bacteriological cabinet. After inoculation, the films were placed in contact with the turkey ham and the samples were stored at 7 °C.
Microbiological analyzes were performed immediately after inoculation (0D) and on days 2 (2D), 6 (6D), and 7 (7D) of refrigerated storage, aiming to simulate the average shelf life of the product, which is approximately one week under refrigeration [30 (link)]. Approximately 10 g of turkey ham samples from treatments C, BM, and BM/ZnO-0.5 were transferred to 90 mL of a 0.85% (w/v) saline solution in sterile sample bags. After homogenization in a Stomacher (Sample Mixer Stomacher, Stomax, São Paulo, Brazil) for 2 min, the solution was diluted in decimal series (10⁻¹ to 10⁻⁴) and plated in duplicate on Baird–Parker Agar with egg yolk tellurite. The plates were incubated at 37 °C for 24 h. Results were expressed as log CFU g⁻¹. S. aureus was also evaluated on the day of processing in samples of inoculated turkey ham slices.

Bacterial strains and plasmids used in this study are listed in S1 Table. We inoculated E. faecalis from single colonies and grew cells statically at 37°C in Brain Heart Infusion (BHI) broth (Acumedia, USA) or agar (1.5%, Difco, US) for all assays unless otherwise stated. E. coli was grown in Luria Bertani (LB) broth (Difco, US) with shaking, or on agar plates at 37°C for DNA isolation and manipulation. To stimulate pilus expression in E. faecalis, strains were grown statically in Trypticase Soy Broth (Oxoid, UK) supplemented with 0.25% glucose (TSBG) and incubated at 37°C [2 (link)]. We used Müller Hinton (MH) broth and 1.5% agar to perform antibiotic susceptibility assays. All inoculations were cultured for 15 to 18 hours unless otherwise stated. When required, antibiotics were added at the following concentrations: for E. coli, Kanamycin (Km) 50 mg/L or Erythromycin (Em) 500 mg/L; for E. faecalis strains, Em 25 mg/L, Km 500 mg/L, Rifampicin (Rif) 25 mg/L, Chloramphenicol (Cm) 10 mg/L. All antibiotics were purchased from Sigma-Aldrich Corporation, USA.

F. nucleatum ATCC 23726 was obtained from the American Type Culture Collection, USA. F. nucleatum was cultured in brain heart infusion (BHI) broth (Acumedia, USA) supplemented with 0.5% yeast extract (Acumedia), 5 μg/mL hemin and 1 μg/mL vitamin K (Sigma-Aldrich, USA), and incubated at 37°C in an anaerobic chamber supplemented with 80% N₂, 10% H₂, and 10% CO₂ (Don Whitley Scientific, USA).

The investigators providing the submissions listed in Table 1 reported serotype. In this laboratory, cultures were streaked on brilliant green (BG) agar (Acumedia; Neogen Corporation, Lansing, MI) and incubated for 24–48 h at 37 °C to obtain well-separated large colonies. One colony was then transferred to brain heart infusion (BHI) broth (Acumedia) and incubated for 16 h at 37 °C with shaking. For submissions that later appeared to have mixed cultures and for those with disagreement between methods, agglutination reactions for single colonies using commercially available absorbed antisera (Difco, BD, Franklin Lakes, NJ) were carried out, and in some cases, isolates were submitted for serotyping (Silliker, South Holland, IL). Thus, single colonies were processed by both ISR and DNAhyb (Check & Trace, Check Points, Wageningen, the Netherlands). In cases of disagreement between methods, a maximum of 10 well-isolated colonies were selected from agar plates and then transferred to BHI broth for individual analysis.

The strains used in this work were obtained from the sources listed in Appendix␣Table S3. The three clinical C. difficile isolates (MS002, MS010, and MS011) were C. difficile NAAT (GeneXpert) positive via admission stool sample and toxin A (tcdA) and toxin B (tcdB) positive via in‐house research PCR. Each patient was diagnosed with and treated for CDI. Single‐use glycerol stocks were prepared as described previously (Clark et␣al, 2021 (link)). The media used in this work are anaerobic basal broth (ABB, Oxoid), clostridial reinforced medium (CRM, Difco), YP broth (Geva‐Zatorsky et␣al, 2015 (link)), and YBHI. YBHI broth recipe: BHI broth (Accumedia), 5 g/l yeast extract (BD Bacto), 1 g/l d‐cellobiose (Chem‐Impex), 1 g/l d‐maltose monohydrate (Sigma‐Aldrich), and 0.5 g/l l‐cysteine (Sigma‐Aldrich).