L. plantarum WCFS1 was used as the parent strain in this study. The strain was maintained in the De Man, Rogosa and Sharpe (MRS) media (Carl Roth GmbH, Germany, Art. No. X924.1). Genetically engineered L. plantarum WCFS1 strains were grown in MRS media supplemented with 10 µg/mL of erythromycin (Carl Roth GmbH, Art. No. 4166.2) at 37 °C and 250 revolutions per minute (rpm) for approximately 16 h. NEB 5-alpha Competent E. coli cells were used (New England Biolabs GmbH, Germany, Art. No. C2987) for the cloning of certain plasmids. This strain was maintained in Luria-Bertani (LB) media (Carl Roth GmbH, Art. No. X968.1). Genetically engineered E. coli DH5α strains were grown in LB media supplemented with 200 µg/mL of erythromycin at 37 °C, 250 rpm shaking conditions for approximately 16 h. The pLp_3050sNuc plasmid, which was used as the backbone vector in this study, was a kind gift from Prof. Geir Mathiesen (Addgene plasmid # 122,030). E. coli Nissle was a kind gift from Prof. Rolf Müller. L. plantarum ϕg1e was a kind gift from Dr. Makiko Kakikawa.
Erythromycin
Manufactured by Carl Roth
Sourced in Germany, Poland
Erythromycin is a macrolide antibiotic compound. It is used in the production and analysis of various pharmaceutical and chemical products.
Automatically generated - may contain errors
Lab products found in correlation
Chloramphenicol, by Carl Roth (3 mentions)
Ampicillin, by Carl Roth (3 mentions)
Neb 5 alpha competent e coli cells, by New England Biolabs (2 mentions)
Streptomycin, by Carl Roth (2 mentions)
Tigecycline, by Merck Group (1 mentions)
Vancomycin, by Merck Group (1 mentions)
Meropenem, by Merck Group (1 mentions)
Spectinomycin, by Carl Roth (1 mentions)
Clindamycin, by AppliChem (1 mentions)
Amikacin, by Merck Group (1 mentions)
Linezolid, by Merck Group (1 mentions)
Penicillin g, by AppliChem (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Ciprofloxacin, by AppliChem (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Tetracycline, by Carl Roth (1 mentions)
Mueller hinton broth (mhb), by Merck Group (1 mentions)
Gentamicin, by Carl Roth (1 mentions)
Luria bertani medium, by Carl Roth (1 mentions)
Kanamycin sulfate, by Carl Roth (1 mentions)
6 protocols using erythromycin
1
Genetic Engineering of Lactobacillus plantarum
2
Antibiotic Susceptibility Testing Protocol
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Antibiotic MICs were determined by the broth microdilution method according to the CLSI standard guidelines M07-A8, M100-S20, and M45-P (50 , 51 , 53 ). Meropenem, amikacin, gentamicin, linezolid, and tigecycline were purchased from Sigma Aldrich (Germany). Vancomycin and tetracycline were obtained from Merck (Germany). Clindamycin, ciprofloxacin, lincomycin, and penicillin G were obtained from Applichem (Germany). Ampicillin, chloramphenicol, spectinomycin, streptomycin, and erythromycin were purchased from Carl Roth GmbH (Germany). MICs of the quality control strains S. aureus ATCC 29213 and E. coli ATCC 25922 were recorded on each day for all tested antibiotics and fell within the acceptable range prescribed by the CLSI.
