Luria bertani medium lb

S. flexneri 2a 301 (Sf301, GenBank accession number AE005674) was kindly provided by Pr. Qi Jin (MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China). The bacterial strains and plasmids used in this study are listed in Table 1. S. flexneri and E. coli were grown in Luria–Bertani medium (LB; Oxoid, Basingstoke, UK) at 37°C. Antibiotics were used at the following concentrations: ampicillin (100 μg/ml), kanamycin (50 μg/ml), tetracycline (10 μg/ml) and gentamicin (50 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA).

Clinical S. aureus isolates were obtained from a comprehensive teaching hospital in Shanghai, China (Renji Hospital, School of Medicine, Shanghai Jiao Tong University). Bacteria were identified as staphylococci by classic microbiological methods, including Gram staining, catalase and coagulase activity on rabbit plasma. S. aureus strains were further categorized by VITEK2 automated systems (BioMérieux, France). CA-SA was defined as previously described (Li et al., 2016 (link)). Isolates were obtained either from an outpatient or from an inpatient <24 h after hospital admission and lacked the following risk factors: contact with the hospital environment in the preceding 6 months, residence in a long-term care facility in the preceding 12 months, S. aureus infection in the preceding 12 months, and presence of a central vascular catheter at the time of infection. All bacterial strains and plasmids used in this study are listed in Table 1. S. aureus was grown in tryptic soy broth (TSB; Oxoid) with 0.25% glucose or on agar plates at 37°C, and Escherichia coli was routinely grown in Luria-Bertani medium (LB; Oxoid). When necessary, antibiotics were used at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 10 μg/ml. All oligonucleotides used in this study are listed in Table 2.

The bacteria and fungi were isolated from clinical specimens obtained from Asir hospital through proper channel for research. These isolated organisms were confirmed for antibiotic resistance by antibiotic sensitivity pattern and were determined by Kirby-Bauer method [23 (link)]. The EOs were individually tested against a panel of microorganisms. Two Gram-positive bacteria, Staphylococcus aureus and Streptococcus pyogenes, and two Gram-negative bacteria, Escherichia coli and Salmonella typhimurium were chosen for the study. The bacterial strains were cultivated in Luria-Bertani Medium (LB) (Oxoid Ltd, UK) at 37°C. Working cultures were prepared by inoculating a loopful of each test bacteria in 3 ml of Muller–Hinton broth (MH) (Oxoid Ltd, UK) and were incubated at 37°C for 12 h. For the test, final inoculum concentrations of 10⁶ CFU/ ml bacteria were used.
Candida albicans was cultured in Sabouraud dextrose broth (SDB) or on Sabouraud dextrose agar (SDA) (Difco, Sparks, MD, USA) for 48 h at 35°C. A standardized inoculum isolate of Candida was propagated in SDB at 35°C for 24 h with 200 rpm agitation. One ml of 24 h old culture in SDB was centrifuged (3900 rpm at 4°C for 1 min), and the pellets were washed twice with 1 ml of physiological saline. Sterile physiological saline was added to give a McFarland turbidity of 0.5 at 530 nm, corresponding to 5 × 10⁶CFU /ml).

The strains used for specificity analysis in this study are listed in Table 1. C. jejuni NCTC 11168 was used as an FQ-susceptible control. The FQ-resistant strain C. jejuni DY01, containing a C257T mutation in gyrA, was used as an FQ-resistant control. C. jejuni DY01 was isolated from a chicken farm in Hubei, China (35 (link)) and kept in our laboratory. For specificity testing, 14 reference or clinical strains, including four C. jejuni strains and 10 strains of species other than C. jejuni were used. C. jejuni and Campylobacter coli strains were grown on Bolton broth (Oxoid, Basingstoke, UK) or modified charcoal cefoperazone desoxycholate agar (mCCDA) plates containing 1% Campylobacter growth and selective supplements (Oxoid) for 48 h at 42°C in air-tight jars containing AnaeroPack (Mitsubishi, Japan) to generate microaerobic conditions. Escherichia coli and Salmonella were grown in Luria-Bertani medium (LB, Oxoid) at 37°C. Pasteurella multocida and Staphylococcus aureus were grown in tryptic soy broth medium (TSB, BD, Sparks, MD, USA) at 37°C. Clostridium perfringens was cultured anaerobically in a Brain-Heart Infusion medium (BHI, BD) at 37°C. Enterococcus faecalis was cultured in a BHI medium at 37°C.

Five E. coli O157:H7 strains and 40 non- E. coli O157:H7 strains (Table 1) were cultured in Luria-Bertani medium (LB, Oxoid, Basingstoke, UK) at 37 °C for 20 h before use. Then 10⁵ CFU/mL of strains were prepared by serial dilutions of cultures in phosphate buffered saline (PBS, Sigma Chemical Company, St. Louis, MO, USA, 0.01 M, pH 7.4). One hundred microliters of the 45 strains were tested by the Au-LFIA test strip for evaluating the specificity of the method. The non–E. coli O157:H7 strains cannot interact with the antibody-labeled AuNPs and no red line develops at the test line.

Bacteria were seeded and cultured in suspension using the following media: E. coli O157:H7, E. coli O6, S. enterica, and P. aeruginosa were cultured in Luria–Bertani medium (LB, Oxoid, Basingstoke, UK) at 37 °C for 20 h; S. aureus and E. faecalis were cultured in Brain-Heart infusion broth (Oxoid, Basingstoke, England) at 37 °C. The concentration of S. aureus was determined by serial dilution with subsequent plating on agar plates and measurement of colony forming units (CFU). The cells were then treated with 0.3% formaldehyde for 24 h. The inactivated bacteria were collected by centrifugation at 4000 rpm and resuspended in 0.01 M PBS (pH 7.4). Finally, these bacteria were serially diluted to the desired concentrations with 0.01 M PBS (pH 7.4) for further use.