Erythromycin

B. cereus BC1 and its derivatives were cultured in tryptic soy broth (TSB) at 37 °C, 200 rpm, or on nutrient agar plates at 37 °C in standing culture. E. coli strains were grown in Luria–Bertani (LB) medium at 37 °C. The following concentrations of antibiotics were added when needed: 10 μg/mL erythromycin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) 50 μg/mL kanamycin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for B. cereus growth, and 100 μg/mL ampicillin (Shanghai Boer Chemical Reagents Co., Ltd., Shanghai, China) for E. coli growth. The strains and plasmids used in this work are listed in Table S1. Primers are listed in Table S2.

Plasmids including pMG36e-RFP-EGFP (358), pMG36e-EGFP-mcherry, pMG36e-penp-OVA, pMG36e-CMV-penp-OVA and pNZ8149-penp-OVA were constructed and kept by Xinjiang Key Laboratory of Biological Resources and Genetic Engineering. pMG36e-RFP-EGFP (358) encodes a green fluorescent protein (EGFP) under the control of a eukaryotic promoter CMV and a red fluorescent protein (RFP) under the control of a prokaryotic promoter penp. pMG36e-EGFP-mcherry encodes EGFP-mcherry under the control of a prokaryotic promoter penp. pMG36e-penp-OVA encodes OVA protein under the control of penp. pMG36e-CMV-penp-OVA encodes OVA protein under the control of CMV and penp. pNZ8149-penp-OVA encodes OVA protein under the control of penp. The L.L was cryopreserved in this laboratory. L.L and recombinant L.L carrying pMG36e were grown statically at 30°C in M17 broth supplemented with 10% glucose (GM17), recombinant L.L of pMG36e was grown in GM17 with 1 mg/ml erythromycin (Solarbio, China). Recombinant L.L carrying pNZ8149 was grown statically at 30°C in M17 broth supplemented with 10% lactin, recombinant L.L of pNZ8149 were grown in M17 with lactin and 50 μg/ml ampicillin (Solarbio, China).

The 10 PPCP standards, namely, sulfadiazine (SDZ), sulfamethoxazole (SMZ), tetracycline (TC), oxytetracycline (OTC), ciprofloxacin (CIP), ofloxacin (OFX), erythromycin (ERY), roxithromycin (ROX), ibuprofen (IBU), and NAX, were purchased from Solar-bio (China). Each PPCP was added into the feedwater at 200 ng/L. PPCPs were first dissolved with methanol and then added to the feedwater. The feedwater and PPCPs were prepared daily and mixed thoroughly in the feed tank. PPCPs were detected using the Waters ACQUITY UPLC H-class-Xevo TQ MS triple quadrupole MS/MS spectrometer equipped with an electrospray ionization source (Waters, USA). The detailed detection process was referred to our previous publication^{3 (link)}.

The standard disk diffusion assay was used to determine the sensitivity or resistance of LAB to conventional antibiotics. Paper disks containing ampicillin (10 μg), penicillin (10 μg), amoxycillin (10 μg), norfloxacin (10 μg), levofloxacin (5 μg), gentamicin (120 μg), streptomycin (10 μg), amikacin (30 μg), and erythromycin (15 μg), which were purchased from Solarbio Technology Co., Ltd. (Beijing, China), were employed for the antibiotic resistance tests (Lee et al., 2014 (link)). From the MRS broth culture of each one of the test strains, 100 μL was mixed with 8 mL of liquid MRS agar, over-layered on a pre-solidified agar plate and allowed to solidify, and then disks were aseptically placed onto the center of plates using sterile forceps. The plates were incubated for 48 h at 30°C in an anaerobic chamber. The results were recorded according to the interpretive category defined by the Clinical and Laboratory Standards Institute (CLSI) (Sharma et al., 2017 (link)). The tests were carried out in triplicate.

The L. monocytogenes isolate used, with designated strain name “100”, was previously isolated from a milk sample. Previous experiments in our laboratory identified this as a serotype 1/2a strain with strong biofilm and suspended aggregate formation ability. R. insidiosa ATCC 49129 was purchased from the American Type Culture Collection (ATCC). Both strains were stored at −80 °C in tryptic soy broth (TSB; Hope Bio-Technology, Qingdao, China) containing 40% glycerol. Before all experiments, frozen cells were activated and separated on tryptic soy agar plates (TSA; Hope Bio-Technology, Qingdao, China) for 24 h at 37 °C. Then one colony was transferred into TSB overnight at 37 °C, and the cell concentration was adjusted to 10⁸ colony-forming units (CFU)/mL. The concentration of bacteria used in each experiment was 10⁸ CFU/mL, unless otherwise specified. E. coli DH5α (Takara Bio Inc., Otsu, Shiga, Japan) was used as the standard plasmid host for cloning procedures and was grown in lysogeny broth (LB; Hope Bio-Technology, Qingdao, China) broth and agar. Ampicillin (100 μg/mL) (Biofroxx, Einhausen, Germany) or erythromycin (3 μg/mL) (Solarbio, Beijing, China) were added to agar media or broth as required.

Nine antibiotics were used in the present study. Tetracycline (active ingredient 95.0%, dissolvent: ddH₂O, TET), ampicillin (USP grade, d: 1 mol/L HCl, AMP), ciprofloxacin (a.i. 90.0%, d: 1 mol/L NaOH, CPFX), sulfadiazine (a.i. 98.0%, d: DMSO, SUD), amikacin (a.i. 67.4%, d: ddH₂O, AMI), oxyTetracycline (a.i. 95%, d: 0.1 mol/L HCl, OTC), kanamycin (a.i. 75.0%, d: ddH₂O, KAN) and erythromycin (a.i. 94.1%, d: ddH₂O, EM) were kindly provided by Beijing Solarbio Science & Technology Co., Ltd., Beijing, China; rifampicin (a.i. 98.0%, d: DMSO, RFD) was generously provided by Wuhan Pytbio Bioengineering Co., Ltd., Wuhan, China. The above antibiotics were dissolved and diluted with the corresponding solvents to 50 mg/mL, a stock solution was prepared, and the mixture was stored at 4 °C for subsequent dilution in this study. Additionally, tryptone soybean agar medium (TSA) was used for the isolation and purification of bacteria; nutrient broth agar medium (NA) was used for the propagation of bacteria; 15% glycerol–nutrient broth medium (15% glycerol–NB) was used for the cryopreservation of bacteria; 0.3% agar–NB was used for the determination of bacterial motility; and Luria–Bertani medium (LB) and Luria–Bertani agar medium (LA) were used for the determination of bacterial resistance to antibiotics.