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. 2020 Jun 2;10:9021. doi: 10.1038/s41598-020-66082-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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(a) Efficacy of 8 MLC Cip, 40 mM D-AA and 40 mM D-AA combined with 1 MLC, 4 MLC and 8 MLC Cip on biofilm density whilst inhibiting and dispersing S. aureus biofilms. Two tailed T-test revealed that D-AA have a significantly (indicated by *) greater anti-biofilm activity (inhibition p < 0.001, dispersal p < 0.0001) as compared to 8x MLC of Ciprofloxacin. One-way ANOVA showed no significant synergistic effect (p > 0.05) on biofilm density between D-AA and when D-AA and Cip are used in combination. a-e) Synergistic effect of using combination of 1 MLC (b,d) or 4 MLC (c,e) Cip with 2.5, 5, 10 and 20 mM D-AA on biofilm inhibition (b,c) and dispersal (d,e). One-way ANOVA showed an overall significant difference for amino D-AA + Cip data (b-e). This was followed by a post-hoc t-test with Bonferroni correction to see which concentration of D-AA + Cip had significantly higher (p < 0.01) anti-biofilm activity as compared Cip on its own. Next one-tail t test was done to see whether this combination of D-AA + Cip had significantly (p < 0.05) improved anti-biofilm activity as compared to the corresponding D-AA on its own. (b) 10 mM D-AA + 1 MLC Cip was significantly (*) able to inhibit greater biofilm formation compared to both Cip and D-AA on their own (p < 0.001 and p < 0.00001 respectively). (c) 20 mM D-AA + 4 MLC Cip was significantly (*) able to inhibit greater biofilm formation compared to both Cip and D-AA on their own (p = 0.00014 and p = 0.014 respectively). (d) No combination was significantly able to disperse greater biofilm compared to both Cip and D-AA on their own (e) 5 mM D-AA + 4 MLC Cip gave significant (*) rise in biofilm dispersal activity compared to both Cip and D-AA on their own (p = 0.0045 and p = 0.018 respectively). (a-e) D-AA = 40 mM D-Aspartic acid and 40 mM D-Glutamic acid; x MLC = x times minimum lethal concentration; Cip = ciprofloxacin; n = 3, ±S.D.