E faecalis atcc 29212

The bacterial strains S. aureus ATCC 29213, S. aureus ATCC 29247, S. aureus ATCC BAA-41, E. faecium ATCC 49624, E. faecium ATCC 700221, E. faecalis ATCC 29212, S. epidermidis ATCC 12228, E. coli ATCC 25922, and K. pneumoniae ATCC BAA-1144 used in the antimicrobial susceptibility assays were purchased from American Type Culture Collection (ATCC, USA). B. subtilis strain 168 was already available in our in-house collection. E. coli JM109 WM647 was kindly provided by Dr. W. Margolin (University of Texas-Houston Medical School). FM 4–64 was purchased from Invitrogen (Eugene, Oregon). All other chemicals and reagents were purchased from Sigma-Aldrich. Stock solutions were prepared in DMSO. The final percentage of DMSO in the assays was 1% (v/v) for all experiments.

A total of
54 S. aureus and 4 E.
faecalis clinical isolates were used in this study,
and these isolates were collected from Shenzhen Nanshan People’s
Hospital (Grade A, level III Hospital, 1500 beds) between January
1, 2019, and December 31, 2021. All clinical isolates were identified
with a Phoenix 100 automated microbiology system (BD, Franklin Lakes,
NJ, USA) and were re-identified with matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (IVD MALDI Biotyper, Germany).
The S. aureus ATCC29213, S. aureus SA113 (ATCC35556), and E.
faecalis ATCC29212 were used as reference strains
and were purchased from American Type Culture Collection (ATCC).
All the strains were grown in tryptic soy broth (TSB) at 37 °C
with shaking of 180 rpm unless otherwise stated. For the antimicrobial
susceptibility test and time-killing assay, strains were grown in
a cation-adjusted Mueller–Hinton broth (CAMHB) at 37 °C
with shaking. Strains were grown in TSBG (TSB with 0.5% glucose) at
37 °C for biofilm assay.

E. faecalis ATCC 29212 and C. albicans ATCC 10231 were purchased from the American Type Culture Collection (ATCC). Microorganisms were stored at −20 °C in 5% glycerol. Unfrozen microbial cells were transferred to Brain Heart Infusion broth (BHI, Kasvi, Brazil) and incubated overnight at 37 °C. Then, one loop of the culture was transferred to Mitis Salivarius agar (Sigma-Aldrich, St. Louis, MO, USA) and Sabouraud agar (Kavsi, Brazil) for E. faecalis and C. albicans, respectively. Cultures were incubated for up to 18 h at 37 °C.

The Gram-positive S. aureus ATCC 6538, E. faecalis ATCC 29212 and B. subtilis ATCC 6633 and the Gram-negative P. aeruginosa ATCC 27853 and E. coli ATCC 8739 were purchased from American Type Culture Collection (ATCC, Virginia, USA). Glycerol stocks were streaked on LB agar to obtain 24-h cultures to be used for all further studies.
Monospecific biofilm development was assessed at two different times of exposure, i.e., 15 min and 24 h. The textile materials were cut in equal circular samples of 8 mm and sterilized by autoclaving at 121 °C for 15 min. The sterile samples were then immersed in 1 mL of microbial suspensions of ~10⁷ colony forming units (CFU)/mL performed in sterile saline and left in contact for 15 min and 24 h, respectively. After this interval, microbial suspensions incubated with the tested samples were vortexed and further serially ten-fold diluted, and 10 µL of each serial dilution were plated in triplicate on LB agar. After 24 h of incubation at 37 °C, viable cell counts were performed, and the number of CFU/mL for each sample was established.

L. plantarum KCTC10887BP was obtained from the Korean Collection for Type Culture (Daejeon, Korea). S. aureus ATCC 29213, Streptococcus pneumoniae ATCC 27336, E. faecalis ATCC 29212, and B. subtilis ATCC 6633 were obtained from the American Type Culture Collection (Manassas, VA, United States). Streptococcus gordonii CH1 was obtained from Professor Paul M. Sullam (University of California at San Francisco). Vibrio harveyi BB170 was obtained from Professor Bong-Kyu Choi (Seoul National University, Seoul, Korea). S. aureus RN4220 was obtained from Professor Bok Luel Lee (Pusan National University, Pusan, Korea). Clinical isolates of S. aureus NCCP14780 and MRSA NCCP14769 were obtained from the National Culture Collection for Pathogens (Osong, Korea). USA300, USA300ΔluxS, and USA300ΔagrA were obtained from the Nebraska Transposon Mutant Library (Omaha, NE, United States). The Texas Red conjugate of concanavalin A (Texas Red-Con A) and LIVE/DEAD Baclight Bacterial Viability Kit were purchased from Molecular Probes (Eugene, OR, United States). Proteinase K and octyl-sepharose beads were purchased from Sigma–Aldrich Chemical Inc. (St. Louis, MO, United States). DNase I was purchased from Roche Molecular Biochemicals (Laval, QC, Canada).

Antimicrobial susceptibility of all 542 Enterococcus spp. was determined using the Kirby–Bauer disc diffusion method on Mueller Hinton II Agar (MHA, Becton Dickinson, Franklin Lakes, NJ, USA) according to the EUCAST (European Committee on Antimicrobial Susceptibility Testing) disc diffusion method ver. 9.0 recommendations [55 ]. The following antibiotic discs were used: ampicillin (2 μg), imipenem (10 μg), norfloxacin (10 μg), vancomycin (5 μg), teicoplanin (30 μg) and linezolid (10 µg) (OXOID, Basingstoke, UK). After 16–18 h of incubation at 35 °C, under aerobic conditions, inhibition zones around the discs were measured. The results were interpreted according to the EUCAST ver. 11.0 breakpoint tables [56 ]. The study included reference strain E. faecalis ATCC^® 29212™ from the American Type Culture Collection (ATCC^®).
The presence of resistance mechanisms was assessed using the disc diffusion method, determining the resistance to selected antibiotics, according to the EUCAST ver. 11.0 breakpoint tables [56 ]. The assessed isolates were considered GRE if they showed resistance to both glycopeptide antibiotics used in the study (vancomycin and teicoplanin), while the strains showing resistance to vancomycin with simultaneous sensitivity to teicoplanin were considered VRE. Enterococcal strains resistant to linezolid were considered LRE.

A total of ten E. faecalis strains were used in this study. E. faecalis ATCC 29212 was acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). Other nine E. faecalis strains (Table 1) were originally isolated from the chicken cloacal swab, swine nasal swab, bovine breast swab, and raw milk. In MIC and MBC testing, all ten E. faecalis strains were employed, while E. faecalis ATCC 29212 was used in all subsequent investigations. For the use in assays, E. faecalis strains stored were streaked on BHI agar and cultivated for 24 h at 37 °C. After that, the colony was inoculated into 30 mL BHI broth and cultured at 37 °C for 16 h.