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Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections
Open accessAbstract
Diabetes mellitus (DM) patients frequently develop diabetic foot ulcers (DFU) which are generally infected by a community of microorganisms, mainly Staphylococcus aureus and Pseudomonas aeruginosa. These bacteria exhibit a multi-drug resistance profile and biofilm-forming ability which represent a hurdle in the treatment of diabetic foot infections (DFI). We aimed to evaluate the potential of Nisin Z, an antimicrobial peptide (AMP), as an alternative treatment for severe DFI. Nisin Z shows antibacterial activity against Gram-positive and Gram-negative bacteria and an increased antibacterial effect against Gram-negatives when added to EDTA. As such, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Minimum Biofilm Inhibitory Concentration (MBIC), and Minimum Biofilm Eradication Concentration (MBEC) were determined for Nisin Z, Nisin Z + EDTA (0.4%), and Nisin Z + EDTA incorporated into guar gum, in order to test its efficacy against S. aureus and P. aeruginosa isolated from the same DFU. Results showed that Nisin Z added to the chelation agent EDTA displayed higher antibacterial and bacteriostatic efficacy against mono and dual co-cultures of S. aureus and P. aeruginosa, and higher antibiofilm efficiency against monocultures. Nisin Z was moderately cytotoxic at 200 µg/mL. Prospect in vivo studies are needed to confirm the potential of Nisin Z supplemented with EDTA to be used as a complement to conventional antibiotic therapy for severe DFI.
Overview
CoChat Summary
TL;DR
Nisin Z supplemented with EDTA (0.4%) was the most promising tested solution against diabetic foot infection isolates of *S. aureus* and *P. aeruginosa*, showing higher antibacterial/bacteriostatic efficacy against mono- and dual planktonic co-cultures and higher antibiofilm efficacy against monocultures. However, biofilm eradication was not achieved at moderate-cytotoxic levels, and in vivo studies are still needed.
Key findings
- Nisin Z alone inhibited *S. aureus* Z25.2 with MIC mean 6.11 ± 2.2 µg/mL and MBC mean 18.8 ± 6.9 µg/mL, while *P. aeruginosa* Z25.1 and the dual DFI co-culture were resistant (MIC and MBC > 400 µg/mL). EDTA alone showed no inhibitory effect from 250 to 8000 µg/mL (MIC/MBC > 8000 ± 0 µg/mL).
- Adding EDTA (0.4%) made all tested planktonic suspensions susceptible: *S. aureus* MIC mean 1.0 ± 1.1 µg/mL and MBC mean 8.6 ± 5.3 µg/mL; *P. aeruginosa* MIC mean 2.5 ± 0 µg/mL and MBC mean 150.0 ± 53.5 µg/mL; dual DFI co-culture MIC mean 2.5 ± 0 µg/mL and MBC mean 100.0 ± 0 µg/mL. The paper classifies Nisin Z + EDTA as bacteriostatic against *S. aureus* Z25.2, *P. aeruginosa* Z25.1, and dual co-cultures.
- For biofilms, *S. aureus* Z25.2 was susceptible to Nisin Z solutions at low MBIC values: Nisin Z MBIC mean 6.7 ± 2.5 µg/mL and MBEC mean 366.7 ± 81.6 µg/mL; Nisin Z + EDTA MBIC mean 0.5 ± 0 µg/mL and MBEC mean 350.0 ± 92.6 µg/mL; Nisin Z + EDTA + guar gum MBEC mean 622.2 ± 210.8 µg/mL. *P. aeruginosa* and dual DFI co-culture biofilms had MBEC > 1250 µg/mL, and guar gum decreased dual DFI co-culture MBIC from 275.0 ± 147.5 to 100 ± 0.0 µg/mL.
- Cytotoxicity against HEKa cells: Nisin Z was non-cytotoxic from 5 µg/mL (90.3% viability) to 25 µg/mL (84.6%), weakly cytotoxic at 50 and 100 µg/mL (78.9% and 68.2%), moderately cytotoxic at 200 µg/mL (56.7%), and strongly cytotoxic at 400 and 1250 µg/mL (2.5% and 0.9%). EDTA-containing solutions were cytotoxic at all tested concentrations (9.2–33.1% with EDTA; 4.6–17.4% with EDTA + guar gum).
Methods
- In vitro study using biofilm-producing DFI strains *S. aureus* Z25.2 and *P. aeruginosa* Z25.1, co-isolated from the same diabetic foot ulcer, plus reference strains *S. aureus* ATCC 29213 and *P. aeruginosa* ATCC 27853.
- Tested three Nisin Z solutions: Nisin Z alone, Nisin Z + EDTA (0.4%), and Nisin Z + EDTA (0.4%) incorporated into 0.75% (w/v) guar gum gel, against mono- and dual planktonic and biofilm cultures.
- MIC/MBC by microtitre broth dilution; MBIC/MBEC using a modified Calgary Biofilm Pin Lid Device; cytotoxicity by MTT assay on adult human primary epidermal keratinocytes (HEKa). Data analyzed with GraphPad Prism, mean ± SEM, one-way ANOVA (p < 0.0001) and Tukey’s multiple comparisons test, with triplicate experiments and at least three independent replicates.
Limitations
- The paper states that in vivo studies are needed to confirm efficacy and safety; Nisin Z at 200 µg/mL was moderately cytotoxic, and EDTA cytotoxicity must be analyzed carefully because it may be associated with specific in vitro conditions and probably does not relate to in vivo cell toxicity.
- The paper notes that after guar gum addition, only dual co-cultures were evaluated for MIC/MBC, and Nisin Z MBIC/MBEC toward *P. aeruginosa* and dual DFI biofilms were not evaluated because those cultures were assumed to remain resistant.
