Isogenic E. coli K-12 strains (see Table S1 in the supplemental material) were obtained from the Keio collection (52 (link)) or constructed using CRISPR genome editing technology (53 (link)). Plasmids and primers used for this work are listed in Table S4. E. coli strains were cultured by shaking at 200 rpm aerobically at 37°C in Luria-Bertani (LB) broth or by plating on LB agar incubated at 37°C. Yeast extract, tryptone, agar, and 5(6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) were obtained from Thermo Fisher Scientific Corp. (Waltham, MA, USA). Ciprofloxacin, ampicillin, and kanamycin were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Vancomycin, erythromycin, lysine monohydrochloride, arginine hydrochloride, histidine hydrochloride, malonate, propidium iodide, proteinase K solution, 5× protein loading dye, dimethyl sulfoxide (DMSO), and 2.2′-bipyridyl were purchased from Sangon Biotech Inc. (Shanghai, China). Lipopolysaccharide (LPS) was bought from Beyotime Biotech Inc. (Jiangsu, China). Ceftriaxone (Roche, Shanghai, China), and meropenem (Shenghuaxi Pharmaceutical Co., Chongqing, China) were gifts from the Zhongshan Hospital (Xiamen, China) pharmacy.
Erythromycin
Manufactured by Sangon
Sourced in China
Erythromycin is a macrolide antibiotic used for laboratory research purposes. It functions as an inhibitor of bacterial protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Chloramphenicol, by Sangon (6 mentions)
Vancomycin, by Sangon (4 mentions)
Tetracycline, by Sangon (4 mentions)
Kanamycin, by Sangon (4 mentions)
Ampicillin, by Sangon (3 mentions)
Clindamycin, by Sangon (3 mentions)
Ofloxacin, by Sangon (3 mentions)
Cefotaxime, by Sangon (2 mentions)
Rifampicin, by Sangon (2 mentions)
Ciprofloxacin, by Sangon (2 mentions)
Trimethoprim, by Sangon (2 mentions)
Amoxicillin, by Sangon (2 mentions)
Penicillin, by Sangon (2 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Tryptone, by Thermo Fisher Scientific (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Sangon (1 mentions)
Propidium iodide, by Sangon (1 mentions)
12 protocols using erythromycin
1
Isogenic E. coli Strain Generation
2
Antibiotic Resistance Profiling of Lactobacillus
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Following the results of the resistance gene analysis, the minimum inhibitory concentrations (MICs) of GE2270A (Adipogen, San Diego, CA, USA) were determined against L. rhamnosus X253 and L. rhamnosus GG. In addition, the MICs of other antibiotics (Sangon Biotech, Shanghai, China)—including ampicillin, chloramphenicol, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin—were determined for these two strains in accordance with the ISO 10932:2010 standard (https://www.iso.org/standard/46434.html , accessed on 30 May 2022). In 96-well plates, suspensions of the two strains were combined with antibiotics at varying doses and incubated anaerobically at 37 °C for 48 h. The optical density at 625 nm was measured using a microplate reader (Thermo Labsystems, Franklin, MA, USA). The threshold values for resistance to each antibiotic were derived from the European Food Safety Authority (EFSA) [32 ].
3
Bacterial Cultivation and Antibiotic Use
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This study complies with all relevant ethical regulations and the bacterial strains and plasmids used in this study are listed in Table S2 . Unless otherwise noted, S. aureus strains were grown in tryptic soy broth (TSB, Difco) at 37 °C with shaking (250 rpm), or on tryptic soy agar (TSA, Difco) at 37 °C. Escherichia coli strains were grown in Luria-Bertani (LB) broth at 37 °C with shaking (250 rpm) or on LB agar plates at 37 °C. For plasmid maintenance, antibiotics were used at the following concentrations where appropriate: for S. aureus, erythromycin (Sangon Biotech) at 10 μg/ml for RN4220 and 80 μg/ml for USA300 LAC and its derivatives, chloramphenicol (Sangon Biotech) at 15 μg/ml; for E. coli, carbenicillin (Sangon Biotech) at 150 μg/ml. For other reagents, tunicamycin and vancomycin (Dalian Meilun Biotech Co., Ltd.), targocil (Shanghai TopScience Co, Ltd.), oxacillin (Shanghai Aladdin Biochemical Technology Co.,Ltd.), imipenem, cefuroxime, ceftizoxime, cefaclor, cefoxitin, and epicatechin gallate from MedChemExpress, cefotaxime, isopropyl β-D-1-thiogalactopyranoside (IPTG), and AIP from Sangon Biotech, anhydrotetracycline (aTc) from APExBIO, Tarocin A1 from Merck, moenomycin complex from GlpBio, and nile red from Yeasen Biotechnology Co., Ltd. were used in this study.
