Columbia blood agar base

At each sampling time (T0, T1, and T2), vaginal discharge samples were collected, using swabs filled with the transport medium Transystem Amies Clear (Biolife, Milan, Italy), and were subjected to culture-dependent analysis according to Pino and co-workers (26 (link), 27 (link)). In detail, after dislodging bacterial cells in sterile phosphate-buffered saline (PBS), 10-fold dilutions were made and plated using the following agar media and conditions: Rogosa SL agar (Biolife, Milan, Italy) for Lactobacillus counts, incubated at 35–37 °C for 40–48 h; Streptococcus Selective Agar (Biolife, Milan, Italy) for the enumeration of streptococci, incubated at 32 °C for 24 h; Columbia Blood Agar base (Oxoid, Milan, Italy), supplemented with Gardnerella Vaginalis Selective Supplement (Oxoid, Milan, Italy), incubated at 37 °C for 40–48 h for Gadnerella spp. count; MacConkey Agar Mug (Biolife, Milan, Italy) incubated at 37 °C for 16–18 h for Escherichia coli; Mannitol Salt Agar (Oxoid, Milan, Italy) incubated at 32 °C for 48 h for the count of staphylococci; Slanetz Bartley Agar (Biolife, Milan, Italy) incubated at 37 °C for 48 h for enterococci; and Chromogenic Candida Agar (Biolife, Milan, Italy), incubated at 35–37 °C for 18–48 h, for the count of Candida spp.
Microbiological count was performed in triplicate and results were reported as mean log cfu/ml and standard deviation.

Samples were plated on Blood Agar Base and Columbia Blood Agar Base with nalidixic acid and colistin sulfate (Oxoid ltd., Hampshire, UK), supplemented with 5% sterile defibrinated sheep blood and incubated both in aerobic and microaerophilic (5% CO₂) conditions at 37 °C for 48 h. Colonies were selected and identified as previously described [7 (link)]. Further biochemical identification was performed using commercial identification galleries (API®Coryne and API®20Strep, bioMérieux, Marcy-l’Etoile, France) according to manufacturer’s instructions. Isolates were identified as a species only if identification scores in the multi-substrate identification systems were excellent, very good or good (90.0–99.9% ID); otherwise, identification was made only at the genus level (spp.). Latex agglutination test (Streptococcal grouping kit, Oxoid ltd, Hampshire, UK) and Christie Atkins Munch-Petersen test (CAMP test) were used for identification according to previous reports [30 (link)]. Pure cultures of each isolate were stored at − 70 °C.

The Helicobacter pylori (H. pylori) American Type Culture Collection (ATCC) 43504 (cagA⁺, vacA⁺) was purchased from the BeNa Culture Collection, Henan, China. H. pylori was grown on Columbia Blood Agar Base (Oxoid, Basingstoke, Hampshire, UK) plates containing 7% defibrillated sheep blood (njbianzhen, Nanjing, Jiangsu, China) in a humidified environment. Moreover, its liquid culture was carried out in BactoTM Brain Heart Infusion (BD Difco, Sparks, MD, USA) supplemented with 10% newborn calf serum (NBCS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Plates and liquid cultures were maintained at 37°C in the microaerophilic environment (5% O₂, 10% CO₂, and 85% N₂). Escherichia coli TOP10 was used for plasmid amplification, and Rosetta-gami (DE3) pLysS (Fineyoungbio, Guangzhou, China) was used for protein expression. These were grown on Luria-Bertani (LB) agar (Solarbio, Beijing, China) or LB broth (Solarbio, Beijing, China) containing Ampicillin (100 μg/ml) at 37°C.

The schematic representation of the methodology is given in Figure 1. For confirmation of Helicobacter pylori infection, fresh biopsy samples from patients with gastric cancer (n = 35), gastric ulcer (n = 53), and gastritis (n = 62) were collected. About 4 mm of tissue samples from the antrum of the stomach were taken in a sterilized Petri plate and cut into pieces using a sterile scalpel blade. Biopsy samples were cultured on Columbia blood agar base (Oxoid, Hampshire, UK), supplemented with 5% sheep blood, using DENT (Oxoid, Hampshire, UK) and CampyGen sachet (Thermo Fischer Scientific, Warrington, UK) and was morphologically and phenotypically identified. Biochemical identification was carried out using oxidase, catalase, and urease tests [23 (link)]. The findings were confirmed through PCR targeting species-specific 16S rRNA genes using primers (F-5′-GCGACCTGCTGGAACATTAC-3′), R (5′-CGTTAGCTGCATTACTGGAGA-3′).

Clinical specimens were grown aseptically on Columbia blood agar base supplemented with 5% of defibrinated sheep blood (Oxoid), chocolate blood agar (GC II agar with IsoVitaleX, BD) and MacConkey agar (Biolife). All plates were incubated at 35–37 °C in aerobic conditions with 5–10% CO₂ for 24–48 h. Identification of isolates was carried out by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass espectrometry (Biotyper System, Bruker Daltonics, Bremen, Germany) as previously described [24 (link)].

Columbia blood agar base, brain heart infusion (BHI), Mueller–Hinton (MH) agar, and H. pylori selective supplement (Dent) SR0147E were obtained from Oxoid, United Kingdom. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), MEM nonessential amino acids, TRIzol reagent, and Live/Dead BacLight Bacterial Viability kits (Molecular Probes) were purchased from Thermo Fisher Scientific, United States. Sheep blood was procured from Pingrui Biotechnology, China. CLR was purchased from Dalian Meilun Biotechnology, China. MTZ and saponin were acquired from Sigma, Germany. Fastking gDNA Dispelling RT SuperMix Kit, Talent qPCR PreMix (SYBR Green) Kit, TIANamp Bacteria DNA kit, and 2 × Taq PCR Mix were purchased from TIANGEN, China. Anti-H. pylori antibody ab20459 was obtained from Abcam, United Kingdom. Alexa Fluor 488 and goat anti-rabbit were bought from Southern Biotech, United States.

A total of 10 clinical Staphylococcus aureus strains (9 MRSA and one MSSA) and S. aureus ATCC 25923 reference strains stored at −80 °C in Brain Heart Infusion (BHI) Broth with 50% glycerol were cultivated on Columbia Blood Agar Base (Oxoid) supplemented with 7% defibrinated sheep blood and incubated at 35 ± 2 °C for 18–24 h. Clinical S. aureus strains have been isolated in a previous study from patients with SSTIs and referred for laboratory investigations and research to “Cantacuzino” National Institute Reference Laboratory⁴ . All subjects provided written informed consent. Clinical strains have been isolated using a protocol approved by the Local Ethics Committee of Cantacuzino Institute (no. CE/31/06.05.2014). All methods used in this study were performed in accordance with the relevant guidelines and regulations in this research field.
The taxonomic affiliation of all 10 isolates has been confirmed by a combination of phenotypic and genotypic tests: bound and free coagulase and PCR for the nuc gene detection⁴ .
Peptide P6 synthesis and characterization were reported in our previous study, and we prepared the peptide solution from the initial batch synthetized^{20 (link)}. All cell cultivation media and reagents were purchased from Biochrom AG (Berlin, Germany). Mueller-Hinton broth, Mueller-Hinton agar and saline solution were purchased from Merck KGaA (Darmstadt, Germany).