- Evident from the text: findings are based on a single DFI strain pair, rely on in vitro visual endpoints for MIC/MBIC/MBEC, and biofilm eradication was not attainable in any tested Nisin Z solution at moderate-cytotoxic levels.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“MIC and MBC of EDTA were evaluated and showed no inhibitory effect in any tested concentration (250 to 8000 µg/mL) towards planktonic DFI and control strains. EDTA exhibited a MIC and MBC mean value of >8000 ± 0 µg/mL.”
§ Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections > 3. Results > 3.1. MIC and MBC
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Therefore, Nisin Z was non-cytotoxic from 5 µg/mL (HEKa cell viability of 90.3%) to 25 µg/mL (HEKa cell viability of 84.6%), weakly cytotoxic at 50 and 100 µg/mL (HEKa cell viability of 78.9% and 68.2%), moderately cytotoxic at 200 µg/mL (HEKa cell viability of 56.7%), and strongly cytotoxic at 400 and 1250 µg/mL (HEKa cell viability of 2.5 and 0.9%, respectively).”
§ Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections > 3. Results > 3.3. Cell Toxicity
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Comparing the results for P. aeruginosa Z.25.1 to those for the reference strain, only Nisin Z + EDTA (0.4%) MIC value was significantly different.”
§ Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections > 3. Results > 3.1. MIC and MBC
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper states — it cites no source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Nisin Z was moderately cytotoxic at 200 µg/mL.”
§ Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections > Abstract
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Rests on an assumption not shown here.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“and higher antibiofilm efficiency against monocultures.”
§ Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections > Abstract
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
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Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Rated major: it would touch a result — a value a result rests on, reported and recomputed p-values on opposite sides of the significance level, two passages that conflict with no sign which is right, or a cited work retracted before this paper was published.
In the paper
§ Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections > 4. Discussion
“The addition of guar gum was beneficial for dual DFI co-cultures because it significantly increased the antibiofilm efficacy to nontoxic Nisin Z levels.”
§ Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections > 3. Results > 3.3. Cell Toxicity
“The addition of EDTA (0.4%), and of EDTA (0.4%) + guar gum gel 0.75% (w/v) to Nisin Z render these solutions cytotoxic in all concentrations tested, with cell viability ranging from 9.2% to 33.1% and from 4.6% to 17.4%, respectively (Table 2).”
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
“MBC values were two to 2.5-fold higher than MIC (MBC mean of 18.8 ± 6.9 µg/mL, ranging from 10 to 25 µg/mL).”
§ Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections > 3. Results > 3.1. MIC and MBC
Reported 2.5 · recomputed 3.076923
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
“MBC was four- to six-fold higher, with a mean of 8.6 ± 5.3 µg/mL, ranging between 1 and 15 µg/mL.”
§ Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections > 3. Results > 3.1. MIC and MBC
Reported 6 · recomputed 8.6
Citations
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Practical Guidelines on the prevention and management of diabetic foot disease (IWGDF 2019 update)
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Research works printed but not linked: 1
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Cited by
Wang, Q., Liu, C., An, J., Liu, J., Wang, Y., & Cai, Y. (2025) · Frontiers in Endocrinology · doi:10.3389/fendo.2025.1657928 · Cited by 77
Derakhshan-sefidi, M., Bakhshi, B., & Rasekhi, A. (2024) · BMC Microbiology · doi:10.1186/s12866-024-03400-7 · Cited by 36
Biofilm in Diabetic Foot Ulcers: A Systematic Narrative Review
Theodorakopoulos, G., & Armstrong, D. G. (2025) · International Wound Journal · doi:10.1111/iwj.70795 · Cited by 30
Antimicrobial Resistance in Pseudomonas aeruginosa before and during the COVID-19 Pandemic
Serretiello, E., Manente, R., Dell’Annunziata, F., Folliero, V., Iervolino, D., Casolaro, V., Perrella, A., Santoro, E., Galdiero, M., Capunzo, M., et al. (2023) · Microorganisms · doi:10.3390/microorganisms11081918 · Cited by 28
Qin, X., Cai, X., Wang, Y., Chen, L., Zhao, J., Zhang, Y., Bi, S., Zhou, Y., Zhu, Q., Cheng, Y., et al. (2024) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2024.134548 · Cited by 24
Metadata
- Authors
- Serrano, I., Alhinho, B., Cunha, E., Tavares, L., Trindade, A., & Oliveira, M.
- Affiliations
- University of Lisbon (PT)
- Corresponding author
- Oliveira, Manuela
- Venue
- Life
- Publisher
- MDPI AG
- Published
- 2023-02-11
- Volume
- 13
- Issue
- 2
- Pages
- 504
- ISSN
- 2075-1729 (ISSN-L)
- PMC
- PMC9964538
- OpenAlex
- W4320491567
- Semantic Scholar
- bd6f58c15995f1306d145b96757788c45b823795
- Cited by
- 33
- References
- 44
- Citations by year
- 2026: 5 · 2025: 14 · 2024: 8 · 2023: 6
- FWCI
- 5.03
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Life (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- FCT—Fundação para a Ciência e a Tecnologia (PTDC/SAUINF/28466/2017)
- Topics
- Life Sciences › Immunology and Microbiology › Microbiology › Antimicrobial Peptides and Activities
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
- Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Antimicrobial agents and applications
- Keywords
- nisin Z, antimicrobial peptides, diabetic foot infection, diabetic foot ulcer, Staphylococcus aureus, Pseudomonas aeruginosa, co-culture, biofilm eradication, EDTA chelation, guar gum, bacteriostatic activity, minimum biofilm inhibitory concentration, minimum biofilm eradication concentration, cytotoxicity, multidrug resistance
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