4
Antibiotic Resistance Profiling of L. sakei
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The predicted antibiotic resistance gene information in the genome was obtained by comparing the amino acid sequences of the strains with the comprehensive antibiotic research database (CARD, http://arpcard.mcmaster.ca , accessed on 24 May 2021) [51 (link)]. The strains were clustered using HemI software [50 (link)].
The microbroth dilution method was used to determine antibiotic resistance of L. sakei according to ISO 10932:2010 [52 ]. The following 11 antibiotics were detected: chloramphenicol, rifampicin, streptomycin, kanamycin, gentamycin, tetracycline, clindamycin, neomycin, erythromycin, ciprofloxacin, and vancomycin (all purchased from Sangon Biotech Co., Ltd., Shanghai, China). OD625 was determined using an enzyme-labeled instrument (Varioskan Lux, Thermo, Waltham, MA, USA) to determine the MIC of strain to antibiotics.
The microbroth dilution method was used to determine antibiotic resistance of L. sakei according to ISO 10932:2010 [52 ]. The following 11 antibiotics were detected: chloramphenicol, rifampicin, streptomycin, kanamycin, gentamycin, tetracycline, clindamycin, neomycin, erythromycin, ciprofloxacin, and vancomycin (all purchased from Sangon Biotech Co., Ltd., Shanghai, China). OD625 was determined using an enzyme-labeled instrument (Varioskan Lux, Thermo, Waltham, MA, USA) to determine the MIC of strain to antibiotics.
5
Bacterial Strain Culture Conditions
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Bacterial strains and plasmids used in this study are listed in Supplemental Table S1 (https: / / doi .org/ 10 . 6084/ m9 .figshare .14890167 .v1; Song, 2021a) . Escherichia coli TOP10 was used as a host for molecular cloning, grown aerobically at 30°C in Luria-Bertani medium (Oxoid) if carrying the pLL-derived plasmids. We cultured L. lactis NZ9000 and its mutants in M-17 medium supplemented with 0.5% (wt/vol) glucose (GM17) at 30°C without shaking (de Ruyter et al., 1996) (link). The L. lactis was grown in semi-defined medium (Kimmel and Roberts, 1998) (link) to explore resistance to 5-fluorouracil (added at 50 µg/mL; Sangon). The antibiotics were supplemented at following concentrations when required: 50 µg/mL kanamycin (Sangon) for E. coli and 10 µg/mL erythromycin (Sangon) for L. lactis NZ9000.
6
Antibiotic Resistance Profiling of Bacterial Isolates
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The bacteria were streaked on separate MRS agar plates containing 200 μg/mL erythromycin (Sangon Biotech, Shanghai, China), and the plates were incubated in an anaerobic workstation at 37°C. After 2 days of culture, single colonies of bacteria were separately activated for two generations in MRS liquid medium containing 200 μg/mL erythromycin at 37°C for 16 h. Subsequently, a 3-5% (v/v) inoculum from an overnight culture was inoculated into 10 mL MRS liquid medium containing 0, 0.5, 2, 16, and 256 μg/mL of vancomycin.
7
Profiling antibiotic resistance in Bifidobacterium bifidum
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B. bifidum genomes were aligned against sequences from the latest version of the Comprehensive Antibiotic Resistance Database [39 (link)], and a conservative threshold (amino acid identity ≥ 30%, comparison hit-bit score ≥ 37.0) was used to predict putative ARGs.
The antibiotic susceptibility of the B. bifidum strains was evaluated using the broth microdilution method, according to ISO 10932:2010 [40 ]. The following 10 antibiotics were tested: tetracycline, erythromycin, clindamycin, ampicillin, amoxicillin, trimethoprim, ciprofloxacin, chloramphenicol, rifampicin, and vancomycin (all purchased from Sangon Biotech Co., Ltd., Shanghai, China). The microbiological breakpoints of Bifidobacterium recommended by the European Food Safety Authority were used to distinguish susceptible strains from resistant strains.
The antibiotic susceptibility of the B. bifidum strains was evaluated using the broth microdilution method, according to ISO 10932:2010 [40 ]. The following 10 antibiotics were tested: tetracycline, erythromycin, clindamycin, ampicillin, amoxicillin, trimethoprim, ciprofloxacin, chloramphenicol, rifampicin, and vancomycin (all purchased from Sangon Biotech Co., Ltd., Shanghai, China). The microbiological breakpoints of Bifidobacterium recommended by the European Food Safety Authority were used to distinguish susceptible strains from resistant strains.