3
Antibiotic Susceptibility of Campylobacter Strains
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The susceptibility of the isolated Campylobacter strains to selected antibiotics was tested by determining the minimum inhibitory concentrations (MIC) in Mueller Hinton broth (Sigma‐Aldrich). MICs were determined by the broth microdilution procedure in 96‐well flat‐bottomed microtiter plates according to Andrews (2001 ). The antibiotics tested were ciprofloxacin (CIP; Fluka), amoxicillin (AML; Fluka), ampicillin (AMP; Roth, Poland), gentamicin (CN; Roth), streptomycin (S; Roth), tetracycline (TE; Roth), erythromycin (E; Roth), and chloramphenicol (C; Roth). The concentrations for the antibiotics ranged from 0.5 to 256 µg/ml for tetracycline and erythromycin; from 0.25 to 128 µg/ml for amoxicillin, ampicillin, chloramphenicol, erythromycin, and gentamicin; and from 0.125 to 64 µg/ml for ciprofloxacin. The plates were incubated for 24 hr at 42°C in microaerophilic conditions. A C. jejuni reference strain (ATCC 33560) was used as a control. All tests were run in triplicate. The Campylobacter spp. isolates were classified as resistant or susceptible according to EUCAST, European Committee on Antimicrobial Susceptibility Testing 2016a , 2016b .
4
Construction of Recombinant Lactobacillus and E. coli
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L. plantarum WCFS1 was used as the parent strain in this study. The strain was grown in the De Man, Rogosa and Sharpe (MRS) media (Carl Roth GmbH, Germany, Art. No. X924.1). Recombinant L. plantarum WCFS1 strains were grown in MRS media supplemented with 10 μg/mL of erythromycin (Carl Roth GmbH, Art. No. 4166.2) at 37°C and 250 rpm shaking for 16 h. For the indirect cloning experiments, NEB 5-alpha Competent E. coli cells were used (New England Biolabs GmbH, Germany,Art. No. C2987). This strain was grown in Luria-Bertani (LB) medium (Carl Roth GmbH, Art. No. X968.1). Recombinant E. coli DH5α strains were grown in LB media supplemented with 200 μg/mL of erythromycin at 37°C, 250 rpm shaking conditions for 16 h.
5
Versatile Bacterial Cultivation Protocol
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The tryptic soy broth (TSB, CASO Buillon, X938.2) was from Carl Roth (Karlsruhe, Germany). The kanamycin sulfate (≥750 I.U./mg, T832.1), ampicillin sodium salt (≥97%, K029.5), chloramphenicol (≥98.5%, 3886.1), geneticin disulfate (Bio-Science grade, 2039.2), and erythromycin (≥98.0%, 4166.1) were from Carl Roth. The tetracycline (≥98.0%, 87128) was from Fluka (Vienna, Austria) and poly-(R)-3-hydroxybuttersäure (quality level 200, 363502) from Sigma-Aldrich (Vienna, Austria). Other chemicals were from Sigma-Aldrich/Fluka or Carl Roth and were of the highest purity available. An enzymatic assay for ammonium (K-AMIAR) was obtained from Megazyme International (Wicklow, Ireland). The strain C. necator H16 DSM 428 (aka ATCC 17699, NCIB 10442) was from DSMZ, Deutsche Sammlung für Mikroorganismen und Zellkulturen.
6
Construction and use of pMAD shuttle vector in Enterococcus faecalis
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The pMAD Gram-positive, temperature-sensitive mutagenesis shuttle vector has been described previously. 14 Enterococcus faecalis 12030 strain was grown in tryptic soy broth (TSB; Carl Roth, Karlsruhe, Germany) medium or on TSA plates (Carl Roth) at 37 C for 18 h. 15 For growth of E. faecalis 12030ÁfabN TSB media or TSA plates were supplemented with 0.1 mM oleic acid (Sigma Aldrich, St. Louis, MO, USA). When required, medium was supplemented with 50 mg/ml erythromycin (Carl Roth). Escherichia coli XL-1-blue (Invitrogen, Carlsbad, CA, USA), containing pMAD, was grown in lysogeny broth supplemented with 100 mg/ml ampicillin (Carl Roth) at 30 C with agitation (200 rpm) for 48 h. For blue/white selection, agar plates were supplemented with 80 mg/ml X-gal (Applichem, Chicago, IL, USA).
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