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SUPPLEMENTARY MATERIAL
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Antimicrobial Activity of Human β-defensins Against Lactic Acid Bacteria
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Xiao-Fang Wang, Fei Tian, Rui-Ming Cao, Jing Li, Sheng-Mei Wu, Xiao-Kui Guo and Tong-Xin Chen
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Abstract In this study we evaluated the antimicrobial activity of human β-defensin-1 (hBD-1), human
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β-defensin-2 (hBD-2) and human β-defensin-3 (hBD-3) against 3 international common probiotic
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strains of Lactic acid bacterium (LAB). Our results indicated that hBD-1, -2 and -3 at the range of
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0.08-10 µg/ml does not have obvious antimicrobial activity against these strains. Viability of
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Bifidobacterium longum (B. longum) JDM301, Bifidobacterium lactis (B. lactis) HN019 and
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Lactobacillus rhamnosus GG (LGG) were still very high even at concentration of 10 µg hBD/ml. Then
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we explored the mechanism of resistance by using carbonyl cyanide 3-chlorophenylhydrazone (CCCP)
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to inhibit efflux pumps. In the presence of CCCP, hBD-1, -2 and -3 exhibited enhanced antibacterial
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effect against B. longum JDM301 and B. lactis HN019, but not against LGG. Efflux pumps in B.
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longum JDM301 and B. lactis HN019 may contribute to their resistance to hBD-1, -2, and -3.
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Keywords: Human β-defensin; Probiotics; Efflux pump; Antimicrobial peptide; Defensin
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Experimental
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Bacterial Strains and Culture Conditions
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All bacteria strains were provided by Department of Medical Microbiology and Parasitology, Institutes
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of Medical Sciences, Shanghai Jiao Tong University School of Medicine. B. longum JDM301, B. lactis
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HN019 and LGG were incubated in de Man-Rogosa-Sharpe broth (MRS broth) (Difco Laboratories,
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Detroit, USA) supplemented with 0.05% (w/v) cysteine hydrochloride in anaerobic conditions using
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Whitley DG250 Anaerobic Workstation (Don Whitley Scientific Ltd., Shipley, England) at 37℃ as
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reported previously (Liu et al. 2009).
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Antimicrobial Assay
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The β-defensins hBD-1,-2 and -3 were purchased from ProSpec-Tany TechnoGene, Ltd. (Rehovot,
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Israel). Different concentrations of hBD working solution were achieved by serial twofold dilution in
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broth. The microbroth dilution method was performed using sterile 96-well microtiter plate, slightly
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modified from the Clinical Laboratory Standards Institute (CLSI 2007). Ten microliters of hBD
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solution and 80 µl MRS broth was added to each well. Then 10 µl bacteria suspension (1×107 CFU/mL)
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was added. The final concentrations of hBDs were 10 µg/ml, 5 µg/ml, 2.5 µg/ml, 1.25 µg/ml, 0.625
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µg/ml, 0.31 µg/ml, 0.16 µg/ml and 0.08 µg/ml. Wells containing 10 µl bacteria and 90 µl MRS broth
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alone were used as positive control. Wells containing 100 µl MRS broth alone were used as negative
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control.
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After incubation at 37°C for 24 h, the optical density (OD) was read at 600 nm using a microplate
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spectrophotometer (DAS, Rome, Italy) and was compared with the positive control. The final results
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are expressed as viability and was calculated as follows: viability (%) = (OD in the presence of
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hBDs-OD of the negative control/OD of the positive control-OD of the negative control)×100. All
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assays were performed in triplicate and the results are from three independent experiments with the
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data presented as mean viability ± standard deviation.
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CCCP inhibiting efflux pump test
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Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma Chemicals, St. Louis, MO) was soluted in
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dimethyl sulfoxide (DMSO) and suspended in MRS Broth. The final concentration of CCCP was
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determined to be 10 μg/ml, referred to previous literatures (Brissette & Lukehart 2007) and verified as
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nontoxic to the testing bacteria in our pre-experiment. Seventy microliters MRS broth and 10 µl
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bacteria were added in microliter wells followed by CCCP solution, and then exposed to hBDs with
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final volume and concentrations the same as above antimicrobial assay. CCCP effect was expressed as
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increased bacterial killing percentage, using the following formula: increased killing (%) = (viability
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without CCCP – viability in the presence of CCCP/ viability without CCCP) ×100.
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Figure S1. Antimicrobial activity of hBDs. Antimicrobial activity is expressed as viability (%). Data
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represents the mean±SD of three independent experiments. hBD, human β-defensin.
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Figure S2. Effect of CCCP on the susceptibility of 3 probiotic strains to hBDs. CCCP capacity was
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expressed as increased killing (%). Data represents the mean±SD of three independent experiments.
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hBD, human β-defensin; CCCP, carbonyl cyanide 3-chlorophenylhydrazone.
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References
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Brissette CA, Lukehart SA. 2007. Mechanisms of decreased susceptibility to beta-defensins by
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Treponema denticola. Infect Immun. 75:2307-2315.
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[CLSI] Clinical and Laboratory Standards Institute. 2007. Methods for antimicrobial susceptibility
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testing of anaerobic bacteria. Approved standard. Wayne, PA: CLSI Publication M11-A7.
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Liu C, Zhang ZY, Dong K, Yuan JP, Guo XK. 2009. Antibiotic resistance of probiotic strains of lactic
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acid bacteria isolated from marketed foods and drugs. Biomed Environ Sci. 22:401-412.