8
Antibiotic Resistance in S. epidermidis
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Referred study and our pre-experiments show that S. epidermidis can be isolated from the human intestine tract and colostrum, and drug resistance is high [23 (link)]. S. epidermidis was selected for traceability of ARGs because of its high abundance, isolation and resistance rate in the samples. S. epidermidis from different sources was tested for resistance to 10 antibiotics using the K-B method or by determining the minimal inhibitory concentration according to the standards issued by CLSI in 2012 (https://clsi.org/ ). The 10 antibiotics were ampicillin, cefotaxime, penicillin, tetracycline, erythromycin, kanamycin, vancomycin, ofloxacin, chloramphenicol and trimethoprim (Sangon Biotech, Shanghai, China).
9
Bacterial Hemolysis and Enzyme Assays
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Hemolysin production was detected using Columbia agar plates supplemented with 5% of sheep blood (Amersco, Solon, OH, USA). The presence of α or β-hemolysis was assessed by the formation of clear or greenish zones around the colonies, respectively.
Enzyme activity was measured using the commercially-available, semi-quantitative API-ZYM system (BioMérieux, Montreal, QC) as previously described. According to the manufacturer’s instructions, cell suspension was adjusted to McFarland standards 5. Then 65 μL of cell suspension were added into each well of the API-ZYM strip and were incubated at 37 °C for 4 h in anaerobic conditions. The results were graded based on the amount of from substrate hydrolyzed on a scale from 0 (no activity) to 40 (or ≥ 40 nM).
For antibiotic susceptibility testing, LAB strains (108 CFU/mL) were inoculated onto MRS soft agar. Commercial antibiotic discs (amoxicillin, penicillin, tetracycline, erythromycin, gentamicin, clindamycin, and ofloxacin, provided by Sangon Biotech (Shanghai) Co., Ltd) were placed onto the agar and incubated at 37 °C for 24 h. Resistance or sensitivity was assessed according to the CLSI/NCCLS standard.
Enzyme activity was measured using the commercially-available, semi-quantitative API-ZYM system (BioMérieux, Montreal, QC) as previously described. According to the manufacturer’s instructions, cell suspension was adjusted to McFarland standards 5. Then 65 μL of cell suspension were added into each well of the API-ZYM strip and were incubated at 37 °C for 4 h in anaerobic conditions. The results were graded based on the amount of from substrate hydrolyzed on a scale from 0 (no activity) to 40 (or ≥ 40 nM).
For antibiotic susceptibility testing, LAB strains (108 CFU/mL) were inoculated onto MRS soft agar. Commercial antibiotic discs (amoxicillin, penicillin, tetracycline, erythromycin, gentamicin, clindamycin, and ofloxacin, provided by Sangon Biotech (Shanghai) Co., Ltd) were placed onto the agar and incubated at 37 °C for 24 h. Resistance or sensitivity was assessed according to the CLSI/NCCLS standard.
10
Antimicrobial Susceptibility Testing Protocol
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According to the requirements of the Clinical and Laboratory Standard Institute (CLSI ), the antimicrobial susceptibility changes of the strains were detected by using Mueller–Hinton (MH ) (hopebiol, Qingdao, China) medium dilution antimicrobial susceptibility tests. The concentration that completely inhibits the growth of the strain is defined as the minimum inhibitory concentration (MIC ). The experiment was repeated 3 times.The stock solutions of the following antibiotics were prepared, and their classes are listed in parentheses: ofloxacin (fluoroquinolone, Sangon) at 20 mg/mL; erythromycin (macrolide, Sangon) at 30 mg/mL; Kanamycin (aminoglycoside, Sangon) at 50mg/mL; penicillin G (β-lactams, Sangon) at 100mg/mL.
Based on previous methods (Yu et al., 2020 (link)), the antibiotic susceptibility of the strains was determined by antibacterial activity assays. The overnight cultured strains were diluted into fresh MH containing 30 μg/mL chloramphenicol and incubate in 37°C for 2 h with shaking. After incubation, different antibiotics (final concentration 1/2MIC) were added separately and incubate for another 2 h. Then, they were diluted continuously by 10-fold and three appropriate dilutions (0.1 mL) were dropped onto LB agar plates and colony counts (CFU/mL) were counted after incubation at 37°C. The experiments were repeated 3 times.
Based on previous methods (Yu et al., 2020 (link)), the antibiotic susceptibility of the strains was determined by antibacterial activity assays. The overnight cultured strains were diluted into fresh MH containing 30 μg/mL chloramphenicol and incubate in 37°C for 2 h with shaking. After incubation, different antibiotics (final concentration 1/2MIC) were added separately and incubate for another 2 h. Then, they were diluted continuously by 10-fold and three appropriate dilutions (0.1 mL) were dropped onto LB agar plates and colony counts (CFU/mL) were counted after incubation at 37°C. The experiments were repeated 3 times.
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